Antimicrobial Peptides (Advances in Molecular and Cellular Biology Series)

Advances in Molecular and Cellular Microbiology 18

Antimicrobial Peptides Discovery, Design and Novel Therapeutic Strategies

Edited by

Guangshun Wang, PhD

Eppley Institute University of Nebraska Medical Center Omaha, Nebraska, USA Advances in Molecular and Cellular Microbiology

Through the application of molecular and cellular microbiology we now recognize the diversity and dominance of microbial life forms that exist in all environments on our planet. These microbes have many important planetary roles, but for humans a major problem is their ability to colonize our tissues and cause disease. The same techniques of molecular and cellular microbiology have been applied to the problems of human and animal infection since the 1990s and have proved to be immensely powerful tools in elucidating how microorganisms cause human pathology. This series has the aim of providing information on the advances that have been made in the application of molecular and cellular microbiology to speciﬁ c organisms and the diseases they cause. The series is edited by researchers active in the application of molecular and cellular microbiology to human disease states. Each volume focuses on a particular aspect of infectious disease and will enable graduate students and researchers to keep up with the rapidly diversifying literature in current microbiological research.

Series Editors

Professor Brian Henderson University College London

Professor Michael Wilson University College London Titles Available from CABI

17. Helicobacter pylori in the 21st Century Edited by Philip Sutt on and Hazel M. Mitchell

18. Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies Edited by Guangshun Wang

Titles Forthcoming from CABI

Stress Response in Pathogenic Bacteria Edited by Stephen Kidd

Lyme Disease: an Evidence-based Approach Edited by John Halperin

Tuberculosis: Molecular Techniques in Diagnosis and Treatment Edited by Timothy McHugh

Microbial Metabolomics Edited by Silas Villas-Bôas and Katya Ruggiero

Earlier titles in the series are available from Cambridge University Press (www.cup.cam.ac.uk). CABI is a trading name of CAB International

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Library of Congress Cataloging-in-Publication Data

Antimicrobial peptides : discovery, design, and novel therapeutic strategies / edited by Guangshun Wang. p. ; cm. -- (Advances in molecular and cellular microbiology ; 18) Includes bibliographical references and index. ISBN 978-1-84593-657-0 (alk. paper) 1. Peptide antibiotics. I. Wang, Guangshun. II. Series: Advances in molecular and cellular microbiology ; 18. [DNLM: 1. Antimicrobial Cationic Peptides. 2. Anti-Infective Agents. 3. Drug Design. 4. Immunity, Innate. QU 68 A6308 2010]

RS431.P37A575 2010 615’.1--dc22

2010016796

ISBN-13: 978 1 84593 657 0

Commissioning editor: Rachel Cutt s Production editor: Tracy Head

Typeset by Columns Design Ltd, Reading, UK. Printed and bound in the UK by CPI Antony Rowe, Chippenham. Contents

Contributors xi Preface xiii Introduction xv Michael Zasloﬀ

Part I: Natural Antimicrobial Peptides: Nomenclature, Classification and Interesting Templates for Peptide Engineering

1 A Database View of Naturally Occurring Antimicrobial Peptides: Nomenclature, Classifi cation and Amino Acid Sequence Analysis 1 Guangshun Wang, Xia Li and Michael Zasloff 1.1 Database Scope and Overview 2 1.2 Nomenclature of Antimicrobial Peptides 3 1.2.1 Peptide-based method 4 1.2.2 Source-based method 4 1.2.3 Source and peptide combined method 4 1.3 Annotation and Classifi cation of Antimicrobial Peptides 5 1.3.1 Source organisms and peptide family classifi cation 5 1.3.2 Classifi cation based on biological activities 8 1.3.3 Classifi cation based on peptide characteristics 9 1.3.4 Peptide-binding targets and mechanisms of action 14 1.4 Amino Acid Sequence Analysis of Antimicrobial Peptides 15 1.5 Concluding Remarks 17

2 Lantibiotic-related Research and the Application Thereof 22 Brian Healy, Jim O’Mahony, Colin Hill, Paul D. Cott er and R. Paul Ross 2.1 Introduction to Bacteriocins 22 2.2 Biosynthesis and Post-translational Modiﬁ cations 23 2.3 Classiﬁ cation 24 2.4 Lantibiotic Subgroups: Biology, Structure and Mode of Action 25 2.4.1 Nisin-like lantibiotics 25 2.4.2 Epidermin-like lantibiotics 26

v vi Contents

2.4.3 Planosporicin- and streptin-like lantibiotics 27 2.4.4 Pep5-like peptides 27 2.4.5 Lacticin 481-like lantibiotics 27 2.4.6 Mersacidin-like lantibiotics 28 2.4.7 LtnA2-like peptides 28 2.4.8 Other type II lantibiotics 28 2.5 Current and Future Use of Lantibiotics for Food Applications 29 2.6 Lantibiotics and Their Medical Applications 30 2.7 Engineering of Lantibiotics 30 2.8 Screening for New Lantibiotics 31 2.9 Concluding Remarks and Perspectives 32

3 Antimicrobial Peptides in Plants 40 Quentin Kaas, Jan-Christoph Westermann, Sónia Troeira Henriques and David J. Craik 3.1 Introduction to Plant Antimicrobial Peptides 40 3.1.1 Lipid-transfer proteins 41 3.1.2 Thionins 43 3.1.3 Plant defensins 44 3.1.4 Chitin-binding peptides 45 3.1.5 Miscellaneous AMPs from plants 48 3.1.6 Non-ribosomal antimicrobial peptides 49 3.2 Cyclotides 49 3.2.1 Diversity of plant cyclotides 49 3.2.2 Detection and isolation of cyclotides 54 3.2.3 Cyclotide biosynthesis 54 3.2.4 Biological activities of cyclotides 56 3.2.5 Cyclotide engineering and mass production 62 3.3 Concluding Remarks and Perspectives 63

Part II: Expanding the Peptide Space: Prediction Methods, Design Strategies and Peptidomimetics

4 Database-aided Prediction and Design of Novel Antimicrobial Peptides 72 Guangshun Wang 4.1 Database-aided Antimicrobial Peptide Prediction 73 4.1.1 Prediction based on mature peptides 73 4.1.2 Prediction based on highly conserved propeptide sequences 75 4.1.3 Prediction based on both propeptides and mature peptides 75 4.1.4 Prediction based on processing enzymes 76 4.1.5 Genomic context-based bacteriocin prediction 76 4.2 Database-aided Peptide Design and Improvement 76 4.2.1 Database screening for HIV inhibitory antimicrobial peptides 76 4.2.2 Sequence shuﬄ ing is a primitive combinatorial approach 77 4.2.3 The hybrid approach and grammar-based peptide design 78 4.2.4 De novo peptide design 78 4.2.5 Database-aided enhancement of peptide anti-HIV activity 80 4.3 Strategies for Improving Cell Selectivity of AMPs 80 4.4 Strategies for Improving Peptide Stability to Proteases 82 4.5 Concluding Remarks 82 Contents vii

5 Discovery of Novel Antimicrobial Peptides Using Combinatorial Chemistry and High-throughput Screening 87 William C. Wimley 5.1 The Interfacial Activity Model of AMP Activity 87 5.2 Combinatorial Chemistry Methods 88 5.2.1 Overview of library synthesis 88 5.2.2 Non-indexed methods 89 5.2.3 Indexed methods 90 5.3 High-throughput Screening 90 5.3.1 Biological assays 90 5.3.2 Selection of broad-spectrum peptide antibiotics 92 5.3.3 Non-biological assays 94 5.4 Accomplishments 97 5.4.1 Beyond high-throughput screening 97 5.5 Future Directions 98

6 Chemical Mimics with Systemic Eﬃ cacy 100 Amram Mor 6.1 Introduction 100 6.2 De Novo-designed Rigid Peptidomimetics 102 6.2.1 Hairpin-shaped peptides 102 6.2.2 Peptoids 103 6.2.3 Arylamides 103 6.3 Acyl-lysyl Oligomers 104 6.3.1 Design 104 6.3.2 Structure–activity relationships 104 6.3.3 Antibacterial properties 107 6.3.4 Antiparasital properties 109 6.4 Concluding Remarks and Perspectives 110

Part III: Biophysics, Structural Biology and Mechanism of Action of Antimicrobial Peptides

7 Biophysical Analysis of Membrane-targeting Antimicrobial Peptides: Membrane Properties and the Design of Peptides Specifi cally Targeting Gram-negative Bacteria 116 Richard M. Epand and Raquel F. Epand 7.1 Diff erences in Chemical Composition and Molecular Organization of Gram-positive and Gram-negative Bacteria 116 7.2 Role of Bacterial Membrane Lipid Composition in Antimicrobial Sensitivity 118 7.3 Antimicrobial Agents that Target the Outer Membrane of Gram-negative Bacteria 119 7.4 Antimicrobial Agents that Promote Clustering of Anionic Lipids 120 7.4.1 Evidence for lipid clustering 120 7.4.2 Bacterial species specifi city of toxicity 121 7.4.3 Putative mechanism of action 124 7.4.4 Properties of antimicrobial agents that favour clustering 125 7.4.5 Sources of energy to account for clustering and entropy of demixing 125 7.5 Concluding Remarks and Perspectives 125 viii Contents

8 Non-membrane Targets of Antimicrobial Peptides: Novel Therapeutic Opportunities? 128 Ju Hyun Cho and Sun Chang Kim 8.1 Introduction 128 8.2 Buforin II: α-Helical AMP with Single Proline Residue Binding to Nucleic Acids 130 8.3 PR-39 and Indolicidin: Mammalian Proline-rich Peptides Inhibiting Macromolecule Synthesis and Cell Division 131 8.4 Apidaecin, Drosocin and Pyrrhocoricin: Short, Insect Proline-rich Peptides Binding to Heat-shock Protein DnaK 133 8.5 Concluding Remarks and Perspectives 136

9 Structural Studies of Antimicrobial Peptides Provide Insight into Their Mechanisms of Action 141 Guangshun Wang 9.1 Human Defensins and Cathelicidins 141 9.2 Bacterial Expression and Puriﬁ cation of Antimicrobial Peptides 143 9.3 Structural Studies of Membrane-targeting Antimicrobial Peptides 146 9.3.1 Membrane-mimetic models 146 9.3.2 NMR methods 147 9.3.3 Three-dimensional structures of antimicrobial peptides 149 9.3.4 Interactions of antimicrobial peptides with bacterial membranes by NMR spectroscopy 155 9.3.5 Structural basis of peptide selectivity 159 9.4 Structure-based Peptide Engineering 159 9.5 Concluding Remarks and Perspectives 160

Part IV: Novel Therapeutic Strategies to Bolster Host Defence

10 Lung Infection: Shift ing the Equilibrium Towards the Free and Active Form of Human LL-37 and the Design of Alternative Antimicrobial Agents 169 Paul A. Janmey and Robert Bucki 10.1 Introduction 169 10.1.1 Electrostatic properties of LL-37 169 10.1.2 Interaction of polyvalent cations with linear polyelectrolytes 170 10.2 Production of Anionic Polyelectrolytes by Host and Microbial Sources 171 10.3 Sequestration and Inactivation of LL-37 by DNA and F-actin 172 10.4 Releasing LL-37 from Polyelectrolyte Bundles 172 10.4.1 Severing polymers with DNase, gelsolin and alginase 173 10.4.2 Destabilizing bundles with small multivalent anions 174 10.5 Designing Antimicrobial Agents Resistant to Inactivation by Polyelectrolytes 174 10.5.1 Cationic steroids: minimizing and redistributing charge 175 10.5.2 Increasing the hydrophobicity of antimicrobial agents 175 10.6 Possible Therapeutic Use 175 10.6.1 Selective use in some body ﬂ uids and not others 175 10.6.2 Stimulation of host cells 176 10.7 Conclusion 176 Contents ix

11 Role of Vitamin D in the Enhancement of Antimicrobial Peptide Gene Expression 181 John H. White and Ari J. Bitt on 11.1 Vitamin D 181 11.2 Early History of Vitamin D 181 11.3 Vitamin D Deﬁ ciency 182 11.4 Vitamin D Signalling and Mechanisms of Action 182 11.5 Overview of Vitamin D and Immune System Regulation 184 11.6 Vitamin D as a Regulator of Antimicrobial Peptide Gene Expression 185 11.7 Antimicrobial Peptides 185 11.8 Regulation of DEFB2/hBD-2 Expression by 1,25D 186 11.9 1,25D Regulation of LL-37 is Human and Primate Speciﬁ c 187 11.10 Broader Physiological and Pathophysiological Implications of the Regulation of LL-37 Expression by 1,25D 188 11.11 Therapeutic Role for Vitamin D Analogues 190

12 Fine Tuning Host Responses in the Face of Infection: Emerging Roles and Clinical Applications of Host Defence Peptides 195 Matt hew L. Mayer, Donna M. Easton and Robert E.W. Hancock 12.1 Mammalian Host Defence (Antimicrobial) Peptides 196 12.1.1 Defensins 196 12.1.2 Cathelicidins 197 12.2 The Role of Endogenous Host Defence Peptides in the Response to Infection 198 12.2.1 Natriuretic peptides 201 12.3 Potential Therapeutic Uses beyond Anti-infective Activity 202 12.3.1 Adjuvant potential 204 12.3.2 Wound-healing activity 205 12.4 Recent Clinical Advances in Therapeutic Application of Host Defence Peptides 206 12.5 Innate Defence Regulators as Anti-infective Therapeutics 208 12.6 Rational Design of Immunomodulatory Peptides 209 12.7 Limitations and Challenges 209 12.8 Conclusions 211

Index 221 This page intentionally left blank Contributors

Bitt on, Ari J., Departments of Physiology and Medicine, McGill University, McIntyre Building, Room 1112, 3655 Drummond St, Montreal, QC, Canada, H3G 1Y6 Bucki, Robert, Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA Cho, Ju Hyun, Department of Biology, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Korea Cott er, Paul D., Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Cork, Ireland. E-mail: paul.cott [email protected] Craik, David J., Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. E-mail: [email protected] Easton, Donna M., Centre for Microbial Diseases and Immunity Research, 2259 Lower Mall Research Station, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4 Epand, Raquel F., Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada, L8N 3Z5 Epand, Richard M., Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada, L8N 3Z5. E-mail: [email protected] Hancock, Robert E.W., Department of Microbiology and Immunology, University of British Columbia, Room 232, 2259 Lower Mall Research Station, Vancouver, BC, Canada, V6T 1Z4. E-mail: [email protected] Healy, Brian, Department of Microbiology, University College Cork, Cork, Ireland Hill, Colin, Alimentary Pharmabiotic Centre, Cork, Ireland. E-mail: [email protected] Janmey, Paul A., Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA. E-mail: janmey@mail. med.upenn.edu Kaas, Quentin, Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia Kim, Sun Chang, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea. E-mail: [email protected] Li, Xia, Eppley Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA Mayer, Matt hew L., Centre for Microbial Diseases and Immunity Research, 2259 Lower Mall Research Station, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4

xi xii Contributors

Mor, Amram, Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Israel. E-mail: [email protected] O’Mahony, Jim, Department of Biological Science, Cork Institute of Technology, Cork, Ireland Ross, R. Paul, Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland Troeira Henriques, Sónia, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia, and Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Egas Moniz, Edifício Egas Moniz, 1649-028 Lisbon, Portugal Wang, Guangshun, Eppley Institute, University of Nebraska Medical Centre, 986805 Nebraska Medical Center, Omaha, NE 68198, USA. E-mail: [email protected] or gwwang1@gmail. com Westermann, Jan-Christoph, Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia White, John H., Departments of Physiology and Medicine, McGill University, McIntyre Building, Room 1112, 3655 Drummond St, Montreal, QC, Canada, H3G 1Y6. E-mail: john. [email protected] Wimley, William C., Department of Biochemistry SL43, Tulane Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA. E-mail: [email protected] Zasloﬀ , Michael, Transplant Institute, Georgetown University Medical Center, Medical/Dental Building NW210, 3900 Reservoir Road NW, Washington, DC 20007, USA. E-mail: maz5@ georgetown.edu Preface

The signifi cance and benefi ts of naturally occurring antimicrobial peptides to human health have recently begun to be appreciated. As eff ector molecules of the innate immune system, they are capable of rapidly eliminating invading pathogens to keep us healthy; as signalling molecules, they elegantly modulate the adaptive immune system to trigger other physiological actions. The purpose of this book is to provide a comprehensive account on current antimicrobial peptide research in two major directions. The fi rst direction delineates the classic path for peptide development, ranging through identifi cation, design, structure and mode of action studies. The second direction describes novel strategies for developing peptide therapeutics based on our knowledge of host defence antimicrobial peptides discovered in living organisms. The book commences with a vivid and inspiring introduction contributed by Professor Michael Zasloff , a distinguished forerunner in the fi eld. Part I provides an overview of nomenclature, classifi cation and bioinformatic analysis of antimicrobial peptides from bacteria, plants and animals. Subsequently, lantibiotics from bacteria and cyclotides from plants are presented. These unique peptide templates with multiple sulfur-mediated bridges are resistant to proteases, rendering them att ractive for engineering new compounds for food preservation, pest control and curing diseases. Part II discusses database-aided peptide prediction and design methods, synthetic combinatorial libraries and peptide mimetics that expand the conformational space of natural antimicrobial peptides. These approaches also allow for the optimization of the desired properties and therapeutic index of the peptide analogues or mimicries. An in-depth understanding of the mode of action of these peptides is essential for drug development. As a consequence, Part III covers the biophysical and structural characterization of antimicrobial peptides and their complexes. While many peptides (e.g. magainin and protegrin-1) target bacterial membranes, apidaecin, buforin II, PR-39 and others can cross bacterial membranes and associate with internal targets such as heat-shock proteins and nucleic acids. Structural determination sheds light on how such peptides recognize membrane or non-membrane targets at atomic resolution, and provides the basis for structure- based peptide design. Finally, Part IV focuses on novel strategies for developing peptide-based therapies. These include liberating LL-37 from the inactive bound state in infected lungs, enhancing the expression of human cathelicidin and human β-defensin-2 by vitamin D, and stimulating immune responses to bacterial invasion by applying peptide analogues that may or may not kill bacteria directly. Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies has been writt en, and anonymously reviewed, by an enthusiastic group of recognized investigators in the fi eld.

xiii xiv Preface

As the editor, I thank all of the authors and reviewers for their contributions. In particular, I am grateful to Professor Zasloﬀ for his excellent introduction and Professors Richard M. Epand, Amram Mor and Donnatella Barra, who recruited the anonymous reviewers for the chapters writt en by the editor and his co-authors. This book is directed to graduate students, postdoctoral fellows, research faculty, principal investigators, educators, clinicians and all others who are interested in the education, research, development and applications of antimicrobial peptides. Guangshun Wang, PhD Introduction

Michael Zasloff

I have been asked by Dr Wang to provide an introduction to this wonderful book on antimicrobial peptides that he and our colleagues have assembled. Dr Wang asked me to explain how I entered the fi eld, to describe my experiences in the area of therapeutic development and to express my thoughts regarding the ‘big questions’ or perhaps ‘the grand opportunities’ that remain in our fi eld. To this day I remain amazed when I examine the cornea of a human eye and see a clear epithelial surface, which I know to be a fragile cellular layer exposed continuously to microbes of unimaginable diversity. Why does the cornea look so healthy? Why does the body not need to call great numbers of phagocytes to defend the surface, which is under continuous microbial assault? What protects that surface? Most of the epithelial surfaces of the body exhibit the almost mysterious, incomprehensible ‘health’ that is so evident in the eye. An endoscopic view of the lumenal wall of a healthy descending colon or rectum reveals a transparent single-celled layer with blood vessels visible beneath, without redness, swelling or signs of infl ammation, despite the direct physical contact between that intestinal wall and faecal bacteria. Or consider the mouth. If we are healthy and we should bite our tongue, the wound heals over the course of a day or so, within an environment far from sterile, populated by an incomprehensible diversity of bacteria, fungi and viruses. How could such a wound heal were the principal antimicrobial defences phagocytic cells att racted to the site (as proposed by Metchnikoff )? My fi rst moment of amazement regarding the ‘mystery’ of our harmonious relationship with the microbes around us came when I saw, as a resident at the Children’s Hospital in Boston, a newborn infant with cystic fi brosis. The infant was not yet 3 days old. The child had been born with meconium ileus, and cystic fi brosis was correctly suspected to be the associated medical condition. What amazed me was that Staphylococcus aureus and Pseudomonas aeruginosa were cultured from the upper respiratory tract of that newborn, before the onset of overt bronchial disease. The prevailing hypothesis at the time was that these organisms infested the airways of individuals with cystic fi brosis because the poorly draining highly viscous bronchial mucous secretions presented a favourable niche for these specifi c microbes. But clearly this explanation made no sense in the case of this newborn. My assumption was that a mechanism of antimicrobial defence existed that protected the bronchial epithelium, but was yet to be discovered. Some years later, while at the National Institutes of Health and involved in studies that required the use of the oocytes of the African clawed frog, I was struck one day that the surgical wounds that I created on these animals in the course of removing their ovaries healed without

xv xvi M. Zasloff

evidence of infl ammation or infection. This ‘ah-ha’ moment was followed by a realization that the wound on the abdomen of the frog could only heal if a potent antimicrobial mechanism existed in the skin of that animal, since these aquatic frogs were healing without diffi culty in tanks that were densely populated with microbes. Within several months I discovered the presence of antimicrobial peptides in the skin of Xenopus laevis, the fi rst being magainin. Some years earlier Hans Boman and colleagues had described the remarkable cecropins in the silk-moth pupa, and Bob Lehrer and colleagues had characterized the defensins in mammalian phagocytic cells. With my observation, three clear examples of the existence of antimicrobial peptides in animals had been described, each operating in a diff erent physiological context. With the help of many colleagues I began to search the tissues of every animal I could study, especially those tissues that seemed to be protected by ‘mysterious’ antimicrobial defences. In addition, I wanted to bett er understand how antimicrobial peptides were used in humans to maintain health, and how their failure to function properly might cause disease. At about the same time I founded Magainin Pharmaceuticals. As a physician scientist, I was thrilled by the opportunity to have the chance to bring a discovery made at the bench to the clinic. In the beginning, most of us were amazed by the antimicrobial properties of these naturally occurring, simple, short amphipathic peptides. They could rapidly kill practically every species of bacteria and many species of fungi, inactivate viruses and lyse protozoa. I can recall a dramatic moment when, following the addition of a solution of magainin into the abdominal cavity of a mouse fi lled with Ehrlich ascites tumour cells, I withdrew some ascites and could see that the peptide had killed most of the tumour cells within minutes of administration. Furthermore, the simplicity of the design of the α-helical peptides and the simplicity of the Merrifi eld method of peptide synthesis fostered an explosion of structure–activity studies. In addition, studies of animals and plants led to the discovery of an enormous diversity of naturally occurring antimicrobial peptides. An understanding of the mechanism of action of antimicrobial peptides has evolved over time. As amphipathic, cationic peptides, it was apparent from the start that antimicrobial peptides targeted the membranes of the microbes they killed, a conclusion consistent with studies utilizing model membranes. Precisely how a particular peptide strikes the fatal blow in a particular microbial target remains the subject of continued and active investigation. We continue to learn more about how antimicrobial peptides participate in defence against microbes and permit multicellular organisms to live in balance with microbes. In general, most healthy epithelial surfaces exposed to microbes express an array of antimicrobial peptides, and a détente of sorts is maintained between the microbes that utilize that surface as a niche and the epithelium that is being inhabited. Upon injury to the epithelium, batt eries of diff erent antimicrobial peptides are generally launched. Of great interest are the details of the scenarios that characterize the responses of various organs to specifi c microbes and the sett ings in which they fail to unfold normally. I believe that the existence of microbial sensors, such as the Toll- related receptors, permits this system of defence to mould an antimicrobial response that is far more microbe-specifi c than might have been otherwise imagined. We have come to learn that antimicrobial peptides, initially discovered on the basis of their antibiotic activity, exhibit other biological properties in the context of injury and infection that promote recovery and healing. The cathelicidins can stimulate epithelial growth and promote angiogenesis. Several of the mammalian defensins and cathelicidins can att ract cells of the immune system such as macrophages, neutrophils and dendritic cells. In the context of injury, as the process of repair unfolds in time, batt eries of antimicrobial peptides are successively expressed, protecting the healing surface in a temporally appropriate context and calling into the healing environment immune cells that complement the process. The relatively low potency of the chemokine activity of most antimicrobial peptides (micromolar) ensures that only those cells within close range of the site of injury will be called to action, as opposed to the more systemic signalling that occurs with the release of classical cytokines. Introduction xvii

The discovery that antimicrobial peptides are inducible has opened up the possibility of new therapeutic opportunities. The remarkable discovery that the human cathelicidin gene is induced by vitamin D, and the association of certain bacterial and viral illnesses with vitamin D defi ciency, has vast implications with respect to public health. Clinical studies suggesting that vitamin D supplementation can reduce the incidence of tuberculosis, upper respiratory infections and infl uenza A have been reported and many other trials are underway. The discovery that simple nutrients and metabolites, such as butyrate and isoleucine, can induce epithelial antimicrobial peptides is being evaluated in studies using the oral administration of these substances in the treatment of bacterial and viral dysentery in humans. Abnormal regulation of the expression of antimicrobial peptides has been linked in recent years to several human diseases. Conditions such as eczema and Crohn’s disease are associated with the depressed expression of specifi c antimicrobial peptides. In diseases such as cystic fi brosis, the milieu (the ionic strength of the airway surface fl uid) in which the antimicrobial peptide is normally designed to operate is disturbed. In these conditions the failure of antimicrobial peptides to protect the epithelial barrier forces the body to support the defence of the barrier by mounting a secondary infl ammatory response that creates the havoc of disease. In diseases such as psoriasis, however, an unexplained over-expression of epithelial antimicrobial peptides appears to excite the infl ammatory arm of the adaptive immune system, creating the skin lesions that characterize this disease. Although humans have one cathelicidin gene, we have numerous copies of the defensin family locus, resulting in diff ering levels of expression of both the α- and β-defensins between individuals. How these genetic diff erences play out in health and disease is of great interest. The development of antimicrobial peptides as human therapeutics remains a challenge. Early on it became clear that peptides such as magainin, although active in the test tube, exhibit a poor therapeutic index when evaluated in the sett ing of an infected animal. Most of the antimicrobial peptides discovered in nature seem to have this characteristic. Unfortunately, we do not know how to re-engineer a peptide to improve its pharmacological activity in such a way that would make it a more eff ective therapeutic. Certain naturally occurring molecules that exhibit the effi cacy and safety in animals expected for a therapeutic – such as plectasin from a fungal organism – have, however, been discovered. Surely the insights molecules such as these will provide will help advance the development of antimicrobial peptides as drugs. In addition, remarkable non-peptide antimicrobial molecules have been created that mimic the activity of antimicrobial peptides. These synthetic molecules exhibit an att ractive therapeutic index, and can be synthesized inexpensively. In other words, it is very likely that antimicrobial peptides, be they of natural or synthetic origin, will surely join the armamentarium of anti- infective agents in the coming years. Many of the contributors to this book have been responsible for establishing the foundations of what we now know about antimicrobial peptides. I hope the knowledge that is shared here in this book will stimulate others to make new discoveries and further advance this exciting and important fi eld. This page intentionally left blank 1 A Database View of Naturally Occurring Antimicrobial Peptides: Nomenclature, Classifi cation and Amino Acid Sequence Analysis‡

Guangshun Wang,* Xia Li and Michael Zasloff

Abstract

The nomenclature and classifi cation of naturally occurring antimicrobial peptides (AMPs) are complex and have not been fully standardized. The Antimicrobial Peptide Database (htt p://aps.unmc.edu/AP/ main.php) is a useful tool that facilitates peptide naming and classifi cation. The names of AMPs are normally derived from peptide properties, source species or a combination of both. In the database, AMPs are classifi ed based on source organisms (protozoa, bacteria, archaea, fungi, plants and animals), biological activities (antibacterial, antifungal, antiviral, antiparasitic, spermicidal and insecticidal), peptide features (charge, length, hydrophobic residue content, chemical modifi cation and three- dimensional structure), binding targets (membranes and non-membranes) and mechanisms of action of the peptides. This database also enables bioinformatic analysis of AMPs that reveals amino acid composition signatures as well as frequently occurring residues.

All organisms possess specialized defence 1981; Selsted et al., 1985; Giovannini et al., systems tailored for survival in a variety of 1987; Zasloff , 1987) stimulated research on environments. Antimicrobial peptides AMPs and led to the isolation and character- (AMPs) bridge this diversity by functioning ization of hundreds of new peptides. as key components of defence systems. In Meanwhile, the global problem of antibiotic invertebrates, AMPs are the major defence resistance further fuelled this research fi eld molecules of innate immunity. In vertebrates, with a hope of identifying new therapeutics. AMPs serve not only as eff ectors in innate The rapid increase in the number of immunity, but also as modulators for adap- AMPs demands a more effi cient data regis- tive immune systems (Shai, 2002; Tossi and tration and management method. In the past Sandri, 2002; Zasloff , 2002; Boman, 2003; several years, 13 databases have been built Brogden et al., 2005; Zanett i, 2005; Hancock for AMPs (Table 1.1). These databases facili- and Sahl, 2006; Amiche et al., 2008; Conlon, tate information management, annotation, 2008). The identifi cation of cecropins, retrieval and peptide analysis. This chapter magainins and defensins in insects, amphib- focuses on peptide nomenclature, classifi ca- ians and humans in the 1980s (Steiner et al., tion and sequence analysis based on the

‡ Chapter editor: Donatella Barra. * Corresponding author. © CAB International 2010. Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies (ed. G. Wang) 1 2 G. Wang et al.

Table 1.1. Chronological listing of major databases dedicated to AMPs. Yeara Database Web site Content 2002 AMSDb http://www.bbcm.univ.trieste.it/~tossi/amsdb.html Plant/animal AMPs 2002 SAPD http://oma.terkko.helsinki.fi :8080/~SAPD Synthetic AMPs 2004 Peptaibol http://www.cryst.bbk.ac.uk/peptaibol/home.shtml Fungal AMPs 2004 APD http://aps.unmc.edu/AP Natural AMPs 2004 ANTIMIC Not active Natural AMPs 2006 PenBase http://penbase.immunaqua.com Shrimp AMPs 2006 Cybase http://research1t.imb.uq.edu.au/cybase Cyclotides 2007 BACTIBASE http://bactibase.pfba-lab-tun.org/main.php Bacteriocins 2007 Defensins http://defensins.bii.a-star.edu.sg Defensins 2007 AMPer http://marray.cmdr.ubc.ca/cgi-bin/amp.pl Plant/animal AMPs 2008 RAPD http://faculty.ist.unomaha.edu/chen/rapd/index.php Recombinant AMPs 2009 PhytAMP http://phytamp.pfba-lab-tun.org/main.php Plant AMPs 2010 CAMP http://www.bicnirrh.res.in/antimicrobial All AMPs a The year when the database was described in a published article. Some databases were built even earlier (see text). updated version of the Antimicrobial Peptide ‘precursor’. For some AMPs such as human Database (APD), which contains AMPs with LL-37, both the precursor protein and mature fewer than 100 amino acid residues (Wang peptide were registered. The database had and Wang, 2004; Wang et al., 2009). also collected 45 antimicrobial proteins. In addition, this database tabulated the AMP literature (1997–2004) alphabetically based on the last name of the fi rst author. Only AMPs 1.1 Database Scope and Overview from pro karyotes were excluded (Tossi and Sandri, 2002). In 2002, a database for synthetic The major databases for AMPs shown in antibiotic peptides (SAPD) was also estab- Table 1.1 can be classifi ed into three lished (Wade and Englund, 2002). Since regis- categories: (i) general databases for natural tration is needed, SAPD is not a freely AMPs (AMSDb, ANTIMIC, APD and accessible tool. CAMP); (ii) specialized databases for natural In 2004, three additional databases were AMPs (Peptaibol, PenBase, Cybase, simultaneously reported in the database BACTIBASE, Defensins and PhytAMP); and issue of the journal Nucleic Acids Research. (iii) specialized databases for non-natural The Peptaibol database, originally created in AMPs (SAPD and RAPD). In the following 1997, contains 307 peptides isolated from soil paragraphs, we briefl y describe the functions fungi (Whitmore and Wallace, 2004). As the of each database in a chronological order. ‘peptaibol’ name implies, such peptides (<20 The fi rst version of the Antimicrobial residues) are rich in non-standard amino Sequences Database (AMSDb) (approxi- acids such as α-aminoisobutyric acid, mately 300 entries) was built by an under- isovaleric acid, ethyl norvaline and the imino graduate as a part of thesis work and placed acid hydroxyproline, which are represented online in 1997 (Alex Tossi, personal communi- by the non-amino acid lett ers U, J, Z and O, cation, 2006). The information was extracted respectively. This is an important feature, as from Swiss-Prot and kept in the same data other databases have collected peptides with format. According to the AMSDb, the number the standard 20 amino acids or their deriva- of AMPs nearly doubled between 1994 and tives. Also published are two general data- 2001. As of 2004, this database hosted 895 bases (APD and ANTIMIC), which cover peptides from eukaryotes, including both AMPs from a variety of biological sources. precursor and mature peptide sequences. These two databases, both created by gradu- Among them, 278 entries con tained the word ate students as part of their master degree Database View of Naturally Occurring AMPs 3

theses, contain registered information as well the peptides collected in AMSDb and Swiss- as some useful tools for AMP research. Prot. Aft er a recent update, BACTIBASE now Unfortunately, ANTIMIC (Brahmachary et hosts 177 bacteriocins. The Defensins data- al., 2004) became inaccessible several years base provides detailed information for vari- ago. The fi rst version of the APD (Wang and ous defensins as well as related literature, Wang, 2004) collected mature AMPs primar- including patents. In 2008, a specialized data- ily from natural sources, ranging from proto- base for recombinant AMPs (RAPD) was also zoa to bacteria, archaea, fungi, plants and built to document peptide expression, host, animals, including humans. Gene-encoded carrier, cleavage method and yield (Li and AMPs that are post-translationally modifi ed Chen, 2008). In 2009, another specialized are also collected, as are a few AMPs synthe- database was established for plant AMPs sized by multienzyme systems. Some (PhytAMP) (Hammami et al., 2009). synthetic peptides of particular interest are Currently, PhytAMP contains 271 peptide included, too. At present, the following entries. In January 2010, CAMP appeared polypeptides are not collected: (i) propep- (Thomas et al., 2010). This database has tides or precursors of AMPs; (ii) genome- collected 1192 peptides with known activi- predicted or isolated peptides, the ties, 1589 peptides from patents and 1084 antimicrobial activities of which have not predicted AMPs, the antimicrobial activities been experimentally valid ated; (iii) peptides of which have not been validated. The data- that have been found to be inactive against base also incorporates a few prediction tools. microbes; and (iv) antimicrobial proteins For raw data and additional information, with greater than 100 amino acid residues. users can consult the original articles, Swiss- These boundaries merely refl ect the current Prot, the Protein Data Bank and PubMed. status of the database, rather than set restric- tions for future database expansion. The fi rst version of the APD collected 525 mature and 1.2 Nomenclature of Antimicrobial active peptides. Among them, 498 were Peptides annotated to have antibacterial activities, 155 antifungal activities, 28 antiviral activities Peptide name search is a common feature of and 18 anticancer activities. The APD all the databases listed in Table 1.1. A unique provides interactive interfaces for peptide aspect of the name search of the updated search, prediction and design. It also APD (Wang et al., 2009) is that it allows for provides statistical data for a selected group multiple-word searches, thereby increasing of peptides or all peptides in the database. search accuracy and fl exibility. Up to three The database has since been updated and search words can be entered into the search expanded signifi cantly. By March 2010, when boxes. The output decreases with increases this chapter was writt en, there were 1528 in the number of search words. For example, peptide entries in the APD. we found 179 AMPs when ‘defensin’ was Subsequently, several other databases, searched; 12 entries appeared when both mostly for special types of AMPs, were built. ‘defensin’ and ‘human’ were searched; and While PenBase is dedicated to shrimp AMPs six human α-defensins appeared when (Gueguen et al., 2006), Cybase is a specialized ‘defensin’, ‘human’ and ‘alpha’ were all database for cyclic polypeptides from searched. Note that other Greek lett ers such bacteria, plants and animals (Mulvenna et al., as β, γ and δ are represented in the APD as 2006). Some cyclic polypeptides such as plant beta, gamma and delta, respectively. A study cyclotides have been found to be antimicro- of the AMP names collected in the APD bial or human immunodefi ciency virus (HIV) reveals a complex picture. Various methods inhibitory (Chapter 3). In 2007, AMPer (Fjell have been employed to name a newly identi- et al., 2007), BACTIBASE (Hammami et al., fi ed peptide. These methods fall into three 2007) and Defensins Knowledgebase (Seebah categories: (i) peptide-based method; (ii) et al., 2007) were also established. AMPer is source-based method; and (iii) source and an AMP prediction tool developed based on peptide combined method. 4 G. Wang et al.

1.2.1 Peptide-based method from termites). Abbreviations of animal names are used to name homologous AMPs. AMPs are named based on a variety of The name of bovine β defensin-1 (bBD-1) is peptide properties. First, the name magainin analogous to human β defensin-1 (hBD-1). is derived from the Hebrew for shield and Other animal source abbreviations include p defensin was derived from ‘defence’, imply- (pigs, e.g. PMAP-36), e (equine, e.g. e-CATH- ing the functional role of this family of 1), s (sheep, e.g. SMAP-29) and oa (ovine, e.g. peptides. Secondly, many AMPs have OaBac5). The sex of an organism is also obtained their names from the amino acid implied in the case of insect andropin (male sequence: human histatins are rich in histi- specifi c). Sometimes the names of organs or dine residues; PR-39 is a 39-residue AMP rich tissues are also used. Examples are human in proline and arginine residues; and LL-37 neutrophil peptide-1 (HNP-1), liver- is a 37-residue cathelicidin starting with two expressed AMP-2 (LEAP-2), dermcidin from leucine residues. Other synonyms for LL-37 skin and human salvic from salivary glands. (most common) used in the literature and collected into the database are LL37, FALL- 39 (old) and leucine, leucine-37 (rare). For 1.2.3 Source and peptide combined plant cyclotides and cyclic dodecapeptide, method ‘cyclo’ or ‘cyclic’ means polypeptide circularization. Thirdly, the word cathelicidin was In many cases, source organisms and peptide coined from the well-conserved ‘cathelin’ features have been combined to assign a domain of the precursor proteins (Zanett i, unique name. The APD has collected 27 plant 2005). Therefore, cathelicidins represent a AMPs that were named by using the fi rst family of AMPs that share the common lett ers of the scientifi c names of the species cathelin domain. In this book, hCAP-18 followed by AMP. For instance, Ib-AMP was (18-kDa human antimicrobial protein) refers abbreviated from Impatiens balsamina AMP. only to the precursor protein of human When multiple similar peptides are found, cathelicidin. Fourthly, peptide targets have they may be named by appending numbers also found their way into AMP names. While (e.g. Ib-AMP1 to Ib-AMP4). When available, bacteri ocins kill bacteria, AFP1 stands for the database gives both full and abbreviated antifungal protein 1. Sometimes both the names. In addition, the peptide part can also structure and activity of the peptide are represent peptide family or peptide activity. implicated in the name. For example, in the While So-D1 was abbreviated from Spinacia name of τ-defensin, τ refl ects the cyclic, oleracea defensin 1 (peptide family), Ee-CBP cysteine-bridged structure and defensin the originated from Euonymus europaeus chitin- activity. binding protein (activity). The chaotic situation with the AMP nomenclature has spurred eff orts in the fi eld to achieve consistency. Based on peptide 1.2.2 Source-based method features and source species names, PenBase proposed a nomenclature for shrimp AMPs The most common approach is to derive the (Gueguen et al., 2006). For example, the peptide name from the name of a source recommended AMP name ‘litvan PEN3-1’ is species. Usually, either the genus or species composed of three parts: name is taken. For example, sesquin is derived from Vigna sesquipedalis and pali- • Litvan was abbreviated from the species courein is taken from Palicourea condensata. name Litopenaeus vannamei. Sometimes, a combination of the scientifi c • Based on the conserved amino acid name is adopted. For instance, Hyfl E is patt ern, it was assigned to penaeidin derived from Hybanthus fl oribundus E. In subgroup 3 (PEN3). other cases, the peptide name is based on the • The number ‘1’ means that it is the fi rst common name of an organism (e.g. termicin AMP found in that species. Database View of Naturally Occurring AMPs 5

Both Amiche et al. (2008) and Conlon 1.3 Annotation and Classifi cation of (2008) proposed a consistent nomenclature Antimicrobial Peptides for amphibian AMPs. This led to the re-naming of some peptides (Amiche et al., 1.3.1 Source organisms and peptide 2008). To facilitate the transition from the old family classifi cation names to the recommended names, the APD has also collected both old and new names, Source organisms and their taxonomy with the preferred name appearing fi rst. It is suggested that the name of the fi rst published AMPs in the APD are classifi ed based on AMP in this genus takes priority. their source organisms. The classic fi ve- Orthologous peptides from the same genus kingdom system proposed by Robert H. and diff erent species can be distinguished by Whitt aker in 1969 has been adopted. The fi ve capitalizing the fi rst lett er of the species kingdoms are Prokaryotae (bacteria and name. Two capitalized lett ers should be used archaea), Protista (protozoa and algae), Fungi if the same lett er has been used by another (fungi), Plantae (plants) and Animalia species (Conlon, 2008). (animals). This classifi cation was enabled in The nomenclature of AMPs is further the database by appending key words such complicated by the discovery of antimicro- as ‘bacteria’, ‘plants’ or ‘animals’ behind bial activities of polypeptides, which were peptide names. For example, partial informa- initially identifi ed and named based on other tion in the ‘name’ fi eld for peptide entry biological functions or medical signifi cances. AP01228 is represented as: ‘Microcin V (MccV, For example, plant-isolated kalata B1 was (old name) Colicin V, ColV; class 2a microcins, used as a uterotonic agent to facilitate child- bacteriocins, Gram-negative bacteria)’. birth in Congo. Its name is derived from a The above text indicates that microcin V native medicine, kalata-kalata (Gran, 1973). (abbreviated as MccV), once called colicin V The discovery of γ-core in cysteine-contain- (or ColV), is a class IIa microcin (one type of ing AMPs such as defensins led to testing the bacteriocin) synthesized by a Gram-negative antimicrobial activity of brazzein (Young and bacterium. This format allows users to Yeaman, 2004), which is a well-known sweet- retrieve AMPs from a specifi c kingdom. The tasting protein (Hellekant and Danilova, number of peptides in diff erent kingdoms is 2005). The peptide name is derived from the given in Fig. 1.1. These numbers were African plant Pentadiplandra brazzeana. It has obtained by database query using search been demonstrated that some neuropeptides words such as ‘plants’ and ‘fungii’ in the and hormones exhibit antimicrobial activity name fi eld. Note that the use of ‘plants’ and and can participate in host defence as well ‘fungii’ for search gave more accurate results, (Brogden et al., 2005). These peptides were since occasionally the names of some AMPs initially named in a diff erent biological contain ‘plant’ or ‘fungi’. Human AMPs are context. Likewise, chemokines such as annotated as an independent group, currently CXCL14 also display wide-spectrum anti- with 54 entries isolated from saliva, skin, bacterial activity (Maerki et al., 2009). eyes, liver and other organs. Furthermore, Chemokines have their own naming rules prokaryotic AMPs have been split into two and are grouped based on the number and groups: bacteria and archaea. This separation arrangement of conserved N-terminal enables a calculation of AMPs from the three cysteine motifs: C, CC, CXC and CX3C, domains proposed by Carl R. Woese in the where ‘X’ is a non-conserved residue (Cole et 1970s: bacteria, archaea and eukarya (Fig. al., 2001). Interestingly, some known AMPs 1.1A). There are 118, two and 1369 peptides such as human defensins and cathelicidin from these domains, respectively. We LL-37 are known to have chemotactic eff ects conclude that AMPs have been identifi ed in (Murakami et al., 2004; Taylor et al., 2008). all life domains or kingdoms. Thus, chemotaxis is an important property of Figure 1.1A shows that 71.3% of peptides AMPs that links the innate and adaptive in the APD originate from animals. The immune systems (Zasloff , 2002). diversity of animal AMPs requires further 6 G. Wang et al.

(A) Bacteria 118 (7.7%) the host–parasite system to represent the Prokaryotes source species of the AMP. AMP Archaea 2 (0.1%) sources Protozoa 5 (0.3%) Fungi 4 (0.3%) Eukaryotes 217 Plants (14.2%) Peptide family classifi cation in different Animals 1089 (71.3%) Humans 54 (3.5%) kingdoms Peptide family classifi cation is enabled, and (B) Fish 50 Amphibians 592 can be searched, in the name fi eld of the Vertebrates Reptiles 8 APD. Bacterial AMPs have a general name of Birds 27 bacteriocins. Initially, those isolated from Animals Gram-positive bacteria were divided into Insects 166 four families based on the original classifi ca- Invertebrates Spiders 24 Molluscs 6 tion scheme of Klaenhammer (1993). A fi ft h Worms 19 group was proposed by Kemperman et al. Crustaceans 11 (2003). Class I peptides are extensively Fig. 1.1. (A) The number of antimicrobial peptides modifi ed aft er translation and are referred to from a variety of kingdoms. (B) The number of as lanti biotics. Examples are nisins, ericins AMPs from selected animal families. Data from the and lacticins. Lantibiotics are complex and Antimicrobial Peptide Database analysed in have been further classifi ed (see Chapter 2). February 2010. (Total AMP number: 1528.) Peptides in class II are composed of normal amino acids without chemical modifi cations. classifi cation. Broadly, they belong to either Class III bacteriocins are peptides conjugated invertebrates or vertebrates. For inverte- to lipid or other moieties, which have not brates, source names such as insects, spiders, been isolated to date. Class IV contains large molluscs, worms and crustaceans are bacteriocins. In the current APD, there are no included behind the peptide names; for verte- members of class III or IV bacteriocins from brates, fi sh, amphibians, reptiles, birds and Gram-positive bacteria. Finally, class V mammals are used as additional classifi ers. contains circular AMPs where the N- and The number of AMPs from select animal C-termini of the polypeptide chain are groups currently annotated in the database is connected by a peptide bond. Enterocin provided in Fig. 1.1B. In particular, amphib- AS-48, gassericin A, circularicin A and clos- ian peptides account for 38.7% of total AMPs, ticin 574 are circular bacteriocins. representing a rich source of AMP discovery. Cott er et al. (2005a) have simplifi ed the Likewise, the APD has collected 166 AMPs classifi cation scheme for bacteriocins from from insects. Gram-positive bacteria. In this approach, The source organism for an AMP is not classes I and II (above) are maintained. While an issue as long as the organism under inves- the original class III is removed, class IV is tigation is ‘pure’. However, problems may re-assigned as a new class III for bacterio- occur when the organism is so large that a lysins larger than 10 kDa. In addition, there microbial species or parasite also lives within are subclasses in class II. While pediocin-like it. For example, cecropin P1 was initially peptide bacteriocins (e.g. leucocin A and thought to be isolated from pigs. Later, it was divercin V41) are placed in class IIa, class IIb found that this peptide originated from a contains bacteriocins with two independent large round worm (nematode) living in pigs polypeptide chains. Plantaricin JK and (Andersson et al., 2003). The origin of an lactocin 705 are examples of class IIb AMP can be more complex in the parasite– members. The above circular class V bacteri- host system. For example, a parasitic tick ocins are re-assigned as class IIc. The remain- generates AMPs in its gut by enzyme diges- ing linear non-pediocin peptides are tion of the haemoglobin protein from catt le combined into class IId (e.g. entericin Q and or rabbit blood (Fogaça et al., 1999; Nakajima MR10). We have adopted this modifi ed et al., 2003). In these cases, it is proper to use approach (summarized in Table 1.2) because Database View of Naturally Occurring AMPs 7

Table 1.2. Classifi cation of bacteriocins. Gram-positive Gram-negative bacteria Defi nition bacteria Defi nition Class I Lantibiotics Class I Microcins <5 kDa Class II Non-lantibiotics Class II Microcins 5–10 kDa IIa Pediocin-like peptides IIa S–S bond-containing IIb Two-chain peptides IIb Linear IIc Circular peptides Class III Colicins >10 kDa IId Non-pediocin-like peptides Class III Large proteins >10 kDa it is similar to that proposed for Gram- In animals, the classifi cation of AMP negative bacteria below. families is more complex and we only high- Recently, bacteriocins from Gram- light a few here. In amphibians, magainins negative bacteria have also been classifi ed and dermaseptins are well known. In (Table 1.2). Small peptides (<10 kDa), referred addition, the following AMP families are to as microcins, were further classifi ed into well established: brevinin-1, brevinin-2, two groups. Class I microcins are small (<5 esculentin-1, esculentin-2, japonicin-1, japon- kDa) and their backbones are extensively icin-2, nigrocin-2, palustrin-1, palustrin-2, modifi ed aft er translation. Class II microcins ranacyclin, ranatuerin-1, ranatuerin-2 and are larger (5–10 kDa). These are further temporin (Conlon, 2008; Conlon et al., 2009). separated into class IIa and class IIb. Class The APD has collected 99 brevinins, 70 IIa peptides contain disulfi de bonds, while temporins, 42 ranatuerins and 20 esculentins. class IIb microcins are linear peptides with a In insects, the well-known families are C-terminal chemical modifi cation (Duquesne linear cecropins, disulfi de-linked defensins et al., 2007). To be complete, we have assigned (e.g. sapecin, SmD1, heliomicin, termicin, large bacteriocins (i.e. colicins >10 kDa) as royalisin and gallerimycin) and Pro-rich class III. The APD has adopted class 1, class 2 peptides (e.g. drosocin, apidaecin IA, and class 3, rather than class I, class II and abaecin, lebocins and pyrrhocoricin) (Bulet class III, to improve database search accur- and Stocklin, 2005). acy. At present, there are three class 1 micro- In marine invertebrates, Otero-González cins and six class 2 microcins in the APD. et al. (2010) have described AMPs from diff er- Based on sequence similarity and ent phyla such as porifera, cnidaria (e.g. cysteine motifs (Egorov et al., 2005), plant aurelin), mollusca (e.g. MGD-1, mytilins, AMPs have been classifi ed into seven fami- Cv-Def and Cg-Def), annelida (e.g. aren- lies (Table 1.3). Cyclotides are treated as a icins), arthropoda (e.g. penaedins, arasins, new group. The number of peptides and tachyplesins, polyphemusins, big defensins examples in each family are given in Table and tachystatins), echinodermata and chor- 1.3. Plant AMPs are discussed in Chapter 3. data (tunicates) (e.g. clavanins).

Table 1.3. Classiﬁ cation of plant AMPs. Group Plant AMPs Number Examples 1 Defensins 44 So-D1 2 Thionins 10 Tu-AMP1, Cp-thionin II 3 Lipid-transfer proteins 0 4 Hevein-like peptides 4 Ee-CBP, WAMP-1 5 Knottin-like peptides 0 6 Glycine-rich peptides 1 Pg-AMP1 7 MBP-1 homologues 1 MBP-1 8 Cyclotides 126 Circulin A, kalata B1 8 G. Wang et al.

In mammals, including humans, the Table 1.4. Peptide classifi cation in the major AMP families are defensins, cath- Antimicrobial Peptide Database based on elicidins and histatins (Zanett i, 2005). In the biological activity. APD, there are 121 defensins and 53 cath- Search Number of elicidins from animals. In addition, there are Peptide activity icon/key peptides 12 human defensins and two human cath- Antibacterial Icon 1180 elicidin peptides (LL-37 and ALL-38) Antifungal Icon 451 encoded by the same cathelicidin gene Antiviral Icon 98 (detailed in Chapter 9). Other well-known Antitumoral Icon 101 human AMPs are dermcidin, LEAP-1 Antiparasitic ZZP 16 (hepcidin) and granulysin. It is evident that Spermicidal ZZS 8 the classifi cation schemes for AMPs from a Insecticidal ZZI 16 variety of biological sources are still in the Haemolytic Icon 175 early stages due to incomplete information. are at least 388 peptides with both antibacterial and antifungal activities, but only 1.3.2 Classifi cation based on biological 41 peptides with both antifungal and anti- activities viral activities. Likewise, 40 peptides have been found to have antibacterial, antiviral Activity spectrum of AMPs and antifungal eff ects. However, merely 12 The APD also divides natural AMPs into peptides show antibacterial, antiviral, anti- subgroups based on antimicrobial activities. fungal and anticancer activities. Because of Some peptides have a narrow activity spec- the partial overlap in activity spectrum, some trum, while others have a wider spectrum. AMPs can be included in diff erent groups. Both defensins and cathelicidins are wide- It is pertinent to point out that the anti- spectrum AMPs (Zasloff , 2002; Zanett i, 2005). microbial activity evaluated using laboratory Bactericocins are characterized by narrow- bacterial strains may not be biologically rele- spectrum activity as well as very low mini- vant in real cases. In addition, for quantita- mal inhibitory concentrations at nanomolar tive structure–activity studies, one should be levels (antimicrobial activity is usually repre- cautious in utilizing the antimicrobial activ- sented in micromoles). At present, the APD ity data obtained from diff erent laboratories allows searching for AMPs with the follow- evaluated under diff erent conditions (e.g. ing biological activities: antibacterial, anti- strains, media and methods). fungal, antiviral, antitumoral, antiparasital, spermicidal and insecticidal. These fi elds can Synergistic effects of AMPs be searched individually, allowing users to retrieve a list of AMPs with the desired prop- A set of AMPs from one specifi c species can erties (Table 1.4). For instance, the database be obtained by searching the database using has collected 1180 peptides with antibacterial the scientifi c name in the ‘AMP source activity. Among these, 206 peptides display species’ fi eld. A search using ‘Odorrana activity against only Gram-positive bacteria grahami’ returned 26 AMPs. In the original and 108 peptides show activity against only proteomic study (Li et al., 2007), 107 peptides Gram-negative bacteria. Antiparasital, were detected from the skin of a single frog spermicidal and insecticidal peptides are O. grahami. Many of these peptides have not searched by entering keys ZZP, ZZS and ZZI, yet been registered into the APD due to a respectively, in the name fi eld. The numbers lack of activity data. Likewise, we found 26, of peptides with diff erent activities are listed 27 and 54 peptides when Viola odorata (an in Table 1.4. In addition, 175 AMPs display a Australian plant), Bos taurus (cow) and Homo cytotoxic eff ect on human cells (e.g. red sapiens (humans) were queried, respectively. blood cells). These activities can also be These numbers should be regarded as searched in combinations. For example, there minima for two reasons. First, scientifi c Database View of Naturally Occurring AMPs 9

names may be absent in the original articles. particular AMP, the net charge can vary Secondly, some peptides are not included slightly depending on the nature of chemical due to a lack of activity data. modifi cation. For example, the charge Scientists have been curious as to why increases by 1 when the C-terminus of the frogs generate such a large number of peptide is amidated. Evidently, the majority peptides. One possibility is that multiple (88.6%) are cationic AMPs with an average peptides are created for diff erent functions net charge of +4.4 per peptide. Figure 1.2 that are not yet fully understood. Another shows the number of AMPs as a function of possibility is that the shear multiplicity of net charge. The distribution for all AMPs environmental microbes requires a variety of (Fig. 1.2A) is relatively smooth with a peak at host defence peptides. Furthermore, some +3. For bacterial AMPs (Fig. 1.2B), two peaks peptides can work together to achieve opti- exist at +2 and +4, while the peaks shift mal protection. Signifi cantly, some peptides slightly to 0 and +2 for plant AMPs (Fig. are ineff ective when evaluated alone, but 1.2C). The amphibian AMPs appear to have a become antibacterial when evaluated in more clustered net charge distribution, with combination (Li et al., 2007). Amphibian the majority in the range of +1 to +4 (Fig. AMPs that show a synergistic eff ect include 1.2D). There are a few outliers not depicted magainin 2 and PGLa (Matsuzaki et al., 1998; in these plots. Oncorhyncin II and OaBac11 Strandberg et al., 2009); magainin 2 and (Anderson and Yu, 2003; Fernandes et al., cecropin A (Cirioni et al., 2008); and tempo- 2004) are the most positive peptides with a rins A, B and L (Rosenfeld et al., 2006). Note net charge of +30. In amphibians, the most that temporins A, B and L were found to positively charged AMP is buforin II with a show a synergistic eff ect in both antimicro- net charge of +13. Current research is primar- bial and antiendotoxin (lipopolysaccharide) ily focused on cationic AMPs, with a favour- activities. Some bacteriocins such as entero- able assumption that they can selectively cin L50 (Cintas et al., 1998) and lichenicidin target negatively charged bacterial (Begley et al., 2009) comprise two independ- membranes. The identifi cation of anionic ent peptide chains, which display higher peptides, however, has further expanded the activity when combined in a 1:1 ratio. AMP spectrum. The two most negatively Peptide synergistic information can now be charged peptides contain a net charge of –6. searched through the ‘additional informa- While the anionic peptide SAAP (sequence tion’ fi eld by using ‘synerg’ or via the name DDDDDD) requires Zn2+ to be antibacterial fi eld by entering the ‘JJsn’ key. By March (Brogden et al., 1996), naegleriapore B is a 2010, the APD had collected 15 such syner- pore-forming peptide from a parasitic proto- gistic pairs. zoon (Herbst et al., 2002). Anionic AMPs have also been found in other sources such as amphibians and humans (Schitt ek et al., 2001; Lai et al., 2002). Anionic dermcidin acts using a diff erent mechanism from cationic LL-37 1.3.3 Classifi cation based on peptide (Senyürek et al., 2009). characteristics AMPs can also be classifi ed based on their length. In the APD and this book, Peptide charge, length and hydrophobic peptides are defi ned as containing fewer residue content than 100 amino acid residues. The amino In the database search interface of the APD, acid sequence of an AMP, in the single-lett er there are search icons for peptide charge, amino acid code (e.g. C, G and R), can be length and percentage of hydrophobic resi- entered in part or full into the search box of dues. Based on the charge, AMPs can be the ‘sequence’ fi eld. This is the most accurate classifi ed into cationic and anionic peptides. method of discovering whether a particular Of a total of 1528 AMPs, 1355 peptides have peptide has been registered in the APD. The a net positive charge, 95 have a net charge of majority of AMPs (98.4%) are listed with a zero and 78 have a net negative charge. For a complete amino acid sequence. Peptides with 10 G. Wang et al.

(A) 250 (B)

200 20

150

100 10 Number of AMPs Number of AMPs 50

0 0 0+1+2+3 +4 +5 +6 +7 +8 +9+10 –4 –3 –2 –1 0+1+2+3 +4 +5 +6 +7 +8 +9+10 –4 –3 –2 –1 Net charge Net charge

30 120

20 80

10 40 Number of AMPs Number of AMPs

0 0 –4 –3 –2 –1 0 +1+2+3+4+5+6+7+8+9+10 –4 –3 –2 –1 0 +1+2+3+4+5+6+7+8+9+10 Net charge Net charge Fig. 1.2. Distribution of antimicrobial peptides in the Antimicrobial Peptide Database versus net charge from (A) all sources, (B) bacteria, (C) plants and (D) amphibians. The total number of peptides in each kingdom is provided in Fig. 1.1. incomplete amino acid sequences can be by Kyte and Doolitt le (1982). For all AMPs, searched in the name fi eld by using ‘BWQ’. the peak is located at 41–50% (labelled as The database also allows searching for AMPs 50%) (Fig. 1.4A). Out of 118 bacterial AMPs in a defi ned length range, such as 11–20 or (Fig. 1.4B), 56 have a hydrophobic residue 21–30 amino acids. The number of AMPs as a content between 31 and 40%, and 110 out of function of peptide length is plott ed in Fig. 217 plant AMPs (Fig. 1.4C) are within the 1.3. When all of the 1528 AMPs in the data- hydrophobic residue content of 31–40%. base (Fig. 1.3A) are analysed, the distribution Nearly all amphibian AMPs (Fig. 1.4D) have plot gives a peak at 30 (i.e. 21–30 residues). a hydrophobic content between 41 and 70%. In both bacteria (Fig. 1.3B) and plants (Fig. The higher hydrophobic content in this 1.3C), most of the AMPs have 21–50 residues, animal family is related to a high content of whereas 97.3% of amphibian AMPs have alanine residues. Three AMPs from bacteria 11–40 residues (Fig. 1.3D). The shortest show hydrophobic contents even greater peptides collected in the database contain than 80%. They are gramicidins that form only fi ve residues, whereas the longest transmembrane ionic pores. These unusual peptides contain fewer than 100 residues due AMPs diff er from the majority of the to the peptide defi nition. peptides collected in the APD in that they are AMPs can also be classifi ed based on synthesized by a non-ribosome multienzyme their hydrophobic residue content. A distri- system. bution of the AMPs in the database versus hydrophobic residue content (hydrophobic residues divided by total residues) is Chemical modifi cations of AMPs presented in Fig. 1.4. In the APD, the following residues are defi ned as hydrophobic: Antimicrobial peptides can also be classifi ed isoleucine, valine, leucine, phenylalanine, based on the types of chemical modifi ca- cysteine, methionine, alanine and tryp- tions. This is the case with bacteriocins from tophan. This was obtained by expanding the Gram-positive bacteria (Table 1.2). Post- original set of hydrophobic residues defi ned translational modifi cations play a critical Database View of Naturally Occurring AMPs 11

(A) (B) 600 30 500 400 20 300 200 10

Number of AMPs 100 Number of AMPs 0 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Peptide length Peptide length (C) (D) 120 300

80 200

40 100 Number of AMPs Number of AMPs 0 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Peptide length Peptide length Fig. 1.3. Distribution of antimicrobial peptides in the Antimicrobial Peptide Database versus peptide length from (A) all sources, (B) bacteria, (C) plants and (D) amphibians. The total number of peptides in each kingdom is provided in Fig. 1.1. Each column represents the number of the peptides in a range deﬁ ned between the two adjacent numbers. For example, ‘20’ means ‘11–20’.

(A) (B) 600 60 500 50 400 40 300 30 200 20

Number of AMPs 100 Number of AMPs 10 0 0 10 20 30 40 50 60 70 80 >80 10 20 30 40 50 60 70 80 >80 Hydrophobic residues (%) Hydrophobic residues (%)

Number of AMPs 20 Number of AMPs 50 0 0 10 20 30 40 50 60 70 80 >80 10 20 30 40 50 60 70 80 >80 Hydrophobic residues (%) Hydrophobic residues (%) Fig. 1.4. Distribution of antimicrobial peptides in the Antimicrobial Peptide Database versus hydrophobic residue (%) from (A) all sources, (B) bacteria, (C) plants and (D) amphibians. The total number of peptides in each kingdom is provided in Fig. 1.1. Each column represents the number of the peptides in a range deﬁ ned between the two adjacent numbers. For example, ‘20’ means ‘11–20’. 12 G. Wang et al.

role in modulating antimicrobial activity or recognizable moiety to the peptide allows other biological functions of the peptides. the peptide to be smuggled into the For example, both aurein 1.2 (unpublished bac terium via the ‘Trojan horse’ trick. Both data) and anoplin (Dos Santos Cabrera et al., microcin C7 (MccC7) and microcin E492 2008) have been found to lose their activities possess such a ferric-binding moiety, which aft er removal of C-terminal amidation. is also important for antibacterial activity. However, not all amidated peptides display MccC7 is cleaved within the cell and the increased activity (Dennison et al., 2009). resulting peptide targets tRNA synthetase Amidation can also improve peptide stabil- and inhibits protein synthesis (Roush et al., ity. Joanne et al. (2009) proposed that amida- 2008). tion might be involved in GraS/GraR Table 1.5 provides an expanded list of (glycopeptide resistance-associated protein the search keys for chemical modifi cations S/R)-mediated AMP sensing at the bacterial with peptide examples. When XX was surface. Enterocin AS-48 is the fi rst circular searched in the name fi eld, 465 peptides bacteriocin identifi ed and an excellent candi- (30.5%) appeared, indicating that post- date as a food preservative. Recent investi- translational modifi cations are common for gation of its linear form revealed that AMPs. Many linear peptides (272 in the circularization is required for peptide struc- APD) are C-terminally amidated (XXA). In ture rather than bactericidal activity natural AMPs, the amidation group is (Montalbán-López et al., 2008). Wilson- derived from a glycine residue. However, 139 Stanford et al. (2009) found that oxidation of peptides (9%) have been found to have a lantibiotics such as nisin A disrupted anti- circular structure (XXC) where the N- and bacterial activity. It was proposed that the C-termini of the peptide are covalently linked oxidized form of sulfur is unable to bind to by forming a peptide bond (Table 1.5). lipid II of the cell wall. Although one may Circular AMPs have been found in bacteria, wonder whether the oxidation occurs in vivo plants and animals. Not all peptides are or during purifi cation, it is good practice to circulated in the same way, however. Microcin prevent oxidation if it inactivates the J25 was originally thought to form a head-to- peptide. Att achment of a bacterial receptor- tail peptide bond. It is now established that a

Table 1.5. Database search keys for AMP chemical modifi cations.a Number of Search key Chemical modifi cation peptides Examples XXA C-terminal amidation 272 Aurein 1.2, indolicidin XXB C-terminal iron-binding moieties 4 MccE492, MccH47 Polypeptide chain cyclization XXC 139 Cyclotides; class IIc bacteriocins between the N- and C-termini XXD D-amino acids 7 Bombinin H4; lactocin S XXE N-terminal acetylation 8 Alpha-MSH, paenibacillin XXF Carboxylic-acid-containing unit 1 Hymenoptaecin XXG Glycosylation (e.g. Thr) 7 Drosocin, heliocin XXH Halogenation (F, Cl, Br) 1 Styelin D XXK Hydroxylation (e.g. Lys, Arg, Tyr) 1 Styelin D XXO Oxidation (e.g. Trp, Cys) 8 Piscidin 4, chrombacin XXP Phosphorylation (e.g. Ser) 2 Human histatin 1 XXQ N-terminally cyclic glutamate 12 Gomesin, Ib-AMP1, heliocin XXS Sulfation 1 Chrombacin XXT Thioether bridge formation 22 Lantibiotics, e.g. nisin A, mersacidin, lacticin 3147 a Some of the keys were originally released in Wang et al. (2009). A key search can be performed in the name fi eld. Database View of Naturally Occurring AMPs 13

ring structure is realized between the back- provide unique tools for peptide engineering. bone amide of residue glycine 1 and the side Cott er et al. (2005b) found an enzyme that chain of glutamic acid 8 (Rosengren et al., converts a dehydrated l-serine to d-alanine. 2003). In addition, some amino acid residues Such enzymes may be harnessed to incorpo- also form a ring structure . N-terminal rate d-amino acids into bacterially expressed glutamine residues can spontaneously polypeptides. The discovery of the broad cyclize to become pyro glutamates. This substrate specifi city of the nisin modifi cation N-terminal blocking makes peptide sequenc- enzymes (Rink et al., 2005) may open the door ing by Edman degradation ineff ective. The to enzyme-mediated introduction of thioether APD has collected 12 such peptides (XXQ in rings into a peptide template for required Table 1.5). They have been isolated from biological activity or structural stability. plants, spiders, insects, amphibians and reptiles, revealing another general modifying mechanism in nature. Three-dimensional structures of antimicrobial d-amino acids were fi rst identifi ed in peptides natural AMPs from amphibians (Simmaco et al., 2009). They have also been found in Protein structures have been classifi ed into α, bacteriocins (Cott er et al., 2005b). The APD β, αβ and α+β families (Murzin et al., 1995). lists seven d-amino-acid-containing peptides Proteins in the α family are composed of (Table 1.5). However, the identifi cation and primarily α-helices, while those in the β characterization of d-amino acids requires family consist of primarily β-strands. In the elegant techniques. Kawai et al. (2004) αβ family, α-helices and β-strands are inter- reported that gassericin A and reutericin 6 spersed. However, the α-helices and β-strands are cyclic bacteriocins with an identical in the α+β family are largely segregated. We amino acid sequence diff ering only in the have adopted this classifi cation scheme for number of d-alanines. A recent revisit of AMPs with two modifi cations. First, the αβ these two AMPs demonstrated that they are family contains all AMPs that have both α identical and there are no d-amino acids and β structures, regardless of whether they (Arakawa et al., 2010). This work indicates are segregated. Secondly, diff erent from the the importance of purifying natural peptides scheme of Murzin et al. (1995), we defi ne a to homogeneity before a full biochemical and non-αβ family that includes all AMPs that biophysical characterization is made. form neither α nor β structures. Thus, we Some AMPs may be chemically modi- classify AMP structures into four families: α, fi ed in multiple ways. For instance, the β, αβ and non-αβ. Representatives from the sequence of styelin D from the sea squirt is four structural families are provided in Fig. extensively modifi ed, including halogenation 1.5. Magainin, an α-helical AMP (Fig. 1.5A), is (XXH) of tryptophan 2 and hydroxylation a typical member of the α family. Lactoferricin (XXK) of arginine, lysine and tyrosine resi- B (Fig. 1.5B), with a pair of β-strands, is used dues (detailed in the database). Such modifi - as a representative of the β family. Heliomicin, cations might be essential for the peptide to with both α-helices and β-strands (e.g. in Fig. remain active even at high salt concentra- 1.5C), is a member of the αβ family. Finally, tions. Indeed, the native peptide is more indolicidin, with neither α nor β structures active than a synthetic unmodifi ed peptide (Fig. 1.5D), is a representative member in the (Taylor et al., 2000). The current version of non-αβ family. These four types of peptide the APD allows a simultaneous search for structures (α, β, αβ and non-αβ) can be three types of chemical modifi cations (Table searched in the ‘structure’ search fi eld by 1.5) in the name fi eld. When XXA (amida- choosing ‘helix’, ‘beta’, ‘combined helix and tion), XXP (phosphorylation) and XXS (sulfa- beta’ and ‘rich in amino acids’, respectively. tion) were searched, only chrombacin Although the word ‘rich’ includes all appeared. peptides rich in certain amino acids, they Understanding the mechanism of chemi- may or may not form a non-αβ structure. cal modifi cation of natural AMPs may When the terms ‘rich’ and ‘NMR’ (nuclear 14 G. Wang et al.

1.3.4 Peptide-binding targets and mechanisms of action

Broadly, AMPs can be classifi ed into membrane targeting and non-membrane targeting. It is assumed that many membrane-targeting AMPs disrupt bacterial membranes by three major mechanisms: carpet, barrel-stave and toroidal models (Ludtke et al., 1996; Shai, 2002). These models are depicted in Fig. 5.1 (Chapter 5). However, there are other possible models (Chapter 7). In addition, AMPs associate with other components on the cell surface. SMAP-29 Representative structures of Fig. 1.5. (sheep myeloid antimicrobial peptide-29) antimicrobial peptides in the Antimicrobial Peptide Database (APD): (A) α-helix (frog magainin; PDB and hRNase 7 may bind to an OprI (outer ID: 2MAG); (B) β-sheet (bovine lactoferricin B; membrane protein I) of Gram-negative PDB ID: 1LFC); (C) αβ with both α-helices and Pseudomonas aeruginosa for activity (Lin et al., β-sheet (insect heliomicin; PDB ID: 1I2U); (D) 2010). While nisin A is known to bind lipid II non-αβ structure (bovine indolicidin; PDB ID: and forms pores in the inner membranes of 1G89). Adapted from the face page of the APD Gram-positive bacteria, short nisins may website (http://aps.unmc.edu/AP). work by blocking cell-wall synthesis due to the deprivation of lipid II (Hasper et al., 2006). For some plant AMPs such as Cy-AMP1, chitin-binding ability is critical for magnetic resonance) are searched simultan- antifungal activity (Yokoyama et al., 2009). eously, only AMPs with non-αβ three-dimen- Some defensins have been shown to bind sional structures are found. Table 1.6 gives specifi cally to carbohydrate moieties of the statistics for the four types of NMR struc- glyco protein 41 (gp41) of HIV-1 and CD4 of tures of AMPs from diff erent sources. It is T-cells to inhibit viral entry into human cells evident that all known AMP structures of (Gallo et al., 2006). In contrast, some amphib- plant origin contain β-strands, while helical ian AMPs are proposed to inhibit HIV-1 structures are common in peptides from infection by disrupting the viral envelope other sources. A further discussion of the (VanCompernolle et al., 2005). Therefore, a three-dimensional structure of AMPs is more inclusive term is cell-surface-binding provided in Chapter 9. peptides. Non-membrane-targeting peptides include all other AMPs that interfere with cell function or survival by binding to intra- Table 1.6. Nuclear magnetic resonance (NMR) structural statistics of AMPs collected in the cellular targets. Broadly, these peptides may Antimicrobial Peptide Database.a be termed cell internal-target-binding peptides. While proline-rich peptides are Kingdom α ˟ α˟ Non-α˟ Total known to interact with heat-shock proteins, Bacteria 13 4 2 0 19 some histone-generated AMPs are capable of Plants 0 12 19 0 31 binding to nucleic acids (Chapter 8). Table 1.7 Animals 69 24 17 3 113 lists the keys for searching AMP binding part- Humans 4 2 3 0 9 ners. It should be emphasized that an illustra- a Information in the table can be obtained by performing a tion of the binding of an AMP to a particular multiple-choice search. By entering ‘bacteria’ into the molecule in vitro does not necessarily mean name fi eld and selecting ‘NMR’ in the structural method fi eld and ‘helix’ in the structure fi eld, one can obtain 13 that the molecule is exactly the target in vivo bacterial AMPs with known helical structures that causes microbial death. Recently, we determined by NMR. started registering the information on Database View of Naturally Occurring AMPs 15

Table 1.7. Keys for the search of binding partners or targets of AMPs.a Number of Key Binding partner peptides Examples BBBh2o Oligomers in water 6 hBD-3; LL-37 BBBm Oligomers in membranes 1 Protegrin-1 BBII Metal ions (e.g. Zn2+) 12 Histatin 5, hepcidin 25 BBW Bacterial cell wall precursors (e.g. lipid II) 3 Mersacidin, lactocin S BBL Lipopolysaccharides 26 Temporin L, LL-37 BBr Bacterial cell surface receptor (protein) 1 SMAP-29, hRNase 7 BBMm Inner membranes (e.g. lipid bilayers) 48 Magainins, LL-37 BBN Nucleic acids (DNA/RNA) 5 Buforin II, indolicidin BBP Proteins (inside cells) 16 Drosocin, pyrrhocoricin BBS Carbohydrates 21 HNP1, AFP1, Ac-AMP2, RTD-1 a Some of the keys were originally published in Wang et al. (2009). A key search can be conducted in the name ﬁ eld.

possible mechanisms of action into the data- (A) 16 base. This is a poorly deﬁ ned zone because the mechanisms of action of AMPs are, in 12 general, not well understood.

% 8

1.4 Amino Acid Sequence Analysis of 4

Antimicrobial Peptides 0 I VLFCMAWGPTSYQNEDHKR

The annotation and classifi cation of AMPs (B) 20 into a variety of families with a common feature enables sequence analysis by 16 bio informatic tools. For example, we have 12 calculated the amino acid compositions of % 8 AMPs from diff erent kingdoms using the database tool. Figure 1.6 shows the average 4 amino acid content (composition signature) 0 for all AMPs from bacteria, plants and IVLFCMAWGPTSYQNEDHKR animals. The types of abundant amino acids (C) (approximately 10% or greater) diff er. We 12 refer to these abundant amino acid residues as frequently occurring residues (Wang et al., 8 2009). In 118 bacterial AMPs, residues alanine % and glycine are frequently occurring residues 4 (Fig. 1.6A). Residues cysteine and glycine are abundant in 217 plant AMPs (Fig. 1.6B), 0 while residues leucine, glycine and lysine IVLFCMAWG P T S YQN ED H KR occur frequently in 1089 AMPs from animals Amino acid (Fig. 1.6C). In addition, there are some amino Fig. 1.6. Average amino acid percentages of acids that are rarely used. For example, antimicrobial peptides from (A) bacteria, (B) plants methionine residues have the lowest percent- and (C) animals. The numbers of peptides age in known plant AMPs. analysed from bacteria, plants and animals were Figure 1.7A presents the average amino 118, 217 and 1089, respectively. Frequently acid content of 548 peptides from frogs. occurring residues are defi ned as those that have Residues leucine, alanine, glycine and lysine a percentage of approximately 10% or greater. 16 G. Wang et al.

β-strands. These abundant residues are important in terms of both the structure and activity of these peptides. While cysteine residues form critical disulfi de bonds that maintain the defensin fold, the glycine residue endows fl exibility to the peptide chain and is an integral part of the γ-core motif (Young and Yeaman, 2004). Arginines are essential for antibacterial and antiviral activities. A similar amino acid composition signature was obtained (Fig. 1.7D) when the 49 members of the αβ-fold family were analysed. The typical examples of this family are human β-defensins, which comprise one α-helix and three β-strands. Diff erent from the β-fold family, where arginine residues are more abundant than lysine residues, β-defensins have an approximate 1:1 arginine to lysine ratio (compare Fig. 1.7C and Fig. 1.7D). The essential role of arginine in α-defensins has been substantiated by Zou et al. (2007). They found that the eff ect of arginine to lysine mutations on antibacterial activity is more pronounced for α-defensins than for β-defensins. We also analysed a set of peptides with relatively well-conserved structures. While the frequently occurring residues in 21 Fig. 1.7. Average amino acid percentages of (A) bacterial lantibiotics were cysteine, threonine 548 frog antimicrobial peptides, (B) 243 helical peptides, (C) 81 AMPs with β structures and (D) and serine (Fig. 1.8A), they were cysteine and 49 AMPs with an αβ fold as found for β-defensins glycine in 126 plant cyclotides (Fig. 1.8B). collected in the Antimicrobial Peptide Database. Interestingly, the most abundant residues in these two families of peptides were identical (cysteine, glycine, threonine and serine) and occur frequently. This list of residues may their amino acid composition signatures contain structural information. As many (overall distribution patt erns) were more or amphibian AMPs such as magainins and less similar. Although cysteine residues are aureins are known to associate with bacterial common in these two peptide families, their membranes and adopt amphipathic α-helices structural roles are entirely diff erent. In lanti- (Haney et al., 2009), we hypothesize that the biotics, they form several three-membered same set of abundant residues also occurs in ring structures that confer stability to the AMPs with known helical structures. A peptides. Such thioether rings are normally statistical analysis of 241 AMPs annotated as formed between cysteine residues and dehy- ‘Helix’ in the APD (based on NMR and circu- drated serine or threonine, making them lar dichroism) reveals that residues leucine, abundant in lantibiotics. In contrast, cysteine alanine, glycine and lysine are indeed abun- residues in cyclotides are known to form dant (Fig. 1.7B). In contrast, the abundant disulfi de bonds, also conferring stability to residues in 81 AMPs with known β struc- the peptide structure. As a consequence, tures are cysteine, glycine and arginine (Fig. these natural templates are att ractive for 1.7C). The typical members of the β-fold developing a new generation of therapeutic family are human α-defensins with three agents. Database View of Naturally Occurring AMPs 17

(A) microbial’, at least according to in vitro microdilution or diff usion assays (Wiegand et al., 2008). The fi nal establishment of a bona fi de AMP requires in vivo data. The success in database expansion and enhancement has validated the fl exibility of our original design. This important feature will enable further growth of this database in the future. For example, how a particular Amino acid AMP is expressed and regulated in response to bacterial invasion is a fundamental ques- (B) tion. Understanding this process promises novel therapeutic strategies. In addition, information such as microbial type, expression cell, translation and post-translational modifi cation enzymes, transport proteins and self-immunity can be entered into the ‘additional information’ fi eld (e.g. see microcin J25, APD ID: 480). It is also clear that the Amino acid biological functions of AMPs are not limited Fig. 1.8. Average amino acid percentages of (A) to antimicrobial activities, as discussed 21 lantibiotics from Gram-positive bacteria and (B) above. In particular, mammalian cath- 126 cyclotides from plants collected in the elicidins and defensins have multiple Antimicrobial Peptide Database (March 2010). biological functions, in particular immune modulation. Functions such as chemotaxis, apoptosis, wound healing and tumour 1.5 Concluding Remarks metastasis can also be entered into the ‘additional information’ fi eld of the APD (e.g. see In summary, the APD is a useful tool for the human LL-37, APD ID: 310). Therefore, this naming, classifi cation and analysis of natur- fi eld can annotate detailed information ally occurring AMPs. By including syno- regarding the biology of AMPs as host nyms, including old and new names, the defence molecules. Detailed classifi cation likelihood of fi nding a peptide in the data- and annotation also enable a more meaning- base increases. This practice also facilitates ful bioinformatic analysis of AMPs. Such the transition to and adoption of newly results are useful in guiding peptide suggested names. We should emphasize that pre diction and design, which we discuss in the classifi cation issue of AMPs is not fully Chapter 4. resolved due to incomplete information as well as the diversity of the peptides. Therefore, accuracy will improve with the refi nement of the classifi cation schemes in Acknowledgement the future. Correct classifi cations of AMPs improve information searches. Our recent This study was supported by the grant updates have led to a substantial increase in award R21AI082689 from the National both peptide entries and search functions. Institute of Allergy and Infectious Diseases For newly established search functions (NIH, USA) to GW. currently under annotation, users should treat the search result as ‘a minimum’ while the annotation is ongoing. Many hypothetical References AMPs, predicted or isolated, have not been collected owing to a lack of activity data. Amiche, M., Ladram, A. and Nicolas, P. (2008) A This practice is in line with the word ‘anti- consistent nomenclature of antimicrobial 18 G. Wang et al.

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Brian Healy, Jim O’Mahony, Colin Hill,* Paul D. Cotter* and R. Paul Ross

Abstract

Since the 1925 discovery that strains of Escherichia coli can retard the growth of neighbouring bacteria, the study of bacteriocins has continuously evolved. During the intervening period, a large and heterogeneous collection of these antimicrobial peptides has been isolated from a myriad of sources and numerous investigations have been carried out with a view to harnessing their potency. The most thoroughly investigated class of bacteriocins, the lantibiotics, is the main focus of this review. These antimicrobial peptides inhibit many human and animal pathogens. They have been the focus of considerable eﬀ orts to maximize the potential of existing lantibiotics, identify new and bett er lantibiotics from nature and utilize bioengineering-based approaches to further improve upon existing well-characterized compounds.

2.1 Introduction to Bacteriocins The identifi cation of the fi rst bacteriocin, a colicin (i.e. an Escherichia coli-associated In order for an organism to survive, thrive bacteriocin), occurred in 1925 (Gratia, 1925). and proliferate in a particular niche, it is This was soon followed by the fi rst report of essential that it competes successfully. In the a bacteriocin produced by a Gram-positive microbial world, many bacteria gain an bacterium, when inhibition of Lactobacillus upper hand by producing antimicrobial bulgaricus by Streptococcus lactis (since compounds that inhibit their competitors. renamed Lactococcus lactis) was noted Bacteriocins, which are bacterially produced (Rogers, 1928). Since then, bacteriocins ribosomally synthesized peptides with produced by lactococci and other lactic acid antimicrobial spectra that can range from bacteria (LAB; i.e. microorganisms with a very narrow to extremely broad, are among long history of safe use in food and of the most potent examples of bacterially considerable industrial importance) have produced antimicrobials (Tagg et al., 1976; continued to be the focus of much att ention Jack et al., 1995; Riley and Wertz, 2002; Cott er (Nissen-Meyer et al., 2009). A number of et al., 2005b). Indeed, bacteriocins frequently diff erent classifi cation systems exist for LAB exhibit antimicrobial activity at nanomolar bacteriocins, but the simplest, and that levels; this contrasts with, for example, the used for this review, was proposed by cationic antimicrobial peptides produced by Cott er et al. (2005b). This system comprises eukaryotic cells, which exhibit similar levels two classes: (i) class I bacteriocins – the of activity only at micromolar concentra tions post-translationally modifi ed lantibiotics (Nissen-Meyer et al., 2009). (Chatt erjee et al., 2005; Cott er et al., 2005a);

* Corresponding authors. © CAB International 2010. Antimicrobial Peptides: Discovery, Design and Novel 22 Therapeutic Strategies (ed. G. Wang) Lantibiotic-related Research and Application 23

and (ii) class II bacteriocins, which are a form Lan and meLan from Dha and Dhb, heterogeneous group of non-lanthionine respectively. Insight into the role of LanC (Lan)-containing peptides. As a consequence was fi rst provided by Meyer et al. (1995) of their unusual structure, unique mechan- who, while working on the lantibiotic Pep5, isms of action and potential as antimicrobials showed that with the removal of the pepC for food, veterinary and clinical applications, gene, the peptide produced contained the lantibiotics have received much att ention dehydrated residues but not Lan or meLan in recent years. They are the focus of this bridges. In addition to being essential for chapter. converting the peptide to an active form, the Lan and meLan structures are also believed to contribute to protease resistance (Kluskens et al., 2005). Unlike the type I 2.2 Biosynthesis and peptides, which are modifi ed by two Post-translational Modifi cations separate modifi cation enzymes, type II peptides such as lacticin 481 and mersacidin The name lantibiotics is derived from are modifi ed by a single protein referred to ‘Lan-containing antibiotics’ and refl ects the as LanM (Willey and van der Donk, 2007). feature that is shared by all of these peptides: Another variation on this theme arises as a intra-molecular rings formed by the thioether consequence of the existence of two peptide amino acids Lan and methylanthionine lantibiotics (e.g. lacticin 3147) that require (meLan) (Sahl et al., 1995). Lantibiotic- the combined activity of two Lan- and associated gene clusters can be located on meLan-containing peptides for optimal chromosomes (including transposable activity. In most cases, the production of elements) or plasmids. In addition to the lantibiotics of this kind relies on the presence genes encoding a structural peptide(s) and of two LanM proteins, each of which is the post-translational modifi cation machinery responsible for the dehydration and that acts thereon, other genes responsible for cyclization of its respective subunit regulation, transport and immunity are (McAuliff e et al., 2000; Lawton et al., 2007b). usually also present (Cott er et al., 2005b). The A third category of peptides, type III, is following paragraphs summarize the steps not described in this review as those involved in lantibiotic biosynthesis. identifi ed to date, while possessing Lan and The lantibiotic prepropeptide contains a meLan structures, lack antimicrobial activity leader region, which is eventually cleaved, (Willey and van der Donk, 2007). Yet another and a propeptide, which is modifi ed to category, modifi ed by a novel group of become the active antimicrobial. The lantibiotic synthetases designated LanL, has corresponding gene is generically designated recently been discovered (Goto et al., 2010). It lanA. Lantibiotics can be further subdivided remains to be established if LanL-modifi ed according to the enzymes that catalyse Lan peptides possess antimicrobial activity, and and meLan formation. Type I prepropeptides thus it has been suggested that the term such as the prototypic lantibiotic nisin A ‘lantipeptides’ be used to describe Lan- undergo dehydration as a result of the containing peptides that lack antimicrobial catalytic activity of the NisB protein activity (Goto et al., 2010). With respect to the (generically referred to as LanB enzymes) lantibiotics, in addition to the LanB, LanC (Karakas Sen et al., 1999). As a result, specifi c and LanM enzymes, a number of other serine and threonine residues are enzymes have been associated with the dehydrated to become the unique amino modifi cation of specifi c peptides (Kupke and acids 2,3-dehydroalanine (Dha) and Gotz, 1997; Peschel et al., 1997; Majer et al., 2,3-dehydrobutyrine (Dhb), respectively. 2002; Cott er et al., 2005c). Some or all of these new residues are then Aft er modifi cation, the peptide is the subject of a LanC-catalysed reaction. transported via an ABC transport system to This results in their interaction with the thiol the cell surface (Fath and Kolter, 1993) and groups of cysteines within the peptide to the leader sequence is proteolytically 24 B. Healy et al.

removed – although not necessarily in that 2.3 Classifi cation order – leading to the production of the active lantibiotic (Havarstein et al., 1995). For A scheme to classify lantibiotics was fi rst type I lantibiotics, these individual steps are proposed by Jung in 1991. This scheme carried out independently by a transporter grouped the peptides into two distinct (designated LanT) (Qiao and Saris, 1996) and categories. The fi rst class, type A, contained a protease (LanP) (Ye et al., 1995; Siezen, the elongated, fl exible, amphipathic peptides. 1996); in the case of type II lantibiotics, both These peptides had a net positive charge and activities are carried out by one enzyme (also were thought to function through the designated LanT) (Rince et al., 1994; Chen et permeabilization of the cell membrane. The al., 1999; Altena et al., 2000). To ensure that second class, type B, contained globular the active antimicrobial does not target the proteins with a net negative or neutral producing cells, immunity proteins are charge. These proteins were thought to produced. Two immunity mechanisms have function by inhibiting sensitive cells through been described. One relies on a single the formation of complexes with specifi c immunity protein, LanI, while the second membrane components. While some of this involves a multicomponent ABC-transporter information continues to be pertinent, the system referred to as LanEFG. Indeed, some use of this system has been made more lantibiotic producers utilize two such diffi cult by investigations that have mechanisms (Draper et al., 2008). established that some lantibiotics act in The remaining group of lantibiotic- multiple diff erent ways, coupled with the associated proteins to which we have yet to identifi cation of new and more diverse refer is the regulators. Initial studies in this lantibiotics. This issue has been addressed by area focused on the regulation of nisin and approaches that refl ect the genes and its related peptide subtilin by two- proteins involved in lantibiotic production. component signal transduction systems, Most recently, this has involved the which consist of a histidine kinase (LanK) fusion of two compatible approaches (Piper and a response regulator (LanR) (Klein et al., et al., 2009a) in which peptides are classifi ed 1993; Engelke et al., 1994; Stock et al., 2000; on the basis of the sequence of the lantibiotic Kleerebezem, 2004). Examination of the propeptide (Cott er et al., 2005a) or the genetic determinants of a subtilin producer composition of the associated modifi cation/ revealed the presence of the spaK and spaR transport systems (Pag and Sahl, 2002; Willey genes. Using a strategy of gene deletion, it and van der Donk, 2007). Using this was shown that mutants lacking these genes combined approach, lantibiotics can be were no longer capable of lantibiotic subdivided into the three types (i.e. types I, production (Klein et al., 1993) A similar II and III, referred to above) and 12 approach also revealed the importance of subgroups. The subclassifi cation of the type I the corresponding nisin genes (Engelke et and II lantibiotics into 12 subgroups is based al., 1994). In both cases, it has been on the alignment of the unmodifi ed amino established that the associated antimicrobial acid sequence of the structural peptide. In peptides also function as pheromones as each case, homology results in the part of a LanRK-mediated quorum sensing classifi cation of the lantibiotic in question, system, which facilitates autoregulation together with the eponymous member of the (Kleerebezem, 2004). While two-component group (i.e. planosporicin, nisin, epidermin, systems play a major role in the production streptin, Pep5, lacticin 481, mersacidin, of a number of other lantibiotics, such as LtnA2, cytolysin, lactocin S, cinnamycin and mersacidin (Guder et al., 2002), other quite sublancin; Fig. 2.1). Thus, for example, the diff erent regulators have also been noted mersacidin subgroup includes RumB, (McAuliff e et al., 2001). plantaricin C, mersacidin, michiganin, Lantibiotic-related Research and Application 25

actagardine, C55a, LtnA1, SmbB, bhtA-α, Plwa, BliA1 and BhaA1, all of which are modifi ed by LanM enzymes (i.e. are of type II) and contain a number of residues that are conserved across the group. It should be noted that problems can arise when trying to add newly purifi ed lantibiotics for which a structure, but not gene sequences, is available to this system. Thus, while it is apparent from its unusual structure and modifi cations that microbisporicin represents a 13th lantibiotic subclass (Castiglione et al., 2008), it will not be possible to satisfactorily incorporate it into the system until the associated biosynthetic genes have been identifi ed and the lanA gene sequenced.

2.4 Lantibiotic Subgroups: Biology, Structure and Mode of Action

2.4.1 Nisin-like lantibiotics

Nisin A is the most thoroughly characterized lantibiotic. This 34 amino acid peptide contains three dehydrated amino acids and fi ve thioether rings (Fig. 2.2) (Gross and Morell, 1971; Buchman et al., 1988). Nisin A is the eponymous member of the nisin-like lantibiotics, which also include nisin Z, nisin F, nisin U, nisin U2, nisin Q, subtilin, ericin S and ericin A. The mechanism of action of nisin A (and nisin Z, which diff ers from nisin A by one amino acid) has been investigated in depth and it is likely that all related peptides function in a broadly similar manner. In addition to a long-standing appreciation of its ability to form pores, it was revealed in the mid-1990s that nisin binds to lipid II, an essential precursor of peptidoglycan, and thus also inhibits cell wall synthesis (Brotz et al., 1998; Breukink et al., 1999). Structural analysis of the lipid II–nisin complex has revealed that the Fig. 2.1. Lantibiotic classifi cation. Lantibiotics N-terminal region of the nisin peptide is can be divided into type I, II and III peptides. The responsible for lipid II binding (Hsu et al., type I and II peptides can be further divided into 2004). The C-terminal end of the peptide, 12 groups on the basis of the sequence of the lantibiotic propeptide. In one representative which is linked to the N-terminus by a three case, the mersacidin-like peptides, each of the amino acid ‘hinge’ region, is the pore- corresponding propeptides is listed and aligned. forming domain (Breukink et al., 1997; Amino acids that are 100% conserved are Wiedemann et al., 2001). The effi ciency of highlighted in black. pore formation is greatly enhanced by lipid 26 B. Healy et al.

II binding (Breukink et al., 1999). A third 2.4.2 Epidermin-like lantibiotics mode of action has also been revealed. It is now apparent that nisin can induce the Epidermin is produced by Staphylococcus autolysis of susceptible staphylococcal epidermidis and is encoded by a structural strains as a consequence of the release of two gene, epiA, which is located on a 54 kb cell wall hydrolysing cationic enzymes plasmid (Schnell et al., 1992). The active during normal autolysis of dividing cells lantibiotic is a 21 amino acid peptide that (Hasper et al., 2004). contains four rings, including three (me)Lans

Fig. 2.2. Some representative lantibiotic structures. Modiﬁ ed residues are shaded or dashed. Ala-S-Ala, lanthionine (dashed); Abu-S-Ala, ˟-methyllanthionine (light grey); Ala, D-alanine (grey dashed); Dha, dehydroalanine (dark grey); Dhb, dehydrobutyrine (black, white text); Asp, ˟-hydroxy-aspartate (grey, white text); Lys-NH-Ala, lysinoalanine (black, grey text). Lantibiotic-related Research and Application 27

and a 2-aminovinyl-d-cysteine (Allgaier et al., 2.4.4 Pep5-like peptides 1986). Aft er epidermin, gallidermin is the most extensively studied epidermin-like Pep5 is a tricyclic lantibiotic consisting of 34 lantibiotic. The two peptides are structural amino acids (including three Lan bridges: analogues that diff er by only one residue (at one meLan and two Lan) (Bierbaum et al., position 6) (Kellner et al., 1988). Nuclear 1994, 1996). It is both screw-shaped and magnetic resonance (NMR) spectroscopy highly cationic, the latt er property being due studies into the structure of gallidermin have to the presence of six lysine and two arginine revealed that the rigid N-terminally located residues in the mature peptide (Pag et al., rings A and B are connected to the C and D 1999). It is produced by Staph. epidermidis rings at the C-terminal end of the peptide by strain 5 (Kalett a et al., 1989), with the a somewhat fl exible region spanning associated genes located on the 20 kb residues A12 to G15 (Ott enwalder et al., plasmid pED503 (Ersfeld-Dressen et al., 1984; 1995). A comparison of the structures of Meyer et al., 1995). epidermin and nisin shows high homology Pep5 also has another less common in the N-terminal, lipid II-binding ends of feature: an N-terminal 2-oxobutyryl group the peptides (Hsu et al., 2004). Their that is thought to be formed through the C-terminal ends, however, diff er con sider- non-enzymatic hydrolysis of N-terminal Dhb ably, thereby explaining the inability of residues (Xie and van der Donk, 2004). Pep5 epidermin to form pores in certain targets exerts its mode of action by forming voltage- (Bonelli et al., 2006). dependent pores in the cytoplasmic membrane of sensitive cells (Sahl et al., 1987; Kordel et al., 1989). These pores cause the leakage of essential metabolites and ATP out 2.4.3 Planosporicin- and streptin-like of the cell, resulting in the cessation of lantibiotics cellular metabolic processes and ultimately causing cell death. Pep5 can also induce the In addition to the nisin- and epidermin-like autolysis of staphylococci through the peptides, two other peptides – planosporicin activation of cell wall hydrolysing enzymes and streptin – resemble the former with (Bierbaum and Sahl, 1985, 1987). respect to their N-terminal domains. Otherwise, they diff er quite considerably from these and from one another. As a result 2.4.5 Lacticin 481-like lantibiotics both are the eponymous (and sole) members of two lantibiotic subclasses. Planosporicin, The type II lacticin 481-like subgroup is the produced by Planomonospora species, contains largest lantibiotic subgroup. Its associated both Lan and meLan residues, which peptides are notable by virtue of lacking generate fi ve intramolecular thioether post-translational modifi cations other than bridges (Fig. 2.2) (Castiglione et al., 2007; the common Dha, Dhb, Lan and meLan Maffi oli et al., 2009). Planosporicin functions residues (Dufour et al., 2007). The Lactococcus primarily through the inhibition of lactis subspecies lactis CNRZ481-produced peptidoglycan synthesis but, unlike nisin eponymous member of this group, lacticin and epidermin, does not bind to the d-Ala-d- 481, is 27 amino acids in length and contains Ala motif of lipid II (Castiglione et al., 2007). two Lans, one meLan and one Dhb residue Streptin is a Streptococcus pyogenes-associated (Fig. 2.2) (Piard et al., 1993; van den Hooven lantibiotic, two major forms of which have et al., 1996). Interestingly, although lacticin been purifi ed. Streptin 1 is the fully mature 481 is quite similar to the related variacin 23 amino acid peptide, while streptin 2 has (fi ve amino acid diff erences), variacin three additional residues at the N-terminal appears to have a much broader target-cell end (TPY) (Karaya et al., 2001; Wescombe and spectrum of activity (Pridmore et al., 1996). Tagg, 2003). Studies with another member of this group, 28 B. Healy et al.

nukacin ISK-1, indicate it to be bacteriostatic on a 60.2 kb conjugative plasmid, pMRCO1, and incapable of pore formation which contains ten open reading frames (Asaduzzaman et al., 2009), whereas the (Dougherty et al., 1998). The mechanism of related streptococcin SA-FF2 peptide causes action of lacticin 3147, which is also lipid II the formation of short-lived pores in target mediated, depends on the presence of both cells (Jack et al., 1994). components (i.e. Ltnα and Ltnβ, derived from the LtnA1 and LtnA2 propeptides, respectively). More specifi cally, Ltnα fi rst 2.4.6 Mersacidin-like lantibiotics binds to lipid II in sensitive cells. This binding is thought to lead to a conformational Mersacidin is a small lantibiotic produced by change that produces a high-affi nity binding Bacillus species (Chatt erjee et al., 1992). The site for the Ltnβ peptide. Cell death occurs biosynthetic gene cluster (12.3 kb in size) through the permeabilization of the cell consists of ten genes and is located on the membrane (Wiedemann et al., 2006), leading bacterial chromosome. This hydrophobic and to the effl ux of potassium ions and phosphate neutral peptide contains three meLans and a and resulting in hydrolysis of cellular ATP. single 2-aminovinyl-2-methylcysteine corres- The related haloduracin peptide has recently ponding to four intramolecular rings (Fig. 2.2) been shown to function in a similar way (Chatt erjee et al., 1992). Mersacidin does not (Oman and van der Donk, 2009). form pores in the cell membrane of sensitive cells, but does inhibit cell wall synthesis through binding with lipid II (Brotz et al., 2.4.8 Other type II lantibiotics 1995). An NMR study carried out by Hsu et al. (2003) showed that mersacidin can change Other type II subgroups include the its conformation depending on whether it is cinnamycin-like, sublancin-like, lactocin in the presence of lipid II. In addition to S-like and cytolysin-like subgroups. While one-peptide lantibiotics such as mersacidin, there are a number of cinnamycin-like the mersacidin-like peptides also contain the peptides, sublancin, lactocin S and cytolysin A1 peptide of a number of two-peptide are the sole members of their respective lantibiotics: lacticin 3147 (i.e. Ltnα and Ltnβ; subgroups. Cinnamycin is a tetracyclic Fig. 2.2) (Ryan et al., 1999b), staphylococcin lantibiotic produced by Streptoverticillium C55 (Navaratna et al., 1998), plantaricin W griseoverticillatum. It is a 19 amino acid (Holo et al., 2001), BHT (Hyink et al., 2005), peptide and contains the unusual residues Smb (Yonezawa and Kuramitsu, 2005), lysinoalanine and 3-hydroxyaspartic acid lichenicidin (Begley et al., 2009; Dischinger et (Fig. 2.2). In addition to its antimicrobial al., 2009) and haloduracin (Fig. 2.2) activity, cinnamycin and related peptides (McClerren et al., 2006; Lawton et al., 2007a). have other potentially useful pharmaceutical These two-peptide lantibiotics only exhibit properties, including the inhibition of optimal activity when the A1 component is phospholipase A2 and angiotensin-converting combined with its A2 counterpart. These enzyme (Fredenhagen et al., 1990; Kalett a et conserved A2 peptides are referred to as the al., 1991; Hosoda et al., 1996). Sublancin, LtnA2-like peptides (see next section). produced from Bacillus subtilis 168, is a 37 amino acid peptide that contains one meLan and two disulfi de bridges. The presence of 2.4.7 LtnA2-like peptides the stabilizing Lan bridges along with relatively weak disulfi de bridges suggests a Lacticin 3147, produced by L. lactis conformational uniqueness that could confer subspecies lactis DPC3147, is the most a selective advantage (Paik et al., 1998). extensively studied two-peptide lantibiotic Lactocin S, produced from Lactobacillus sake (Ryan et al., 1996). The genes responsible for L45, is a 37 amino acid lantibiotic (Mortvedt the production of the lantibiotic, and of the et al., 1991; Skaugen et al., 1997). This associated immunity proteins, are encoded lantibiotic is noteworthy as a consequence of Lantibiotic-related Research and Application 29

containing d-alanine residues (Skaugen et al., The Netherlands) or ‘ingredient’ DURAFresh 1994). Among lantibiotics, only lacticin 3147 (Kerry Bio-Science, Co. Kerry, Ireland). shares this trait. At a neutral pH, lactocin S Other than nisin, lacticin 3147 and exhibits a net neutral charge (Rawlinson et lacticin 481 are the two most extensively al., 2002). Finally, cytolysin is a two-peptide studied LAB lantibiotics. Both exhibit traits lantibiotic (CylLL and CylLS) produced by that suggest they have commercial value, enterococci. It is unique by virtue of its and a few selected examples of potential ability to target both eukaryotic and applications are presented here. In the case prokaryotic cells (Booth et al., 1996). of lacticin 3147, it has been established that lactococcal producers of lacticin 3147 can be employed as starter cultures for the manufacture of Cheddar cheese (Ryan et al., 2.5 Current and Future Use of 1996). Lacticin 3147 producers successfully Lantibiotics for Food Applications reduce the pH of milk to 5.2, while also generating suffi cient quantities of the The most prominent event in the majority of lantibiotic to control the adventitious food fermentations is the conversion of non-starter LAB (NSLAB) over a 6-month sugars to lactic acid by LAB. In addition to ripening period (Ryan et al., 1996). This is lactic acid, LAB can produce other signifi cant as NSLAB can be the cause of metabolites with antimicrobial activity such fl avour defects and calcium lactate formation as hydrogen peroxide, diacetyl, acetoin and (Thomas and Crow, 1983). The use of lacticin bacteriocins, including a number of 481-producing strains as adjunct cultures in lantibiotics such as nisin A. Highlights in the cheese production has also been mooted industrial application of nisin A as a food (O’Sullivan et al., 2003). This lantibiotic, preservative include its initial use by the which is produced by L. lactis strains (Piard food industry in 1953 and its approval by the et al., 1992), demonstrates a higher ability to World Health Organization, European Union lyse sensitive lactococci than lacticin 3147 and US Food and Drug Administration in when used in combination with the starter 1969, 1983 and 1988, respectively. Currently, culture Lactococcus HP. The associated nisin A is approved for use in over 48 benefi ts are the release of intracellular countries worldwide (Delves-Broughton et enzymes, thereby speeding up the ripening al., 1996; Cott er et al., 2005b; Deegan et al., process, and a reduction in the numbers of 2006). Nisin can be added to a food in NSLAB (O’Sullivan et al., 2003). Studies number of ways. These include the direct investigating the use of a lacticin 3147-based application of the peptide, in a highly powder as a biopreservative have also purifi ed form if necessary, as an ‘additive’ yielded interesting results (Morgan et al., (being one of only two authorized natural 2001). Like nisin, a fermentate containing food antimicrobials, the other being the anti- lacticin 3147 provides an alternative means mould additive natamycin); the introduction of introducing the lantibiotic into food. of a nisin-producing bacteria, as a starter or Incorporation of a whey powder (10%), an adjunct culture, to a fermented food (and which was fermented with a lacticin 3147 the subsequent production of nisin in situ); producer, was found to bring about a 99.9% and using the producer to make a food-grade reduction in Listeria monocytogenes in natural fermentate, which can be dried to make a yoghurt and an 85% reduction in pathogen powdered ‘ingredient’ that can be numbers in a cott age cheese sample within a incorporated into either fermented or 2-h time frame. It has also been established non-fermented foods. In the case of nisin, all that an 80% reduction in Bacillus cereus three alternatives are employed, for example numbers occurs within 3 h when 1% powder ‘Additive’ Nisaplin (Danisco, Copenhagen, is added to soup. For a comprehensive Denmark), Nisin-producing cultures (from review on the use of bacteriocins as biological culture providers such as Chr. Hansen, agents for food safety, see Deegan et al. Horsholm, Denmark, and CSK, Leeuwarden, (2006). 30 B. Healy et al.

2.6 Lantibiotics and Their Medical infection (Kruszewska et al., 2004) and nisin Applications F, both alone and when used in combination with lysozyme and lactoferrin, can The possibility of using lantibiotics to control successfully treat respiratory tract MRSA or treat multidrug-resistant forms of infections in mice (De Kwaadsteniet et al., pathogens such as Staphylococcus aureus, 2009). Trials investigating the use of Enterococcus species and Clostridium diffi cile lantibiotics to control the microorganisms has gained increased att ention in recent years responsible for dental plaque, halitosis, due to a number of positive results obtained ‘strep’ throat (Hillman, 2002; Burton et al., by researchers in the fi eld (for a 2006; Dierksen et al., 2007) and even bovine comprehensive review, see Piper et al., mastitis (Ryan et al., 1999a; Twomey et al., 2009a). In vitro, many lantibiotics, including 2000) have all been successful. lacticin 3147, mutacin B-Ny266, nisin and mutacin 1140, show activity against clinical targets such as methicillin-resistant Staph. 2.7 Engineering of Lantibiotics aureus (MRSA), vancomycin-resistant Entero- coccus faecalis (VRE), penicillin-resistant Lantibiotics are gene encoded. Advantage Pneumococcus , Propionibacterium acne, Strepto- can be taken of this trait to engineer novel coccus mutans, Strep. pyogenes, Strep. variants of the parent peptide. Such variants pneumoniae, C. diffi cile, Listeria and Bacillus have been used to study structure–function species (Severina et al., 1998; Galvin et al., relationships and, in some cases, engineering 1999; Mota-Meira et al., 2000; Brumfi tt et al., strategies have led to the generation of 2002; Rea et al., 2007; Ghobrial et al., 2009; peptides with enhanced antimicrobial Piper et al., 2009b). It is also interesting that activity. Lantibiotic engineering can take both Pep5 and epidermin successfully inhibit place in vivo (i.e. by manipulating the original the adhesion of staphylococcal cells to the producing strain or expressing the genes surfaces of catheters (Fontana et al., 2006). It heterologously in an alternative host) or in is important to note, however, that these vitro (i.e. by harnessing the activity of represent just a selection of the studies that purifi ed forms of the individual components have highlighted the effi cacy of lantibiotics of biosynthetic machinery outside of a host). against Gram-positive clinical pathogens. It The application of engineering to lantibiotic is anticipated that the number of studies in research commenced in 1992. Although this area will continue to increase as a initial nisin-focused investigations did not consequence of the further investigation of lead to the production of variants with existing lantibiotic peptides and the enhanced activity (Kuipers et al., 1992), they continued identifi cation of new forms of clearly demonstrated the power of this these antimicrobials. Two recent examples of technology. During the same year, the note are the two-peptide lantibiotic extreme consequences of making single, lichenicidin, which exhibits antimicrobial deliberate amino acid changes were activity against MRSA and VRE strains demonstrated when it was established that a (Begley et al., 2009), and microbisporicin, single residue change in subtilin resulted in a which is active against MRSA, VRE and 57-fold increase in its biological and chemical clinical streptococci (Castiglione et al., 2008). stability (Liu and Hansen, 1992). This While the in vitro success of a technology has continued to be applied, and chemotherapeutic agent does not always in 2006 the fi rst alanine-scanning correspond to in vivo effi cacy, a number of mutagenesis of a lantibiotic, lacticin 3147, studies have indicated that this may not be a was completed (Cott er et al., 2006a). In this major failing of lantibiotics. It has been study, alanine (or glycine in cases where an revealed that mutacin B-Ny266 can be as alanine was already present) was introduced active as vancomycin against MRSA in vivo in place of the 59 amino acids, in turn, and (Mota-Meira et al., 2005), mersacidin can be the impact on the antimicrobial activity of employed to eradicate a nasal MRSA the associated producing strain was Lantibiotic-related Research and Application 31

quantifi ed. The data generated highlighted KFI and KSI (the lett ers indicate the amino specifi c areas of the peptides in which subse- acids present at positions 4, 5 and 6), quent site-specifi c mutagenesis approaches displayed increased antimicrobial activity might be benefi cial. This strategy was taken against a number of bacteria such as a step further when site-saturation Leuconostoc mesenteroides, Lactobacillus mutagenesis was employed to engineer both johnsonii and Lactococcus lactis. KFI, and nukacin ISK-1 (Islam et al., 2009) and another variant, VFG, also inhibited the mersacidin (Appleyard et al., 2009). Both outgrowth of B. subtilis 168 spores more studies provided an in-depth insight into the eff ectively than the wild type. structure–function relationships within the It should be noted that these and other respective peptides. In the case of nukacin (bio)engineering-related strategies have also ISK-1, two variants displaying a twofold been used for a variety of other purposes, increase in specifi c activity were identifi ed such as increasing lantibiotic production (Islam et al., 2009). (Cott er et al., 2006b; Heinzmann et al., 2006), The use of engineering to study or introducing Lans into class II bacteriocins improve nisin has also continued at pace. (Majchrzykiewicz et al., 2010) and even post- Since the 1990s, this lantibiotic has been the translational modifi cation of other bioactive subject of a number of engineering-based peptides (Kuipers et al., 2004; Kluskens et al., strategies, which have employed site- 2009; Rink et al., 2010). In addition to these directed, site-saturation and random approaches, the ever-improving ability of mutagenesis. While various diff erent regions chemists to generate lantibiotic-like peptides of the peptide have been engineered, the through synthetic chemistry (Cobb and N-terminal and ‘hinge’ regions have received Vederas, 2007; Arnusch et al., 2008; Ross et al., the greatest att ention. The benefi ts of 2010) is particularly exciting and has already manipulating the hinge (consisting of Asn20- facilitated the creation of potent nisin– Met21-Lys22) have been particularly notable vancomycin hybrids (Arnusch et al., 2008). (Yuan et al., 2004; Field et al., 2008). Yuan et al. (2004) employed a site-directed approach whereby either positively or negatively 2.8 Screening for New Lantibiotics charged amino acids were introduced into the hinge. These studies demonstrated that While scientists are continuing with their specifi c changes (i.e. N20K and M21K) eff orts to further improve known lantibiotics, increased the activity of the peptide against there is still considerable merit att ached to Gram-negative bacteria such as Shigella, identifying new peptides. It has also become Pseudomonas and Salmonella species. In the apparent in recent years that bioinformatics case of Field et al. (2008), screening of a bank can be a very useful means of screening for of random mutagenized nisin variants such novel lantibiotics. The availability of revealed that a K22T variant displayed databases, including both general (NCBI) enhanced activity against the mastitic patho- and dedicated (BAGEL and BACTIBASE) gen Streptococcus agalactiae. This prompted systems (see Chapter 1, Table 1.1, for more) the use of site-saturation mutagenesis for (de Jong et al., 2006; Hammami et al., 2007, each of the individual hinge residues that, 2010), has facilitated the use of in silico when coupled with a larger selection of approaches to lantibiotic screening. The target strains, led to the identifi cation of a two-peptide lantibiotic lichenicidin (Begley number of peptides with enhanced activity et al., 2009) was identifi ed using such an against Strep. agalactiae, Staph. aureus and L. approach. Here, the highly conserved nature monocytogenes. Yet another study, focusing on of the lanM gene was exploited to screen the rings A and B at the N-terminal end of nisin ever-increasing number of bacterial genome A, showed that the various activities of nisin sequences that are publicly available. Initial A can be altered by changing the amino acid screening revealed 89 lanM genes, of which arrangement in this region of the peptide 61 had not previously been associated with (Rink et al., 2007). Two mutants, designated lantibiotic production. One of the potential 32 B. Healy et al.

novel lantibiotic producers identifi ed, Bacillus therapeutics that can target the multidrug- licheniformis ATCC 14580, was selected and resistant Gram-positive clinical pathogens, from it lichenicidin was isolated (Begley et including the particularly problematic al., 2009). A similar approach was previously pathogens MRSA, VRE and C. diffi cile. A employed to identify another two-peptide number of these peptides are particularly lantibiotic, haloduracin, which is produced att ractive as a consequence of research that by Bacillus halodurans C-125 (McClerren et al., has established that they have mechanisms 2006; Lawton et al., 2007a). Bioinformatics of action and target binding sites that are has also been of considerable use when distinct from those of non-lantibiotics. This, designing engineered lantibiotics and novel coupled with their high potency and Lan-containing peptides. A study by Rink et generally non-cytotoxic nature, could lead to al. (2005) used bioinformatics to predict the these compounds having clinical applications impact of fl anking amino acids on the in the future. The possibility of engineering dehydration of serine and threonine residues, new and improved lantibiotics, producing and subsequent Lan and meLan formation, novel chemothera peutics through the fusion in lantibiotic peptides. An in silico of lantibiotics with antibiotics, introducing comparison of known lantibiotics found that lantibiotic-associated modifi cations into the majority of modifi ed serines and non-lantibiotics and chemically synthesizing threonines were fl anked by hydrophobic new lantibiotic-like peptides – as well as the residues. In silico models predicted the likely ongoing use of traditional and in silico impact of specifi c residues on modifi cation strategies to fi nd novel compounds – all when located adjacent to hydroxyl-amino bring this potential to a new level. In acid residues. The subsequent creation, and addition to these new markets, it should not investigation, of these peptides validated this be forgott en that a lantibiotic, nisin, has been theory. successfully employed by the food industry The discovery of the lantibiotics micro- for over a half century. Other lantibiotics bisporicin and planosporicin was achieved have the potential to be similarly employed using a more traditional screening method and, as a consequence of the limited activity (Castiglione et al., 2007, 2008). This method of nisin against certain target strains and involved 120,000 broth extracts obtained by species, together with its poor activity at fermenting 40,000 actinomycetes. The neutral pH, there are obvious niche-markets microbial products were screened to assess that these can fi ll. The commercial potential their activity against Staph. aureus before of lantibiotics and lantibiotic-related selecting those that retained activity technology and the cutt ing-edge funda- following exposure to a β-lactamase cocktail mental science that underpins lantibiotic or d-alanyl-d-alanine affi nity resin (i.e. they research will ensure that these peptides were neither β-lactams nor vancomycin-like continue to att ract great att ention in the glycopeptides). Those that retained anti- coming years. microbial activity aft er these steps were selected for further investigation. This strategy yielded 35 lantibiotics, of which fi ve Acknowledgement showed litt le or no similarity to any known lantibiotics. The authors would like to thank Des Field for contributing to fi gure preparation.

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Quentin Kaas, Jan-Christoph Westermann, Sónia Troeira Henriques and David J. Craik*

Abstract

This chapter provides an overview of plant antimicrobial peptides. It mainly focuses on one particular class of plant defence peptides, namely the cyclotides, which have been discovered over the last decade in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. Cyclotides have a head-to-tail cyclized peptide backbone and a cystine knot motif formed from their six conserved cysteine residues, which makes them exceptionally stable. This chapter describes their isolation and characterization, structure and biosynthesis, and applications. The structural stability of cyclotides makes them excellent scaﬀ olds for the engineering of novel therapeutic proteins. Advances in methods for the production of cyclotides and their potential clinical applications are also described.

3.1 Introduction to Plant pharmaceutical activities of plant natural Antimicrobial Peptides products, readers are referred to other reviews (O’Keefe, 2001; da Rocha Pitt a and Unlike animals, plants lack the ability to Galdino, 2010). evade the att ack of predators or pathogens To set the parameters of this chapter, we through movement. With the ‘fi ght or fl ight’ will defi ne a peptide as a structure consisting defence response not available to them, of <100 amino acid residues, and we will not plants have evolved an impressive array of cover the literature for proteins of >100 defence molecules to confer protection once amino acids. A search for the term ‘anti- physical barriers have been compromised. microbial plant peptide’ in the UniProtKB This chapter describes a subset of this database (Bairoch et al., 2005; UniProt defence arsenal, namely peptide-based Consortium, 2010) yields >400 sequences, but molecules produced by plants as anti- many of these have >100 residues and are microbial agents directed against fungi, thus not the subject of this chapter. AMPs bacteria or viruses. Peptides that protect isolated from endophytes (Yu et al., 2010) against att ack by herbivores such as insects living in symbiosis with plants are also not have been reviewed elsewhere (Ryan, 2000; covered here. In summary, we focus on Kessler and Baldwin, 2002; Gruber et al., peptides of <100 amino acids produced 2007a; Ryan et al., 2007; Howe and Jander, endogenously by plants to protect themselves 2008; Craik, 2009). Many antimicrobial against bacteria, viruses or fungi. peptides (AMPs) from plants were originally Most plant AMPs act by compromising discovered through screening programmes the structural integrity of the most prominent directed at a variety of pharmaceutical target in microbes, their outer membrane, activities. For a broader perspective on the which has dramatic consequences for the * Corresponding author.

microbe. This mechanism of action is (Hammami et al., 2009). As of February 2010, achieved by either non-specifi c targeting of this database contains just over 270 sequences. microbial membrane lipids or through In addition to the peptide families noted targeting specifi c macromolecular com- above, PhytAMP recognizes the cyclotides, ponents in the membrane. As shown in Table snakins, β-barrelins and impatiens peptides as 3.1, many plant AMPs are positively charged, plant AMPs. With the exception of cyclotides, which is thought to play an important role in which are documented in a dedicated their interaction with membrane lipid head database, CyBase (Mulvenna et al., 2006a; groups. In addition to their positively Wang et al., 2008a), these miscellaneous charged primary structure, plant AMPs peptide families have only a few members display the typical range of secondary and BLAST searches against the UniProtKB structure motifs seen in proteins, (i.e. database return only a few (<30) protein hits α-helices, β-strands, β-turns and loops). As from the plant kingdom. Several other well as having well-defi ned secondary databases also contain information about structures, they typically have well-defi ned plant AMPs. These include the Antimicrobial tertiary structures, oft en as a result of spatial Peptide Database (htt p://aps.unmc.edu/AP/ restraints from disulfi de bonds or in some main.php) (Wang and Wang, 2004) and the cases via cyclization of the peptide backbone. AMSDb (Antimicrobial Sequences Database) Both of these restraint types are combined in from the Anti-infective Peptides Laboratory of the cyclotide class of peptides (Craik et al., Allesandro Tossi at the University of Trieste 1999), which incorporate three disulfi de (htt p://www.bbcm.univ.trieste.it/~tossi/pag1. bonds and a cyclized peptide backbone, htm). Table 3.1 gives an overview of the conferring remarkable structural stability to largest families of AMPs from plants, these molecules. Cyclotides are a major focus indicating the size, charge and number of of studies in our laboratory and hence we disulfi de bonds, as well as information on devote a substantial section of this chapter to secondary structures. them. Most classical AMPs from plants can be divided into four main classes: the lipid- 3.1.1 Lipid-transfer proteins transfer peptides, the thionins, the plant defensins (previously named γ-thionins) and Non-specifi c lipid-transfer proteins (LTPs) the hevein/knott in-type chitin-binding pep- from plants typically contain >90 amino tides (Broekaert et al., 1997). Additional acids. They are the largest peptides miscellaneous plant AMPs do not fi t into considered in this chapter, falling just below these categories. PhytAMP is a curated online our arbitrary cut-off of 100 amino acids. They database of plant AMPs that focuses on AMPs have an overall positive charge and, as their shown experimentally to be expressed name suggests, they bind lipids in a

Table 3.1. Overview of structural features of AMPs from plants. Size Number of Secondary No. entries in Peptide class (residues) Chargea disulﬁ de bonds structureb UniProtKBc Lipid-transfer proteins 90–100 +8 4 h 940 Thionins 45–47 +7 or +10 3 or 4 h, s 266 Defensins 40–50 +6 4 h, s 418 Chitin-binding peptides Hevein-like ~50 –1 4 h, l, s 545 Knottins ~30 +3 3 h, l, s Cyclotides 28–37 –2 to +3 3 h, l, s 172 a Charge of side chains at neutral pH; values are for prototypic examples from each family. b h, helix; l, loop or turn; s, sheet. c Number of entries in UniProtKB based on a search with the relevant peptide class keywords (e.g. ‘thionin’) and the word ‘plant’. 42 Q. Kaas et al.

non-specifi c manner, without a preference and plant growth and development are for a particular type of lipid. In vitro, these linked with LTPs (Yeats and Rose, 2008). peptides have been shown to transfer lipids Membrane targeting of LTPs from plants has from one membrane to another. In an early been shown to inhibit some bacterial (Molina example, transfer of radioactively labelled et al., 1993) and fungal (Terras et al., 1992) phosphatidylcholine from artifi cial vesicles plant pathogens in vitro. Terras et al. (1992) to chloroplasts from spinach was shown to showed that increasing the ionic strength of be mediated by an LTP isolated from spinach the assay medium abrogates antifungal leaves (Miquel et al., 1988). Similarly, electron activity, indicating a charge-dependent mech- paramagnetic resonance and fl uorescence anism of action, most likely involving experiments have been used to show that an interactions with the charged head groups of LTP from maize seeds was able to shutt le lipids in the fungal membrane. lipids containing spin labels from artifi cial The fi rst crystal structure of a plant LTP vesicles to membranes from human was for an example from Zea mays in erythrocytes and fl uorescently labelled lipids complex with palmitate (Shin et al., 1995). from artifi cial vesicles to bovine chromaffi n The solution structures of LTPs from Triticum granules, respectively (Geldwerth et al., aestivum (wheat) (Gincel et al., 1994), Hordeum 1991). In that study, negatively charged vulgare (barley) (Heinemann et al., 1996) and lipids with phosphatidylcholine, phospha- Z. mays were early examples solved by tidylethanolamine, phosphatidylserine and nuclear magnetic resonance (NMR) and are phosphatic acid head groups were found to shown in Fig. 3.1A (Gomar et al., 1996). The be transferred to the target membrane at structures of LTP from barley in complex similar rates; thus, a direct charge interaction with palmitate-coenzyme A (Lerche et al., between positively charged LTP and 1997) and palmitate (Lerche and Poulsen, negatively charged lipid head groups 1998) were also solved by NMR. The global appears to be important. This lipid-transfer fold of LTPs reveals a bundle of four helices activity has been implicated in plant cell that cage lipid chains in the hydrophobic development (Nieuwland et al., 2005), but core formed between them (Fig. 3.1B and Fig. generally a variety of bioactivities, including 3.1C). Only minor changes in the C-terminal defence against pathogens, cuticle synthesis region of LTPs have been reported when

Fig. 3.1. Structural features of lipid-transfer proteins (LTPs). (A) The apo (lipid-free) form of LTP from Zea mays comprises four α-helices (Protein Data Bank (PDB) access code: 1afh). (B) Palmitate (black, stick representation, carboxyl group on top) is deeply buried in the cage formed by the four helices of LTP from barley (PDB: 1be2). (C) Palmitate-coenzyme A (CoA) conjugate binds to barley LTP in a similar manner; the lipid (black) is buried inside the peptide, whereas CoA is located on the surface of the peptide (PDB: 1jtb). Note that these are two complexes solved for the same LTP; for sake of clarity, C is rotated by 180° in the view shown. AMPs in Plants 43

binding to lipids (Lerche et al., 1997; Lerche The fi rst two thionins to be discovered and Poulsen, 1998). were identifi ed (Balls et al., 1942a,b) and In general, plant AMPs are of much isolated (Fisher et al., 1968) from T. aestivum interest, not only for the control of plant (wheat) germ. Their names, α- and diseases (Montesinos, 2007), but also due to β-purothionins, refl ect that they are wheat their ability to att ack human pathogens, peptides (Greek: puro = wheat) and that they including Candida and Aspergillus species have a high sulfur content (Greek: thio = (Thevissen et al., 2007). These properties, sulfur). Mixtures of α- and β-purothionins, together with the growing problem of resist- as well as purifi ed peptides, were tested ance to conventional antibiotics (Hancock against a range of phytopathogenic bacteria. and Sahl, 2006), make plant AMPs interesting Activity was shown against Pseudomonas as novel human therapeutic leads. However, solanacearum, Xanthomonas phaseoli, Corynebac- as far as LTPs are concerned, such applica- terium michiganense and others, but not tions need to be evaluated carefully because against Pseudomonas savastanoi (Fernandez de some plant LTPs have been identifi ed as Caleya et al., 1972). Tests with purifi ed allergens in food (Zuidmeer and van Ree, purothionins showed that α-purothionin was 2007). For example, studies have reported more active against X. phaseoli than that allergy to hazelnut (Flinterman et al., β-purothionin, which in turn was more active 2008) and wheat (Inomata, 2009) may be against P. solanacearum (Fernandez de Caleya associated with these peptides. et al., 1972). These complementary activities of α- and β-purothionins exemplify how the diversity of AMPs in a single plant 3.1.2 Thionins confers resistance to a diverse spectrum of pathogens. Thionins comprise 45–47 residues. They are The antimicrobial activity of thionins is divided into fi ve classes depending on the thought to be enabled via an interaction number of disulfi de bonds, number of basic with the head groups of lipids in membranes residues and overall charge. Type I thionins and has been studied in detail. Hughes et al. have four disulfi de bonds and are highly (2000) showed that the presence of basic, with a charge of +10. Type II thionins negatively charged phosphatidylserine in also have four disulfi de bonds, but have a artifi cial lipid mixtures resulted in the reduced basic character with a charge of +7. formation of membrane pores acting as ion Type III and IV thionins both have three channels. However, NMR and infrared disulfi de bonds. Type III thionins have a charge of +7, whereas type IV thionins are spectroscopic evidence exists that in the neutral. Type V thionins appear to be absence of negatively charged lipids, truncated variants of other thionins thionins are also capable of binding both (Bohlmann and Apel, 1991; Stec, 2006). In neutral lipids and those with positive general, it appears that antimicrobial activity charges (Richard et al., 2002, 2005). This is correlated with the overall charge: the broad binding ability may explain their higher the positive charge, the higher the broad antibacterial spectrum. activity (Stec, 2006). Overall, thionins are a widely studied Thionins adopt an overall shape similar class of plant defence peptides. They to the lett er L (Stec, 2006). The structure potentially have applications as drug leads, consists of a β-strand followed by two refl ecting their presence as active compounds α-helices (helix-turn-helix motif) and a in traditional medicines from plants used second β-strand completing a double- since prehistoric times (Stec, 2006). Their stranded β-sheet. Several structures have activities against phytopathogenic microbes been found to accommodate lipid moieties in have also led to their incorporation in the groove formed by the two α-helices and transgenic plants to increase or confer two β-sheets (Fig. 3.2) (Stec et al., 1995; resistance to plant pests (Carmona et al., Debreczeni et al., 2003). 1993; Chan et al., 2005). 44 Q. Kaas et al.

charge, cysteine content and size. This resemblance resulted in their original assignment as γ-thionins (Colilla et al., 1990; Mendez et al., 1990). This terminology was abandoned when it became apparent that they shared more characteristics of their primary and tertiary structures with insect and mammalian defensins than with other classes of thionins (Terras et al., 1995). Plant defensins typically have a well-defi ned three- dimensional structure that comprises a triple-stranded β-sheet and an α-helix between the fi rst and second β-strand. The overall structure is maintained by four disulfi de bonds. Two are formed between the helix and the third β-sheet, while one tethers the C- and N-terminus for Rs-AFP1 (Raphanus sativus (radish) antifungal protein 1) (Fant et al., 1998) and Psd1 (Pisum sativum (pea) defensin 1) (Almeida et al., 2002). The remaining disulfi de bond links the loop between the fi rst β-strand and helix and the C-terminal end of the second β-strand. In contrast to LTPs and thionins, which non-specifi cally target membranes, many defensins, including Dm-AMP1 from Dahlia merckii (dahlia) (Thevissen et al., 2000a,b, 2003), Rs-AFP2 (Thevissen et al., 2004) and Fig. 3.2. The structure of type III ˟-purothionin Psd1 (Lobo et al., 2007), have specifi c from Triticum aestivum determined by X-ray molecular targets in the plasma membrane of crystallography (Stec et al., 1995). (A) Two fungi. extended ˟-sheets are stabilized by two disulfi de bonds (stick representation), one between the Dm-AMP1 is active against the sheets and a second linking the fi rst ˟-strand and fi lamentous fungus Neurospora crassa and the the C-terminus. A short third ˟-strand is located at unicellular fungus Saccharomyces cerevisiae. the C-terminus. A single disulfi de bond links the Binding of Dm-AMP1 to both fungi is α-helices. The structure shows the complex of the saturable, indicating that a specifi c target thionin with glycerol (sphere representation) in the with a fi nite concentration in the membrane groove between the helices and sheet. (B) For an exists (Thevissen et al., 2000b). Dm-AMP1 overview, the perspective is rotated by 90° (top binding competes with that of highly view of A). homologous peptides, but not with other membrane-active defensins of lower homology. In S. cerevisiae it has been reported 3.1.3 Plant defensins that sphingolipids are determinants of sensitivity towards Dm-AMP1 (Thevissen et Plant defensins are basic cysteine-rich al., 2000b). Evidence for this comes from the peptides of 45–54 residues (Thomma et al., fact that fungal strains of S. cerevisiae lacking 2002; Lay and Anderson, 2005; Pelegrini and the gene that encodes the enzyme required Franco, 2005). They are primarily recognized in the fi nal step in the synthesis of the as potent antifungal agents and exert their sphingolipid mannose-(inositol-phosphate)2- activity at the membrane of fungi, typically ceramide show increased resistance against inhibiting fungal growth (Lay and Anderson, Dm-AMP1 (Thevissen et al., 2000a). The 2005). They resemble thionins in their basic mechanism of action probably involves the AMPs in Plants 45

direct interaction of Dm-AMP1 with In an experiment with rat retinal neuroblasts sphingolipids, as sensitivity to Dm-AMP1 is (a model system to study progression not altered by the disruption of glycosyl through the cell cycle), it was shown that phosphatidylinositol-anchored peptides, Psd1 inhibition of cyclin F blocks transition which may be stabilized in the membrane by from the S to G2 phase during the cell cycle sphingolipids. Enhancement of binding in (Lobo et al., 2007). The localization of Psd1 to the presence of the fungal sterol ergosterol the fungal nucleus may occur as a result of also suggests the involvement of lipid raft s in an interaction of Psd1 with the fungal the interaction of Dm-AMP1 with membrane, with subsequent internalization. membranes (Thevissen et al., 2003). The interaction of Psd1 with artifi cial Rs-AFP2 is inactive against S. cerevisiae, membranes was recently studied by NMR but is highly active against Candida albicans (de Medeiros et al., 2010). Changes in and Pichia pastoris. The lack of glucosyl- chemical shift s, an indicator for changes in ceramide, a type of glucosphingolipid, in the chemical environment, were monitored resistant species is believed to be the for Psd1 in solution in the presence and functional diff erence between sensitive and absence of lipids. The interactions observed resistant species. Following the strategy were then mapped on to the three- exemplifi ed in the Dm-AMP1 example, dimensional structure of Psd1 (Fig. 3.3) sensitivity to Rs-AFP2 has been mapped to (Almeida et al., 2002). glucosylceramide; C. albicans and P. pastoris Plant defensins are att ractive candidates strains lacking the gene coding the enzyme in in the search for novel antimicrobial the fi nal step of glucosylceramide synthesis compounds (Thomma et al., 2003; Thevissen are resistant to the peptide (Thevissen et al., et al., 2007; Carvalho and Gomes, 2009). They 2004). The interaction of Rs-AFP2 and have been shown to be readily expressed as glucosylceramide is independent of sterol recombinant peptides in common expression content of the membrane and selective for systems, even allowing isotopic labelling as glucosylceramide from fungi, as plant and demonstrated, for example, for Psd1 mammalian glucosylceramides do not interact expression in P. pastoris (de Medeiros et al., with Rs-AFP2 (Thevissen et al., 2004). The fact 2010). Plant defensins have also been used in that sterol content does not have a role in this transgenic plants to increase resistance to interaction indicates a mechanism of action phytopathogens, as reviewed by Lay and diff erent from that of Dm-AMP1 (Thevissen et Anderson (2005). al., 2004). Recently, it has been shown that Rs-AFP2 induces the generation of reactive oxygen species (ROS) in C. albicans (Aerts et 3.1.4 Chitin-binding peptides al., 2007). This eff ect was linked to the antifungal activity as the presence of ascorbic The fungal membrane is predominantly acid, a scavenger of ROS, inhibited production (>90%) composed of polysaccharides (Latge, of ROS and abolished antifungal activity. 2007), including chitin, a polymer of β1,4- It was recently shown that defensin linked N-acetylglucosamine (Fig. 3.4A). Psd1, from P. sativum, may interact with the Chitin and its parent carbohydrate moiety, cell-cycle-related protein cyclin F from N. monomeric N-acetylglucosamine, are targeted crassa (Lobo et al., 2007). Identifi cation of by chitin-binding peptides, a subclass of cyclin F as the molecular target was achieved carbohydrate-binding lectins. in two steps. First, potential binding targets Hevein, isolated from the latex of Hevea were identifi ed from a yeast two-hybrid brasiliensis (rubber tree), was the fi rst system. The initial screen identifi ed nine cysteine-rich chitin binding protein from potential protein targets, including eight plants shown to inhibit fungi in vitro (Parĳ s proteins localized in the nucleus and one in et al., 1991); however, the activity was only the plasma membrane. Cyclin F was modest. Two homologues of hevein, confi rmed as a target in a glutathione Pn-AMP1 and Pn-AMP2, were isolated from S-transferase-tagged pull-down experiment. Ipomoea nil (Japanese morning glory) and 46 Q. Kaas et al.

Fig. 3.3. Structure of Rs-AFP1 (Raphanus sativus antifungal protein 1; Protein Data Bank (PDB) access code: 1ayj) and active residues of Psd1 (Pisum sativum defensin 1; PDB: 1jkz). (A) Rs-AFP2 adopts the typical fold of plant defensins. The N- and C-termini are in close proximity due to a disulfi de bond (ball and stick representation) from C3 to C51. (B) Backbone trace of Psd1. Residues of loop 1 (A7–N17) and turn 3 (H36–W38) and C35 are highlighted by stick representation. (C) Residues that experience chemical shift perturbation in the presence of phosphatidylcholine vesicles are shaded. (D) In the presence of 10% monohexosylceramide in the phosphatidylcholine vesicles, the distribution of residues affected by chemical shift perturbations changes signifi cantly. Loop 1 is affected from G12 to N17, as is W38 in turn 3 and the neighbouring K39 (N17, L30 and W38 are not labelled for clarity). showed much higher activity to a set of fungi They also have some activity against Gram- in vitro. The mode of action is most likely positive bacteria, although to a lesser extent. disruption of the fungal cell membrane; it Their amino acid sequence bears some was shown using fl uorescently labelled resemblance to other chitin-binding peptides peptide and confocal microscopy, in but they lack the C-terminal domain, which conjunction with electron microscopy, that includes two cysteine residues (Broekaert et Pn-AMP1 accumulates in the septa of fungal al., 1997). Both peptides show marked affi nity hyphae (Koo et al., 1998). to chitin at neutral pH. In other recent Antifungal activity has also been evidence demonstrating the importance of observed for two small peptides, Ac-AMP1 chitin binding, mutations in the chitin- and Ac-AMP2, from Amaranthus caudatus binding protein Cy-AMP1 (Cycas revoluta, (pendant amaranth) (Broekaert et al., 1992). cycad) that decrease affi nity for chitin also AMPs in Plants 47

decrease antimicrobial activity against fungi non-natural amino acids in complex with but not Gram-positive and Gram-negative chitotriose, that was solved in solution using bacteria (Yokoyama et al., 2009). This indi- NMR (Chavez et al., 2005). In the synthetic cates that binding to chitin is essential for the peptide, phenylalanine in position 18 and antimicrobial activity of chitin-binding tyrosine in position 20 were both substitu- peptides. However, the detailed mechanism ted with 4-ﬂ uorophenylalanine. The complex of membrane disruption is still unknown. reveals the interaction of the aromatic side Figure 3.4 shows the structure of a chain of residues 18 and 20 with the rings of mutant of Ac-AMP2, incorporating two the central N-acetyl glucosamine residue and

Fig. 3.4. Chitin and chitin-binding peptides. (A) ˟1,4-linked N-acetylglucosamine (top) is the building block of chitin. Chitotriose (bottom) is a deﬁ ned oligomer of N-acetylglucosamine. (B) An overlay of the solution structures of hevein (grey, Protein Data Bank (PDB) access code: 1hev) (Andersen et al., 1993) and Ac-AMP2 (Amaranthus caudatus; dark grey, PDB: 1mmc) (Martins et al., 1996) reveals their structural similarity. The C-terminus of hevein is highlighted in light grey to clarify the extension of hevein relative to Ac-AMP2. The C-termini are labelled. (C) Solution structure of Ac-AMP2. (D) Solution structure of a synthetic Ac-AMP2 analogue (in the same orientation as in C in complex with chitotriose (dark grey)). The synthetic peptide incorporates two non-natural para-ﬂ uorophenylalanine (Pff) residues in positions 18 and 20 (arrows) to stabilize the complex with the carbohydrate through aromatic interactions. The rings of the side chains of residues 18 and 20 are aligned with the plane of the carbohydrate rings (PDB: 1znt). 48 Q. Kaas et al.

the residue on the non-reducing end, binding lectins have been shown to be toxic respectively, a common recognition motif in to insects (Hegedus et al., 2009). carbohydrate binding (Jimenez-Barbero et al., 2006). In general, the broad importance of chitin-binding proteins arises from the 3.1.5 Miscellaneous AMPs from plants ubiquitous importance of their targets. Complex carbohydrates are essential com- In addition to the major classes of plant AMPs ponents of cell surfaces and glycoproteins in noted above, there are several peptides that many organisms, including pathogens and do not fall into large families, but none the humans (Gabius et al., 2004). Lectins with less have important activities or novel high specifi city for certain carbohydrate structures. For example, a series of AMPs that motifs, including N-acetylglucosamine and are expressed as a single precursor peptide its oligomers, are valuable research tools and and processed into their fi nal form occur in have potential as therapeutics (Gabius et al., Impatiens balsamina (rose balsam) (Tailor et al., 2004; André et al., 2009). For example, lectins, 1997). These relatively short peptides, including chitin-binding lectins, have been comprising approximately 20 residues, are investigated for their ability to inhibit human highly homologous and are stabilized by two immunode fi ciency virus (HIV) (Balzarini, disulfi de bonds to form well-defi ned 2006) and cancer cells (Liu et al., 2010). These structures (Patel et al., 1998). As can be seen in studies have mainly focused on proteins, but Fig. 3.5, the Ib-AMPs are expressed as the results may encourage further research precursors that contain six highly homol- into chitin-binding peptides to exploit their ogous repeats. Ib-AMP1 is present in three therapeutic and pharmaceutical advantages copies (1a, 1b and 1c), whereas the other over proteins (da Rocha Pitt a and Galdino, peptides appear only as single copies. The 2010). Chitin-binding ability is linked to plant precursor protein comprises a signal peptide protection against herbivore insects. The and seven propeptide regions of approxi- insect gut is lined with the peritrophic mately 28 residues located before each membrane, which contains chitin, and chitin- mature peptide region.

Fig. 3.5. Organization of Ib-AMP precursor (from Impatiens balsamina) and mature AMPs. (A) The precursor contains six repeats encoding four individual peptides. Ib-AMP1 is present in three identical copies (a–c). The mature peptide regions are preceded by an acidic propeptide region that is proteolytically cleaved during the maturation process. (B) The sequence alignment of the mature Ib-AMPs reveals the high homology and identity of the three copies of Ib-AMP1. The disulﬁ de connectivity is indicated. ‘*’ indicates identical residues and ‘:’ indicates highly similar residues. AMPs in Plants 49

MiAMP1 (Macadamia integrifolia AMP1), 3.2 Cyclotides as its name suggests, was originally isolated from macadamia nuts and has antifungal Cyclotides are a structurally fascinating activity (Marcus et al., 1997). It has three family of plant cyclic proteins. They have a disulfi de bonds and a three-dimensional wide range of activities, including against structure that is unique among plant AMPs plant pests and pathogens. Cyclotides fi rst (Fig. 3.6A) (McManus et al., 1999). The came to notice in the early 1970s (Gran, 1970, structure comprises two classic Greek key 1973a) when a peptide was identifi ed as the motifs (Hutchinson and Thornton, 1993) active component of a traditional medicine made up of two β-sheets consisting of four used in Africa. A decoction of an African strands. Each β-sheet has three strands as herb, Oldenlandia affi nis – the vernacular part of one Greek key motif, whereas the last name of which is ‘kalata kalata’ – is used to strand belongs to the other Greek key motif accelerate contractions during childbirth. The (Fig. 3.6B). The arrangement of the two active ingredient was found to be a 29 amino Greek key motifs forms a global structure acid peptide, which was named kalata B1 referred to as a Greek key β-barrel (Fig. 3.6B), (Gran, 1973a). This peptide att racted further and the name β-barrelin has been proposed interest because of its unusual physical to designate this fold (McManus et al., 1999). properties: it can resist high temperatures for So far, no other AMPs presenting this fold an extended period of time (Gran et al., 2000) have been identifi ed. The overall structure and is impervious to the action of chemical has twofold axis symmetry, and the structural chaotropes and proteases (Colgrave and similarity led to the discovery of mild Craik, 2004). sequence homology between the fragments Kalata B1 features two post-translational of the fi rst and second Greek key motifs (Fig. modifi cations that are at the origin of its 3.6C). stability: a cyclized head-to-tail backbone and compact cystine knot. Together, these are termed the cyclic cystine knot motif (Fig. 3.8) 3.1.6 Non-ribosomal antimicrobial (Saether et al., 1995; Rosengren et al., 2003). peptides This interesting protein architecture is the signature motif of the cyclotide protein Cyclopeptide alkaloids diff er from all of the family (Craik et al., 1999). other peptides mentioned so far in this Cyclotides are a major focus of chapter in that they originate from investigation in our laboratory. Strategies to non-ribosomal synthesis. They contain a isolate cyclotides have been established and number of non-coding amino acids and an have led to a broad estimate of the organic moiety, which facilitates cyclization. population of the family at about 50,000 Evidence of antimicrobial activity is only peptides (Gruber et al., 2008). Cyclotides are available for a few compounds. A review by active against various pests and pathogens Tan and Zhou (2006) gives a comprehensive and have activities benefi cial to human list of cyclopeptide alkaloids and their health (Daly et al., 2009; Henriques and biological activity. The molecular mechanism Craik, 2010), including anti-HIV activity at the heart of their antimicrobial activity is (Gustafson et al., 2004). Cyclotides are also unknown, although these compounds are of interesting in the protein-engineering fi eld, much interest due to their diverse biological as the stability of the cyclotide scaff old can activities (Joullié and Richard, 2004; Tan and be exploited to enhance the bioactivity of Zhou, 2006). A report by Morel et al. (2005) peptide drugs (Henriques and Craik, 2010). highlights some of the structure–activity relationships encountered in this class of peptides. The composition, stereochemical 3.2.1 Diversity of plant cyclotides confi guration and arrangement of the residues have a great infl uence on activity Cyclotides are widespread in the plant (Fig. 3.7). kingdom and are present in high abundance 50 Q. Kaas et al.

Fig. 3.6. Structural features of MiAMP1 (Macadamia integrifolia AMP1; Protein Data Bank access code: 1c01). (A) Three-dimensional structure. The disulfi de bonds are shown in a ball and stick representation with N- and C-termini indicated. The Greek key motifs are in light grey (motif 1) and dark grey (motif 2). The representations are rotated by 180° for clarity. (B) Schematic showing the two ˟-sheets in MiAMP1 and the ˟-strand ‘swap’ between Greek key motif 1 (white fi lling) and motif 2 (grey fi lling). (C) An alignment of Greek key motif 1 (residues 1–38) and motif 2 (residues 39–76) reveals a weak sequence identity, but high similarity in the arrangement of the ˟-strands (arrows). Similarity: ‘*’, identical; ‘:’, highly similar; ‘.’, similar. The symmetrical disulfi de bonds within the motifs are indicated by solid lines on the top and bottom, respectively, and the disulfi de bond tethering the two motifs together is indicated as a dashed line between the sequences. AMPs in Plants 51

Fig. 3.7. Cyclopeptide alkaloids from plants. The arrows indicate the stereochemistry at position 3, which differs in scutianine M (right) compared with condaline A (left) and scutianine E (middle). The stereocentre occurs as a result of a cross-link to the ˟-carbon of phenylalanine or valine, respectively. Condaline A and scutianine M both have an isoleucine residue (grey highlight), whereas scutianine E features the unusual amino acid ˟-phenylserine at this position. The dashed outline highlights N-methyl phenylalanine. The breadth of the activity spectrum and activity decreases from left to right (Morel et al., 2005). in at least two plant families, the Violaceae tides, but some individual cyclotides have (violet) and Rubiaceae (coff ee) families been discovered in multiple plants. For (Gruber et al., 2008). Most plants express instance kalata S, also known as varv A non-overlapping suites of diff erent cyclo- (Göransson et al., 1999), has been isolated from six diff erent Violaceae species (Viola odorata, Viola tricolor, Viola arvensis, Viola baoshanensis, Viola yedoensis and Viola bifl ora) and one Rubiaceae species (O. affi nis). Thirty- four cyclotides have been isolated from the Rubiaceae family and 142 cyclotides from the Violaceae family. Two cyclic proteins, MCoTI-I and MCoTI-II, having the same structural topology but with unrelated sequences to other cyclotides, are found in the Cucurbitaceae family (Momordica cochinchinensis) (Hernandez et al., 2000). Because of their plant origin and cyclic cystine knot motif we classify them as cyclotides, but they are also referred to as cyclic knott ins (Heitz et al., 2001). Fig. 3.8. The three-dimensional structure and CyBase is a database established to sequence of the prototypical cyclotide kalata B1. catalogue the sequences and structures of Cyclotides are characterized by a cyclic cystine naturally occurring and engineered cyclic knot motif, which includes a head-to-tail cyclic proteins, including cyclotides (Wang et al., backbone and knotted arrangement of three 2008a). It currently provides information on cystines. The cystine side chains are represented 158 diff erent sequences of wild-type cyclo- in a ball and stick format in the structure and the tides. Figure 3.9 shows a screenshot of a web disulfi de connectivities are shown by black thick page from this database. lines above the sequence. The half-cystines are An inspection of all the sequences numbered using Roman numerals. Kalata B1 has a currently in CyBase shows that cyclotides small ˟-sheet, represented by arrows on the structure. range from 27 to 37 amino acids in size. They 52 Q. Kaas et al.

Fig. 3.9. Alignment of cyclotide sequences carried out with CyBase. This database incorporates specifi c tools for the study of cyclic proteins, including the alignment of cyclotide sequences, as shown here. The cysteine residues of the cyclotide cystine knot motif are aligned and gaps are inserted into the middle of the inter-cysteine regions. The vertical menu on the left provides access to search pages, lists and tools of cyclic protein and nucleic acid sequences. invariably contain six cysteines forming the its conformation is relatively conserved (Fig. three disulfi de bonds that constitute their 3.10) despite a high level of sequence characteristic cystine knot motif, as shown in variability (Fig. 3.11). Loop 6 accommodates an overlay of structures in Fig. 3.10 (Craik et the longest loop sequence seen so far in al., 1999). The inter-cysteine regions are cyclotides (i.e. ten amino acids). By contrast, referred to as loops and have diff erent ranges loops 1 and 4 are short and conserved in of amino acid lengths and sequence length, with three and one amino acids, variability (Fig. 3.11). The loops with the respectively. Some positions seem to be greatest sequence diversity are loops 3, 5 and crucial for cyclotide folding, including the 6. Loop 3 is particularly interesting because cysteines and a glutamate in the second AMPs in Plants 53

Fig. 3.10. Overlay of cyclotide structures. Cystine side chains are represented in dark grey and the cyclotide backbones are in light grey, except for the backbone of kalata B1, which is in black. The cystine knot motif is highly conserved and the cystine side chain positions align well. The conformations of loops 1, 2, 3 and 4 are highly conserved, whereas loops 5 and 6 are more variable. The structures shown are: circulin A (Protein Data Bank access code: 1bh4), circulin B (2eri), cycloviolacin O1 (1nbj), cycloviolacin O2 (2kcg), cycloviolacin O14 (2gj0), kalata B1 (1nb1), kalata B2 (1pt4), kalata B7 (2jwm), kalata B8 (2b38), palicourein (1r1f), tricyclon A (1yp8), varv peptide F (1k7g), vhl-1 (1za8) and vhr 1 (1vb8). position of loop 1, which forms hydrogen family (Simonsen et al., 2005; Burman et al., bonds with the backbone amides of the ﬁ rst 2010), but their occurrence in Rubiaceae two positions in loop 5 (Rosengren et al., species is more sparse, with <10% of 2003; Göransson et al., 2009). The penultimate examined species of this family expressing position of loop 5 is also important as it is cyclotides (Gruber et al., 2008). The Violaceae oft en occupied by a cis-proline, which confers and Rubiaceae belong to the monophyletic a conceptual twist in loop 5, leading to the groups – rosids and asterids, respectively – analogy of a Möbius strip (Craik et al., 1999). which are both part of the eudicot class. The Depending on the presence or absence of this rosids and asterids are thought to have cis-proline, cyclotides have been divided into diverged 100–150 million years ago (Yang et Möbius and bracelet structural subfamilies al., 1999). To explain the gaps in cyclotide (Craik et al., 1999). The impact of sequence expression in the Rubiaceae and also the variations on cyclotide activities is discussed occurrence of cyclotides in both the Violaceae in Section 3.2.4. and Rubiaceae, it has been hypothesized that Methods that detect cyclotide peptide the current cyclotide distribution results expression in plants (see Section 3.2.2) and from convergent evolution in diﬀ erent plant the isolation of transcripts using molecular families (Gruber et al., 2008). This hypothesis biology techniques have facilitated an is supported by the fact that the enzymes increase in knowledge on cyclotide diversity that appear to be responsible for the (Simonsen et al., 2005; Gruber et al., 2008). cyclization process are ubiquitous in plants Screening has been carried out in many (discussed in Section 3.2.3). In support of the tissues from hundreds of plant species convergent evolution hypothesis, cyclotide- (Simonsen et al., 2005; Gruber et al., 2008) and like sequences have been detected in the it is clear that cyclotides are present in all Poaceae family (grass), which includes parts of plants. Cyclotides appear to be important cereal crop species such as wheat, expressed in all species from the Violaceae maize and rice (Mulvenna et al., 2006b). 54 Q. Kaas et al.

Fig. 3.11. Sequence variability of cyclotide loops. Each loop is identiﬁ ed on the three-dimensional structure at the centre of the ﬁ gure. The number of different sequences is indicated. For loops 2, 3, 5 and 6, a bar graph represents the number of loops with a given length. For a given loop length, the loops having identical sequences are counted independently. Therefore, the total number of sequences represented in the graph is 158, which is the total number of cyclotide sequences in CyBase. Loops 1 and 4 do not vary in length, and their length and number of different sequences are provided as text.

3.2.2 Detection and isolation of cyclotides The isolation of transcripts having similarities with known cyclotides is an A recently developed rapid cyclotide alternative strategy that has also been widely screening strategy (Gruber et al., 2008) has employed (Simonsen et al., 2005; Herrmann facilitated cyclotide discovery. First, diﬀ erent et al., 2008; Trabi et al., 2009; Zhang et al., parts of a target plant are ground and 2009; Burman et al., 2010), even though it is incubated in dichloromethane/methanol. not yet possible to predict if the translated Water is then added to separate the methanol peptide will be post-translationally modiﬁ ed layer, which is fractionated and tested for to include a cyclic cystine knot motif. peptide content, with a focus on peaks Nevertheless, sequencing of cyclotide cDNAs displaying similar hydrophobicity and mass has revealed a conserved organization of the to known cyclotides. Fractions that contain regions of cyclotide precursors, and this peptides in the mass range of 2.5–4.0 kDa are conservation has provided insights into selected, and then reduced and alkylated to cyclotide biosynthesis (Jennings et al., 2001). detect mass shift s corresponding to the reduction of the three cyclotide cystines into six alkylated cysteines. Cyclotides detected 3.2.3 Cyclotide biosynthesis in initial screening can be subsequently characterized using a well-established pro- Cyclotides are gene products and their cDNA tocol (Colgrave and Craik, 2004; Trabi and transcripts reveal a conserved organization Craik, 2004; Colgrave et al., 2005) and of their precursors (Jennings et al., 2001). This sequenced by nanospray tandem mass comprises a signal sequence that directs the spectrometry. precursor to the endoplasmic reticulum, a AMPs in Plants 55

pro-region and one or several repeats, each an aspartate at the position usually occupied comprising an N-terminal repeat, a cyclotide by the conserved asparagine. AEP has domain and a C-terminal region (CTR) (Kaas reduced catalytic activity at an aspartate and Craik, 2010). The structure of the O. residue (Müntz et al., 2002) and, consequently, affi nis kalata B2 precursor is shown in Fig. other enzymes might be involved in cyclotide 3.12. backbone cyclization. Interestingly, during a An asparagine endopeptidase (AEP) has small-scale expressed sequence tag project, a been shown to be involved in the backbone transcript coding for an asparaginase was cyclization of cyclotides (Saska et al., 2007; isolated from O. affi nis (Qin et al., 2010). Gillon et al., 2008). This enzyme cleaves the Asparaginases catalyse the hydrolysis of peptide bond aft er a conserved asparagine asparagine into aspartate. Therefore, the that occupies the last position of the cyclotide unusual aspartate could have been formed domain. It has been postulated that before by post-translational modifi cation of the cleavage occurs, the fi rst positions of the CTR conserved asparagine aft er cyclization (Qin could bind in pockets on the surface of AEP et al., 2010). (Gillon et al., 2008). Aft er cleavage and the A protein disulfi de isomerase (PDI) from release of the CTR, the fi rst positions of the O. affi nis has been shown to interact with a cyclotide domain, which have similar linear kalata B1 precursor protein and sequences to the fi rst positions of the CTR, improve its oxidative folding (Gruber et al., occupy the pockets. The N- and C-termini of 2007b). In the absence of the PDI, a stable the peptide would then be in close proximity intermediate in the folding pathway of kalata and could be ligated by AEP, therefore B1 in vitro does not lead directly to the native cyclizing the protein (Gillon et al., 2008). product and requires disulfi de shuffl ing for Twenty-three of the 158 cyclotide eventual formation of the cystine knot (Daly sequences currently recorded in CyBase have et al., 2003). Thus, PDI activity in vivo might

Fig. 3.12. Oldenlandia affi nis kalata B2 precursor. The precursor comprises an endoplasmic reticulum (ER) signal sequence (dark grey background), a pro-region (white background) and three N-terminal repeats (NTRs, light grey background), three kalata B2 domains (black background) and three C-terminal regions (CTRs, diagonal stripes). An NTR, a domain and a CTR consecutive in the sequence form a repeat unit. At the bottom of the fi gure, the three repeats in the kalata B2 precursor are aligned and the positions presenting sequence variability are on a grey background. Whereas the NTR is highly conserved between repeats, the CTR is more variable. The three fi rst positions of the CTR (underlined in the last repeat) have been identifi ed as important for the enzymatic cyclization process and are homologous to the fi rst three amino acids of the mature domain (also underlined). The last position of the domain is usually an asparagine but sometimes an aspartate, as is the case for kalata B2. This last position and the second position of the CTR are crucial for enzymatic cyclization to occur and are marked with a black background. 56 Q. Kaas et al.

be important to prevent a signifi cant number Helicoverpa are among the most polyphagous of cyclotides being trapped into intermediates and cosmopolitan pests. with non-native disulfi de bonds. Moreover, Cyclotides also have potent activity similarities between hydrophobic-solvent- against Haemonchus contortus and Tricho- assisted folding and folding using O. affi nis strongylus colubriformis, two economically PDI suggest that the chaperone activity of important gastrointestinal nematode para- PDI might also improve the folding of sites of livestock. This shows that the cyclotides (Gruber et al., 2007b). Cyclotides pesticidal properties of cyclotides are not have a hydrophobic patch on their surface specifi c to Helicoverpa (Colgrave et al., 2008). that might interact with a hydrophobic Cyclotides also have molluscicidal activity domain of the PDI. The cyclotide hydro- against Pomacea canaliculata, a serious pest of phobic patch has also been shown to be rice in South-east Asia, with a comparable important for the binding of cyclotides to potency to a commercial pesticide (Plan et al., dodecylphosphocholine micelles, used as 2008). P. canaliculata causes billions of dollars model membranes (Shenkarev et al., 2006, worth of damage on rice plantations each 2008; Wang et al., 2009). year (Sin, 2003) and there may be a role for cyclotides as a novel class of molluscicidal agents. Cyclotides seem to act by a mechanism 3.2.4 Biological activities of cyclotides that aff ects cell membrane integrity. Upon ingestion of kalata B1, disruption of the gut Cyclotides fi rst came to notice for their membrane of caterpillars has been observed uterotonic activity employed in a native by light scanning microscopy and trans- medicine application (Gran, 1970), but they mission electron microscopy (Barbeta et al., have a range of other bioactivities including 2008). The integrity of a hydrophobic patch haemolytic (Daly et al., 1999), cardiotoxic on the surface of the kalata B1 structure was (Gran, 1973b), antitumour (Herrmann et al., found to be important not only for 2008), antifungal (Tam et al., 1999), anthel- insecticidal activity, but also for membrane- mintic (Colgrave et al., 2009), anti-HIV binding affi nity, as revealed by alanine- (Gustafson et al., 1994, 2004; Daly et al., 2006; scanning mutagenesis (Simonsen et al., 2008; Wang et al., 2008b) and, reportedly, anti- Huang et al., 2009). This suggests that the bacterial activities (Tam et al., 1999), as well membrane-binding and insecticidal activities as inhibition of trypsin (Hernandez et al., of cyclotides are correlated. 2000) and neurotensin (Witherup et al., 1994). In this section, we focus on their purported antimicrobial properties. First, however, we Anti-HIV activity give a brief overview of their pesticidal Initially two cyclotides, circulin A and activities, because we believe that this host- circulin B, were discovered in the course of defence activity is the primary biological screening for anti-HIV natural products in a function of cyclotides. drug discovery programme at the US National Cancer Institute (Gustafson et al., 1994). In the anti-HIV assay, plant extracts Pesticidal activity were added to cultured T cells and The natural function of cyclotides appears to subsequently exposed to infectious virus. be to defend their host plants from insect The number of surviving cells was quantifi ed pests (Jennings et al., 2001, 2005; Gruber et al., aft er 6 days of incubation and compared 2007a; Craik, 2009; Daly et al., 2009). Kalata with uninfected cells and untreated cells B1 is a potent inhibitor of the growth and (Gustafson et al., 2004). With this protocol, development of Helicoverpa species (Jennings compounds that inhibit early steps of the et al., 2001), moths whose larvae feed on a HIV infection could be detected. In the wide array of plants, including a range of primary anti-HIV screen, extracts from agricultural plants (maize and cott on). Chassalia parvifolia were found to be active, AMPs in Plants 57

and circulin A and circulin B were identifi ed insights in the antiviral mode of action of as the bioactive compounds (Gustafson et al., cyclotides. 2004). Following this study, several other The natural cyclotides tested so far for native cyclotides were reported to have anti- their anti-HIV activity have a low in vitro HIV activity (Daly et al., 2004, 2006; therapeutic index (i.e. the ratio of their Gustafson et al., 2004; Chen et al., 2005; therapeutic eff ects to toxic eff ects) (Gustafson Ireland et al., 2008; Wang et al., 2008b). et al., 1994), which has limited their pro- The anti-HIV activity of cyclotides is gression to the clinic as candidates for anti- interesting, but the mechanism of action is HIV therapeutics. Nevertheless, other natural still unclear. It has been proposed that cyclotides have been shown to possess higher cyclotides could prevent HIV from inter- therapeutic indices (i.e. 44 for cycloviolacin acting or fusing with host cells (Henriques Y5) (Wang et al., 2008b), thus reviving the and Craik, 2010). For effi cient viral infection, possibility of cyclotide-based anti-HIV drugs. viruses must deliver their genomes into the The possibility of decreasing the toxic eff ects interior of target cells. Before virus entry into of cyclotides by a single mutation (Simonsen the cell, the HIV infection process is initiated et al., 2008; Huang et al., 2009), together with by HIV receptor recognition at the surface of a more complete understanding of the host cells, followed by fusion of the viral and mechanism of action, might accelerate the host cell membranes (Cooley and Lewin, process. 2003). The cytoprotective eff ect of cyclotides has been reported to be associated with a Antibacterial and antifungal activity decreased level of infectious virions (Gustafson et al., 1994), whereas no eff ect on Cyclotides have distinct hydrophobic and HIV reverse transcriptase activity was hydrophilic patches (Fig. 3.13), which detected (Gustafson et al., 2004). Such resemble the amphipathic nature of AMPs. observations support the hypothesis that These properties led Tam et al. (1999) to cyclotides exert their eff ect before the entry of hypothesize that cyclotides might exhibit the virus into the cell, inhibiting membrane antimicrobial activity. Four cyclotides were targeting and/or membrane fusion. tested against selected bacteria and fungi, Cyclotide bioactivities seem to correlate and selective antimicrobial activity was with membrane-binding properties, as reported, as summarized in Table 3.2. supported by biophysical studies with model Although low micromolar minimal inhibitory membranes (Shenkarev et al., 2006, 2008; concentrations (MICs) were reported for Huang et al., 2009; Wang et al., 2009) and by some microbes (e.g. the MIC against cell membrane disruption observed in the Staphylococcus aureus was 0.26 μM in the insecticidal studies referred to above (Barbeta absence of salt), the antimicrobial activity et al., 2008). As a hydrophobic patch has been was ablated in the presence of physiological found to be crucial for the interactions with salt concentrations. Salt-dependent activity is membranes (Huang et al., 2009), it is reason- oft en seen for AMPs, as high salt levels can able to propose that cyclotides modulate aff ect electrostatic interactions with bacterial their protective eff ect by a membrane- membranes (Bals et al., 1998; Wei et al., 2007). binding mechanism inhibiting HIV entry into Currently, is not possible to determine if an the cell (Henriques and Craik, 2010). increase in antimicrobial activity correlates Specifi cally, anti-HIV effi ciency shows a with increased electrostatic att ractions correlation with the extent of a hydrophobic between cyclotides and bacterial membranes, patch on the surface of cyclotides (Ireland et as the eff ect of salt concentration on cyclotide al., 2008). Nevertheless, at this stage it is membrane affi nity has not been reported. premature to hypothesize that cyclotides Nevertheless, a lack of activity at physio- exert their eff ects by interacting with either logical conditions brings into question the the HIV membrane or the host cell antimicrobial value of the cyclotides tested, membrane, or with both. Investigation of probably accounting for the fact that since direct ‘virucidal’ activity would give more the study in 1999, no follow-up has been 58 Q. Kaas et al.

Fig. 3.13. Surface characteristics and charges of wild-type cyclotides with known structure. The surfaces of all known wild-type cyclotides are represented by two views that differ by a rotation of 180° along the vertical axis. Structures located below each other have similar orientations (alignment of the cystine knot). The surfaces are darkened according to the properties of the amino acid under the surface: hydrophobic (white), hydrophilic but not charged (grey) and charged (black). The charge of the amino acids is shown on the surface. The number of positive and negative charges is given in columns 4 and 5, respectively, and the total charge of the cyclotide is provided in the last column. Protein Database numbers are shown between the structures. AMPs in Plants 59

Fig. 3.13. Continued. 60 Q. Kaas et al. were were –1 Kalata B7 99 b Kalata B2 ±± Activity reported et al . Gran by Kalata B1 99 ±± ±± ±± ±± ±± ±± >500 c a C for 3 days. Antibacterial 3 days. incubated at 37 ° C for peptides were solution to agar plates; peptide stock –1 C for 1 day (Gran et al ., 2008). (Gran 1 day uenzae and incubated at 37 ° C for were evaluated by adding peptide directly to the growth medium. Peptide concentrations of 2, 4, 8 or 16 mg l concentrations Peptide medium. adding peptide directly to the growth by evaluated uenzae were Circulin B Cyclopsychotride Kalata B1 . (MIC ( μ M)) et al . Tam Activity reported by Circulin A Salt No salt Salt No salt Salt No salt Salt No salt Salt No salt Salt No salt Salt No salt >500>500 18.6>500 19.4 >500 >500 >500 >500 29.0 >500 >500 48.0 >500 >500 14.0 56.5 >500 >500 >500 >500 >500 21.4 >500 >500 >500>500>500 54.6 >500 48.0 >500 25.5 15.6>500 6.80 50.2 8.20 >500>500 0.19 13.5 >500 >500 13.2 13.2 >500 >500 5.80 >500 13.5 >500 >500 >500 >500 >500 >500 54.8 39.0 48.0 >500 >500 0.26 40.4 >500 >500 >500 0.41 >500 1.55 >500 Antimicrobial activity measured for cyclotides under conditions with (100 mM NaCl) and without salt. Comparison of results obtained in two cyclotides under conditions with (100 mM NaCl) and without salt. Antimicrobial activity measured for uenzae Candida kefyr Candida tropicalis Candida albicans Pseudomonas aeruginosa Proteus vulgaris Klebsiella oxytoca Haemophilus in fl Staphylococcus aureus Micrococcus luteus Escherichia coli was tested after addition of 10 on Escherichiaμ l of 5 mg ml was coli The antibiotic effect incorporated inoculated with either S . aureus or H in fl and plates were l of each concentration was incubated at 37°C for 3 h +16–24 h. The bacteria clear zones were The bacteriawere clear zones 3 h +16–24 h. incubated at 37°C for was 5 μ l of each concentration without salt (no salt) or with 100 mM NaCl (salt); 10 mM phosphate buffer et al. , 1999). determinedmeasured and MICs were from the dose–response curves (Tam and Haemophilus in fl aureus on Staphylococcus effects gel containing 1% agarose and with underlay diffusion assay of peptide in a radial concentrations inhibitory Minimum determined (MICs) were concentrations after testing seven shading highlights contradictoryindependent studies. the two Grey results between Fungi Gram-positive Organism Gram-negative Table 3.2. 3.2. Table adapted from its original reported form Henriques (2010). and Craik independent studies; by a b c AMPs in Plants 61

published apart from one recent report (Gran (Giangaspero et al., 2001). Although et al., 2008). The results obtained by the only cyclotides have distinct hydrophobic and two reports on the antibacterial activities of hydrophilic patches, most are not highly cyclotides are compared in Table 3.2. positively charged; the total charge is close to Kalata B1 is the only cyclotide that is zero and most of the currently known common to both studies and contradictory cyclotides have a charge between –1 and +2 results were obtained. Tam et al. (1999) (see Figs 3.13 and 3.14). In addition, kalata B1 reported that kalata B1 is inactive against does not have a preference for negatively Escherichia coli and active against Staph. charged membranes at physiological ionic aureus, whereas Gran et al. (2008) reported strength (Huang et al., 2009). Overall, cyclo- the opposite. In these two studies, activity tides have a poor profi le as antibacterial was assessed using diff erent experimental peptides. set-ups. Tam et al. incubated the bacterial Some cyclotides have a global charge of strains with diff erent concentrations of kalata +3 (Fig. 3.14), but they have not yet been B1 and determined the MIC in a radial tested for their antimicrobial properties. The diff usion assay, and concluded that kalata B1 limited studies on the antibacterial activity of is inactive against E. coli (MIC >500 μM in the cyclotides make it diffi cult to judge the presence or absence of salt). On the other potential applications of native cyclotides as hand, Gran et al. concluded that kalata B1 antimicrobial drugs; however, many more was active against E. coli based on the eff ect cyclotides wait to be discovered and new of 10 μl of 5 mg ml–1 kalata B1 (approximately examples might reveal diff erent char- 1700 μM) added to the agar plate. The very acteristics, including higher charge and high concentration used in the Gran et al. potentially stronger antimicrobial activities. study might explain the diff erence of activity The cyclotides so far reported have observed for E. coli between the two studies. revealed the remarkable plasticity of the Regarding the results obtained against cyclotide framework. Their tolerance for Staph. aureus, Tam et al. concluded that kalata substitution suggests that cyclotides can be B1 was active against Staph. aureus as the used as a scaff old and engineered to include MIC was found to be 0.26 μM in conditions a foreign sequence with a desired activity without salt, but inactive in conditions with (Craik et al., 2006a). As already mentioned, salt (MIC >500 μM). In the study by Gran et al., a lack of activity against Staph. aureus was concluded based on incorporation of kalata B1 into the bacterial growth medium, whereby 16 mg ml–1 of kalata B1 (approximately 5.5 μM) did not aff ect bacterial growth. The reasons for the diff erences in the two studies are not fully understood. Classic antibacterial peptides selectively target negatively charged bacterial membranes over neutral mammalian cells and kill microbes through a membrane disruption mechanism (Yeaman and Yount, 2003). An amphipathic structure, with well-defi ned and distinct hydrophobic and positively charged patches, has been identi fi ed as a determinant for bacterial membrane targeting by antibacterial peptides (Dathe et al., Fig. 3.14. Total charge of cyclotides based on 158 1997). The most frequent global charge cyclotide sequences in CyBase. Arginines and identifi ed in classical antibacterial peptides is lysines are positively charged, while aspartates +5 or +6, emphasizing the importance of the and glutamates are negatively charged. Histidines net positive charge for antimicrobial activity are taken as neutral. 62 Q. Kaas et al.

the bioactivities of cyclotides seem to broadly and thus optimization is sometimes required correlate with membrane-binding affi nity. to fi nd the most appropriate combination of Whereas the disruption of the hydrophobic graft ed sequences and sites (Gunasekera et patch in kalata B1 leads to loss of function al., 2008). Studies investigating the tolerance and a decrease in membrane-binding proper- of the cyclotide framework to substitution ties (Huang et al., 2009, 2010), the insertion of and graft ing have been recently reviewed positive charges in selected locations (Henriques and Craik, 2010). increases bioactivity and membrane leakage The next step towards the development properties (Huang et al., 2010). These of cyclotides as pharmaceuticals will be to properties suggest that cyclotides can be discover or design examples that have high modulated so that toxic properties can be potencies and selectivities for proven eliminated and antimicrobial activity therapeutic targets. Assuming that such improved. In this sense, cyclotide frame- results are forthcoming in the near future, it works – with their circular structure and will then be necessary to achieve high-yield high resistance to thermal, enzymatic and production for scale-up testing. Unfortu- chemical degradation – are a potential new nately, the plant-based production of class of antibiotics that could be designed to engineered cyclotides is problematic as have both high stability and membrane- transgenic plants have so far only produced disrupting properties. low yields (Saska et al., 2007; Gillon et al., 2008). The most probable explanation for the low yields is that the transgenic plants lack 3.2.5 Cyclotide engineering and mass optimized enzymes required for the correct production processing of cyclotides. At least three alternative cyclotide production strategies Despite being rapidly degraded by proteases have been att empted, including solid-phase in vivo and having generally low stability, synthetic chemistry, production in bacteria peptides have generated much interest in and plant cell culture. drug design because they bind to their Solid-phase peptide synthesis is a molecular targets with high affi nity and classical and straightforward method to specifi city. The cyclotide cyclic cystine knot produce peptides, but it has a high cost that motif confers high resistance to diff erent is incompatible with mass production. A kinds of denaturants and can potentially biomimetic strategy based on chemoenzym- overcome the stability and degradation atic cyclization by an immobilized protease problems of peptides (Craik et al., 2006a, has been developed to complement this 2009; Henriques and Craik, 2010). The fact method (Thongyoo et al., 2007), and has that a range of loop sizes is tolerated by the shown improved yields over the usual native cyclic cystine knot framework suggests that chemical ligation/cyclization reaction. More cyclotides are potentially amenable to recently, Austin et al. (2009) developed an substitution by a range of foreign epitopes in ingenious strategy to biosynthesize a protein engineering and drug design studies cyclotide-based library in E. coli cells. E. coli (Craik et al., 2006b). Indeed, cyclotides may lacks the required enzymes to cyclize be thought of as a natural combinatorial proteins, but an alternative approach based library that is expressed on an ultra-stable on a modifi ed protein-splicing unit, or intein, structural scaff old (Craik et al., 2001). As was put into practice and directly employed explained previously, loops 2, 3, 5 and 6 are inside living E. coli cells (Fig. 3.15). This the most naturally variable and are the method generally produced good yields of preferential sites for graft ing biologically cyclic proteins. active epitopes. Because there is a degree of Plant cell-culture technology is a cooperatively between sequences in the promising strategy because cyclotides can be diff erent loops of the cyclotide scaff old (Daly directly cultivated in cells from cyclotide- et al., 2006; Gunasekera et al., 2009), not all producing plants (Dörnenburg, 2009). This graft ed peptides can be successfully folded should overcome the limitations of cyclotide AMPs in Plants 63

Fig. 3.15. The cyclization strategy used by Austin et al. (2009) to produce cyclotides in Escherichia coli. An intein was used as a protein-splicing unit and the last step of cyclization was performed by an intramolecular native chemical ligation reaction. A cellulose-binding domain (CBD) was fused at the C-terminal end of the intein region. Figure adapted from Austin et al. (2009). MCoTI-I, Momordica cochinchinensis trypsin inhibitor I. production in model plants such as tobacco. laboratory are focused primarily on one class Figure 3.16 illustrates cyclotide production of these molecules, the cyclotides, the using this approach. principal natural function of which is thought to be as insecticidal agents. In addition to this presumed native function, 3.3 Concluding Remarks and however, cyclotides have been reported to Perspectives have a variety of antimicrobial properties. Most extensively studied have been their As is clear from this chapter, plants produce antiviral properties, with a large number of a variety of structurally diverse peptides that cyclotides showing signiﬁ cant activity they use for defence purposes against against HIV. Although they have promising microbes and other pests. Studies in our potencies, their therapeutic index is not at 64 Q. Kaas et al.

Fig. 3.16. Oldenlandia affi nis cell culture growth and production kinetics of the batch cultivation process. Kalata B1 accumulation is maximized after 7 days, corresponding to the beginning of the stationary phase. DW, dry weight. Figure adapted from Dörnenburg (2009). present suffi ciently high to justify their One of the major unsolved problems in clinical use as antiviral agents in humans. cyclotide research is the mechanistic basis of Nevertheless, with a large number of their action. They have an impressive array cyclotides predicted to be discovered over of biological activities and it would be the coming years, it is possible that natural simplistic to imagine that all of these examples with bett er therapeutic indices may activities can be accounted for by a single be found. Furthermore, with several routes mechanism. However, if there is a common to their synthesis available, the design of theme, then membrane binding appears to synthetic analogues with improved potency be a central player. Although it is equivocally and reduced toxicity is a promising area for established that cyclotides bind to future investigation. membranes and some aspects of their Cyclotides have been widely reported in binding have been delineated, the way in the review literature to have antibacterial which they form pores in membranes activity. However, as we have indicated here, remains a puzzle. Another mystery is how a careful examination of the literature shows plants themselves are unaff ected by the that only two original research papers have membrane-disrupting properties of cyclo- reported this activity and to an extent they tides. Cyclotides are probably stored inside present confl icting results. Thus, there is a membrane-containing organelles within question about the signifi cance and relevance plants, yet they do not harm the producing of the reported antibacterial activity of plants. Thus, the systematic study of the cyclotides. This is an area that needs further interactions of cyclotides with a range of investigation and, in particular, pathogens diff erent plant membranes is an important relevant to plants should be examined. The area of investigation. studies so far have focused on human Finally, it will be of interest to determine pathogenic bacteria, rather than on bacteria why individual plants produce so many that might be typically encountered by cyclotides – in some cases, up to 100 plants. cyclotides per plant. The variation from one AMPs in Plants 65

cyclotide to another within a producing plant (UniProt). Nucleic Acids Research 33, is oft en not particularly large and raises the D154-D159. question of why a plant would devote a Balls, A.K., Hale, W.S. and Harris, T.H. (1942a) A signifi cant amount of its energy resources to crystalline protein obtained from a lipoprotein of wheat fl our. Cereal Chemistry 19, 279–288. producing a suite of similar peptides. Per- Balls, A.K., Hale, W.S. and Harris, T.H. (1942b) haps some of the questions raised here are Further observations on a crystalline wheat related, and the pore-forming ability of protein. Cereal Chemistry 19, 840–844. cyclotides might involve a mix-and-match Bals, R., Wang, X., Zasloff, M. and Wilson, J.M. interaction between diff erent cyclotides. At (1998) The peptide antibiotic LL-37/hCAP-18 is this stage this is speculation, but it is an expressed in epithelia of the human lung where interesting area for future studies. The it has broad antimicrobial activity at the airway studies of membrane binding of cyclotides surface, Proceedings of the National Academy reported so far show that purifi ed individual of Sciences of the USA 95, 9541–9546. cyclotides can certainly disrupt membranes, Balzarini, J. (2006) Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral but we do not yet know how mixtures of Research 71, 237–247. cyclotides, as occurs naturally in plants, Barbeta, B.L., Marshall, A.T., Gillon, A.D., Craik, might aff ect membranes. D.J. and Anderson, M.A. (2008) Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proceedings of the National Academy of Sciences of the USA 105, 1221– References 1225. Bohlmann, H. and Apel, K. (1991) Thionins. Annual Aerts, A.M., Francois, I.E., Meert, E.M., Li, Q.T., Review of Plant Physiology and Plant Molecular Cammue, B.P. and Thevissen, K. (2007) The Biology 42, 227–240. antifungal activity of RsAFP2, a plant defensin Broekaert, W.F., Marien, W., Terras, F.R., De Bolle, from Raphanus sativus, involves the induction M.F., Proost, P., Van Damme, J., Dillen, L., of reactive oxygen species in Candida albicans. Claeys, M., Rees, S.B., Vanderleyden, J. and Journal of Molecular Microbiology and Cammue, B.P. (1992) Antimicrobial peptides Biotechnology 13, 243–247. from Amaranthus caudatus seeds with Almeida, M.S., Cabral, K.M., Kurtenbach, E., sequence homology to the cysteine/glycine-rich Almeida, F.C. and Valente, A.P. (2002) Solution domain of chitin-binding proteins. Biochemistry structure of Pisum sativum defensin 1 by high 31, 4308–4314. resolution NMR: plant defensins, identical Broekaert, W.F., Cammue, B.P.A., De Bolle, M.F.C., backbone with different mechanisms of action. Thevissen, K., De Samblanx, G.W. and Osborn, Journal of Molecular Biology 315, 749–757. R.W. (1997) Antimicrobial peptides from plants. Andersen, N.H., Cao, B., Rodriguez-Romero, A. Critical Reviews in Plant Sciences 16, 297– and Arreguin, B. (1993) Hevein: NMR assign- 323. ment and assessment of solution-state folding Burman, R., Gruber, C.W., Rizzardi, K., Herrmann, for the agglutinin-toxin motif. Biochemistry 32, A., Craik, D.J., Gupta, M.P. and Göransson, U. 1407–1422. (2010) Cyclotide proteins and precursors from André, S., Kozár, T., Kojima, S., Unverzagt, C. and the genus Gloeospermum: fi lling a blank spot in Gabius, H.-J. (2009) From structural to functional the cyclotide map of Violaceae. Phytochemistry glycomics: core substitutions as molecular 71, 13–20. switches for shape and lectin affi nity of Carmona, M.J., Molina, A., Fernandez, J.A., Lopez- N-glycans. Biological Chemistry 390, 557–565. Fando, J.J. and Garcia-Olmedo, F. (1993) Austin, J., Wang, W., Puttamadappa, S., Shekhtman, Expression of the alpha-thionin gene from A. and Camarero, J.A. (2009) Biosynthesis and barley in tobacco confers enhanced resistance biological screening of a genetically encoded to bacterial pathogens. The Plant Journal 3, library based on the cyclotide MCoTI-I. 457–462. Chembiochem 10, 2663–2670. Carvalho, A.D. and Gomes, V.M. (2009) Plant Bairoch, A., Apweiler, R., Wu, C.H., Barker, W.C., defensins – prospects for the biological functions Boeckmann, B., Ferro, S., Gasteiger, E., Huang, and biotechnological properties. Peptides 30, H., Lopez, R., Magrane, M., Martin, M.J., Natale, 1007–1020. D.A., O’Donovan, C., Redaschi, N. and Yeh, L.S. Chan, Y.L., Prasad, V., Sanjaya, Chen, K.H., Liu, (2005) The Universal Protein Resource P.C., Chan, M.T. and Cheng, C.P. (2005) 66 Q. Kaas et al.

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Guangshun Wang

Abstract

The isolation and characterization of potent antimicrobial peptides from natural sources has opened a new avenue to the development of future antimicrobials. To further expand the peptide space, new antimicrobial peptides can be predicted or designed based on natural peptide templates. By using the knowledge derived from mature peptides, propeptides, or both (i.e. precursor proteins), a variety of peptide prediction tools have been developed. Furthermore, bacteriocins are predicted under the genomic context by considering not only the peptide itself, but also the accompanying genes for its expression, processing and export. This chapter also discusses database-aided peptide design based on database screening, sequence shuﬄ ing, grammar-based design and de novo design. Finally, major strategies for improving peptide selectivity and stability are highlighted.

The majority of the known antimicrobial of prediction programs by other laboratories peptides (AMPs) were isolated from natural (Nagarajan et al., 2006; Lata et al., 2007, 2010). sources by using classic chromatographic The output of these programs is ‘yes’ methods. Recently, genomic and proteomic (antibacterial) or ‘no’ (not antibacterial). For methods have also been applied to peptide a more quantitative structure–activity rela- identiﬁ cation in a more eﬃ cient manner tionship (SAR) evaluation, a clean activity (Conlon et al., 2004; Li et al., 2007). As the dataset for AMPs is required. By ‘clean’, we isolation and characterization of new AMPs mean that the antimicrobial activity data is time-consuming and labour-intensive, should be obtained under the same other strategies have been used to expand conditions (e.g. the same laboratory, bacterial the peptide repository. Based on common assay method, strain, plate-reading method, features in the AMP expression and pro- data processing). Some laboratories have cessing machinery, bioinformatic approaches compiled or generated such activity datasets have predicted additional AMPs not yet for a large number of AMPs against the same isolated from natural sources (Schutt e et al., target bacterial strain. These datasets have 2002; De Jong et al., 2006; Fjell et al., 2007; then been utilized to train the programs to Torrent et al., 2009). This chapter summarizes predict the activity of new candidate database-aided peptide prediction and sequences (Cherkasov et al., 2009; Juretić et design. Because the Antimicrobial Peptide al., 2009). The establishment of the method Database (APD) (Wang and Wang, 2004) for rapid synthesis of small peptides in contains a well-registered set of mature laboratories laid a solid foundation for peptides, it has motivated the development designing new peptides and testing their

‡ Chapter editor: Amram Mor.

effi cacies (Merrifi eld et al., 1995). The earlier work contained the germ for modern large- scale peptide screening and design. The APD also fostered research eff orts in peptide design (Loose et al., 2006; Duval et al., 2009; Wang et al., 2009, 2010). In this chapter, we describe these new developments ranging from peptide prediction to design. These methods expand the peptide sequence space of natural AMPs. Subsequent chapters further expand the peptide space by describing combinatorial peptide libraries Fig. 4.1. Information used in antimicrobial peptide (Chapter 5) and novel chemical mimics of prediction. (A) Methods in category A involve only natural AMPs (Chapter 6). mature peptides. (B) Methods in category B utilize the high similarity of propeptide sequences. (C) Methods in category C use information from both propeptides and mature peptides. (D) Methods in 4.1 Database-aided Antimicrobial category D take advantage of the sequence Peptide Prediction similarity of processing enzymes of lantibiotics. (E) BAGEL provides a more sophisticated approach The prediction of AMPs is a non-trivial by including all useful information in predicting problem due to diversity in sequence, potential AMPs in the bacterial genome. structure and function. The establishment of databases (Table 1.1, Chapter 1), however, has spurred research activities in this direction. charge, hydrophobic residues content and For example, the well-registered AMPs in the amino acid composition (Fig. 4.1A). If the APD (Wang et al., 2009) have been utilized as calculated properties of an input sequence a known positive dataset to train and test a match with those of any known AMPs in the variety of prediction algorithms and database, the program will inform the user approaches. The similarity between the that ‘Your input sequence has been found in trained peptides and the unknown constitutes our database’. Next, a prediction is made the basis for prediction. The AMP prediction based on the amino acid composition that methods fall into fi ve categories depending on determines the possible class of the peptide the amount of information used in the candidate (β-sheet, residue-rich or α-helix). computer programs. The fi rst method (Fig. The amino acid percentages of AMPs from 4.1A) involves only the amino acid sequence bacteria, plants and animals are listed in information of mature peptides. The second Table 4.1. If the properties of the peptide are method (Fig. 4.1B) utilizes the amino acid out of the defi ned ranges in the database, it sequences of propeptides and mature AMPs. will be judged as being less likely to be The third method (Fig. 4.1C) uses both antimicrobial. In addition, the prediction propeptides and mature peptides. For interface can also perform sequence bacteriocins, the method involves either using alignment to identify AMPs with similar processing enzymes (Fig. 4.1D) or using sequences (Wang and Wang, 2004). This context information related to the AMP interface will be upgraded in the next expression and pro cessing (Fig. 4.1E). release.

The Fourier transformation method 4.1.1 Prediction based on mature peptides Based on the mature peptides in the APD (Wang and Wang, 2004), Nagarajan et al. Knowledge-based prediction (2006) derived an indexing method for peptide properties such as hydrophobicity, The prediction protocol in the APD is based charge, polarity, cysteine content and amino on simple calculations of peptide length, acid distribution. Fourier transformation 74 G. Wang

Table 4.1. Amino acid properties useful for AMP prediction and design. o Residue ΔG tf AAa AAb mass BacP (%)c PlaP (%)d AniP (%)e (kcal mol–1)f PVg AASIh I Ile 113.16 6.25 4.29 6.72 2.45 0.198i 1.97 V Val 99.13 6.52 4.7 5.97 1.66 0.200 2.37 L Leu 113.16 5.8 3.31 10.43 2.31 0.246 1.74 F Phe 147.18 2.66 2.82 4.5 2.43 0.246 1.53 C Cys 103.14 5.00 17.96 5.28 2.09 0.165 1.73 M Met 131.20 1.61 0.51 1.18 1.67 0.265 2.50 A Ala 71.08 11.35 3.9 8.86 0.42 0.307 1.89 W Trp 186.21 3.64 1.61 1.27 3.06 0.172 2.00 Y Tyr 163.18 3.49 3.64 1.99 1.31 0.185 2.01 G Gly 57.05 15.04 11.29 11.89 0.0 0.265 2.67 P Pro 97.12 2.92 5.81 4.94 0.98 0.327 0.22 T Thr 101.11 5.57 7.43 3.39 0.35 0.242 2.18 S Ser 87.08 7.18 7.64 5.15 –0.05 0.281 2.14 Q Gln 128.13 2.13 1.91 2.29 –0.30 0.248 3.05 N Asn 114.10 5.88 5.18 3.13 –0.82 0.240 2.33 E Glu 129.12 1.86 3.39 2.12 –0.87 0.449 3.14 D Asp 115.09 2.03 2.34 2.23 –1.05 0.479 3.13 H His 137.14 1.99 1.21 1.97 0.18 0.202 3.00 K Lys 128.17 6.77 6.15 10.86 –1.35 0.111 2.28 R Arg 156.19 2.05 5.27 5.84 –1.37 0.106 1.91 a Amino acid in the single-letter code. b Amino acid in the three-letter code. c,d,e Amino acid percentage of AMPs from bacteria (BacP), plants (PlaP) and animals (AniP), respectively (Wang et al., 2009). Slight variations in these values are anticipated with increases in the number of AMPs in the database (Wang et al., 2009). f Hydrophobic parameter, as represented by the transfer of free energy of an amino acid side chain from the octanol phase to water (Fauchere and Pliska, 1983). An application of this was illustrated in Wang et al. (2005). g Bactericidal propensity value (PV) (Torrent et al., 2009). h Amino acid selectivity index (AASI) (Juretic´ et al., 2009). i Some outstanding values are in bold. analysis revealed a distinct peak in the power similar to the human brain. The artifi cial spectrum of peptide sequences. This fi nding neural network (ANN) is a powerful forms the basis for identifying potential machine-learning method that is insensitive AMPs by scanning the protein sequences to noise and correlated inputs. The ANN can derived from the genomic data. As a test, this be trained using carefully registered known Fourier transformation fi lter found three AMP data and perform predictions based on positive hits from 10,000 randomly generated the extracted rules. The support vector sequences of 16 residues each. The machine (SVM) approach includes a set of antimicrobial activities of those peptides related supervised learning methods used have not yet been evaluated experimentally. for classifi cation and regression. In conjugation with peptide terminal sequence analysis, the authors reported an accuracy of Machine-learning methods 88% for ANN and 92% for SVM. A recent Also based on the positive AMP dataset of update of the APD (Wang et al., 2009) led to the APD (Wang and Wang, 2004), Lata et al. the improvement of this prediction program. (2007) applied machine-learning algorithms By including new peptide entries and source to AMP classifi cation and prediction. Neural information annotated in the APD, the networks handle information in a way overall prediction accuracy reached 98.95% Prediction and Design of Novel AMPs 75

(Lata et al., 2010). We made some test runs of discovery. While the amino acid sequences the program (www.imtech.res.in/raghava/ for the mature AMP region are highly antibp2/submit.html) using the newly variable, the peptide sequences prior to the registered AMPs, which are not likely to be mature peptide portion (propeptide) are part of the training set. When ‘SVM’ and ‘full remarkably similar. This observation laid sequence composition’ were selected, the the basis for identifying novel cathelicidins program gave no prediction for AMPs with (Fig. 4.1B). In analogy to swine PR-39, a <15 residues. Among 17 AMPs with >15 human version sequence was initially pre- residues, 12 were correctly predicted as dicted as FALL-39 (Agerberth et al., 1995). antibacterial (i.e. 71% accuracy). Subse quently, Sørensen et al. (2001, 2003) isolated LL-37 from human neutrophils and ALL-38 from the reproductive system. In AMP sequence motifs this case, the experimentally elucidated Based on a manually compiled list of protease cleavage sites on the precursor disulfi de-containing AMPs such as defensins protein diff ered slightly from those from plants and animals, Yount and Yeaman predicted. This example illustrates that the (2004) identifi ed a well-conserved ‘GXC’ precursor sequence of each AMP is also sequence motif. This motif is common in useful for AMP prediction. Indeed, the defensins, ranging from plants to bacteria, precursors of ranid AMPs also possess a fungi and animals. In the three-dimensional common and highly conserved pro-region, structures of peptides, this sequence motif usually acidic and terminated at a typical forms a ‘γ-core’. Using this signature model, processing signal lysine/arginine. This fi nd- the authors were able to identify previously ing has been utilized as a basis for unidentifi ed AMPs such as brazzein and identifying AMPs on a large scale in charybdotoxin that exerted direct antimicro- amphibians (Li et al., 2007). bial activity against bacteria and Candida albicans. This motif does not apply to the cysteine-bridged Rana box at the C-termini of some amphibian AMPs (Conlon et al., 4.1.3 Prediction based on both 2004). propeptides and mature peptides Structural motif-based prediction has also been performed at the gene level. Based In the AMPer prediction model, Fjell et al. on the conserved six-cysteine β-defensin (2007) considered both propeptide sequences structural motif, Schutt e et al. (2002) were and mature peptides (Fig. 4.1C). Data from able to identify 28 new human and 43 new the Antimicrobial Sequences Database mouse β-defensin genes in fi ve syntenic (AMSDb) (Tossi and Sandri, 2002) were chromosomal regions by combining the validated and expanded as a training dataset. HMMER and BLAST analysis tools. HMMER The basic idea was to classify the peptides in (htt p://hmmer.janelia.org/) is a bioinformatics the database into multiple groups (or tool based on the hidden Markov models. clusters) and then calculate the property These newly discovered genes were missed profi les as a basis for subsequent prediction. in previous annotations of human and mouse Fjell et al. (2007) identifi ed 146 clusters for genomes. Future studies will elucidate when, mature peptides and 40 clusters for pro- where and why particular defensins are peptides (186 clusters in total). The prediction expressed or silenced. was accomplished by matching the profi les of the unknowns with those of the known 186 clusters. The mutual overlap between the 4.1.2 Prediction based on highly matched pairs should be >90%. In this conserved propeptide sequences manner, they identifi ed an additional 229 mature peptides from the 230,133 peptides The sequence alignment of cathelicidin collected in the Swiss-Prot database. Most of precursor proteins has led to an interesting these peptides could be associated with 76 G. Wang

known activity data in the literature. This 4.2 Database-aided Peptide Design tool achieved a high prediction accuracy of and Improvement 99%. 4.2.1 Database screening for HIV- inhibitory antimicrobial peptides 4.1.4 Prediction based on processing Natural AMPs show inhibitory activities enzymes against human immunodefi ciency virus (HIV). Examples are insect melitt in and It has been illustrated that sequence cecropin, amphibian AMPs, mammalian similarities among polypeptide modifi cation cathelicidins, defensins and plant cyclotides enzymes such as LanM are useful in (Wachinger et al., 1998; VanCompernolle et identifying novel lantibiotics. Because this al., 2005; Jenssen et al., 2006; Lehrer, 2007; approach does not utilize polypeptide Ireland et al., 2008). Because eff ective HIV information of either AMPs or their pre- vaccines are not yet available, there is a need cursors, it can be regarded as a fourth to develop topical microbicides that prevent method (Fig. 4.1D). Both haloduracin and the sexual transmission of HIV (Turpin, 2002; lichenicidin were identifi ed in the genomes Buckheit et al., 2010). Currently, <5% of the using this approach (McClerren et al., 2006; AMPs in the APD are known to be HIV Begley et al., 2009). inhibitory. We have hypothesized that a large number of AMPs in our database (Wang and Wang, 2004), with a variety of sequences, 4.1.5 Genomic context-based bacteriocin contain useful templates for developing prediction novel agents that may prevent HIV transmission. To test this hypothesis, we recently A possible reason for missing AMPs during conducted a database-based peptide screen gene annotations is the small size of their against HIV type 1 (HIV-1). In total, 30 open reading frames (ORFs). De Jong et al. natural peptides were selected by considering (2006) developed BAGEL (htt p://bioinform peptide length, charge, cysteine content, atics.biol.rug.nl/websoft ware/bagel) to detect toxicity to mammalian cells and uniqueness AMPs in a bacterial genome. This program of the candidate sequences (Wang et al., incorporates a few published ORF prediction 2010). The well-accepted and highly tools, including Glimmer/RBSfi nder, Zcurve standardized cytopathic eff ects inhibition and GeneMark, for the detection of small assay in CEM-SS cells was utilized to ORFs for AMPs. In addition, the program evaluate the effi cacy and toxicity of candidate also considers the existence of gene clusters peptides. The therapeutic index (TI) is that encode proteins for processing, modi- defi ned as the ratio of TC50 to EC50, where fi cation, transport, regulation and/or EC50 is the concentration required for 50% immunity of bacteriocins. Finally, the inhibition of viral replication and TC50 is the program compares the candidate with those concentration required for a 50% reduction in the knowledge-based bacteriocin database in cell viability. We found that 11 peptides generated by the authors. Because this displayed an EC50 of <10 μM. These include approach is of an integrated nature and an uperin 7.1 mutant, temporin-PTa, clavanin diff ers from all the other methods discussed B, ponericin L2, spinigerin, piscidin 3, above, we shall name it as ‘genomic context- brevinin-2-related, maculatin 1.3, ascaphin-8, based AMP prediction’ (Fig. 4.1E). Using melectin and temporin-LTc. Of these peptides, BAGEL, the authors found one additional frog temporin-PTa, temporin-LTc, insect possible bacteriocin from the genomic ponericin L2 and spinigerin had a TI of >10. sequences of both Streptococcus pneumoniae For longer peptides such as human TIGR4 and R6. This online tool has also been cathelicidin LL-37 and bovine BMAP-27, we used to identify putative bacteriocin genes also identifi ed the major HIV-inhibitory (Majchrzykiewicz et al., 2010). regions (Wang et al., 2008). In the case of Prediction and Design of Novel AMPs 77

LL-37, FK-13 was identifi ed as the smallest that the hybrid peptide became less HIV-inhibitory region; KR-12, which diff ers antimicrobial aft er sequence reversal. In from FK-13 by just one N-terminal phenyl- another case, sequence reversal did not cause alanine (Table 4.2), turned out to be inactive. a selective loss in haemolytic activity Among the series of peptides tested, the (Subbalakshmi et al., 2001). We found that the LL-37-derived GI-20 displayed one of LL-37-derived core AMP (FK-13) was active the highest TIs. Likewise, BMAP-18, the against both Escherichia coli and HIV. Aft er N-terminal 18 residues of BMAP-27, was sequence reversal, retro-FK13 retained its found to have a TI of >20 (Table 4.2). We are bactericidal activity against E. coli (Li et al., now developing anti-HIV microbicides based 2006b), but lost its HIV-inhibitory activity on these peptide templates. (Table 4.2). These examples indicate that the We found excellent overlap between the sequence order of AMPs determines the minimal antibacterial region (Li et al., 2006a) activity spectrum. Sequence reversal, however, and the smallest anti-HIV region of human is only a special case of sequence shuffl ing or LL-37 identifi ed experimentally (Wang et al., rearrangement (Fig. 4.2B), since the latt er can 2008). Torrent et al. (2009) developed a produce numerous new sequences (i.e. a theoretical method to spot active regions in miniature combinatorial library). antimicrobial proteins. A bactericidal pro- To evaluate the eff ect of sequence pensity index value (PV) was calculated for shuffl ing on anti-HIV-1 activity, we created each amino acid (Table 4.1). The PVs for some new peptides based on an aurein 1.2 residues arginine, lysine, cysteine, trypto- analogue, where phenylalanine 13 of aurein phan, tyrosine and isoleucine are ≤0.2 and 1.2 was mutated to tryptophan 13 (sequence: are favoured in AMPs. A potentially active GLFDIIKKIAESW). The peptides were region should have a low PV value on generated by rearranging the 13 amino acid average. This method achieved a prediction residues of peptide 1 and by modelling accuracy of 85%. known helical AMPs. These peptides were subjected to HIV-1 inhibition evaluation as described above. Among the eight peptides 4.2.2 Sequence shuffl ing is a primitive synthesized, two displayed reduced TIs, combinatorial approach three showed litt le variation and two showed improved TIs. Hence, sequence shuffl ing Sequence reversal infl uences the activity of provides an approach for generating bett er AMPs (Fig. 4.2B). Merrifi eld et al. (1995) found HIV-inhibitory peptides (Wang et al., 2010).

Table 4.2. Select HIV-1-inhibitory peptides identiﬁ ed from the APD. Adapted from Wang et al. (2008, 2010). a a a Name Peptide sequence EC50 (μM) TC50 (μM) TI AZTb – 0.009 >0.5 >55.6

KR-12 KRIVQRIKDFLR-NH2 >63.5 >63.5 –

FK-13 FKRIVQRIKDFLR-NH2 3.4 10.4 3.1

Retro-FK13 RLFDKIRQVIRKF-NH2 >58.1 33.7 <0.58

GF-17 GFKRIVQRIKDFLRNLV-NH2 0.98 8.9 9.1

GI-20 GIKEFKRIVQRIKDFLRNLV-NH2 1.08 22.7 21 BMAP-18 GRFKRFRKKFKKLFKKIS 0.35 8.45 24.1 GLK-19 GLKKLLGKLLKKLGKLLLK >47.5 25.1 <0.53 GLR-19 GLRRLLGRLLRRLGRLLLR 4.4 25.7 5.8 DRS S9 GLRSKIWLWVLLMIWQESNKFKKM 31.6 >32.9 >1.04 DRS S9r3 GLRSRIWLWVLLMIWQESNRFKRM 1.25 >32.1 >25.7 a EC50, the concentration required for 50% inhibition of viral replication; TC50, the concentration required for a 50% reduction in cell viability; TI, therapeutic index. b Azidothymidine (AZT; also known as zidovudine) is a Food and Drug Association-approved anti-HIV drug that is used to prevent HIV transmission from infected pregnant women to their children. 78 G. Wang

were tryptophan-rich (discussed in Chapter 9), whereas poorly active candidates contained no or only one tryptophan. This approach nicely illustrates the feasibility of discovering novel AMPs from a computer- generated virtual library.

4.2.3 The hybrid approach and grammar- based peptide design

A classic approach for generating new templates is the hybrid method. In this approach, a new AMP is obtained by combining the parts of amino acid sequences from two diff erent AMPs (Fig. 4.2C). Fig. 4.2. Methods for antimicrobial peptide design Merrifi eld et al. (1995) synthesized various based on databases such as the APD. These peptide hybrids based on a cecropin and a include (A) database screening, (B) peptide melitt in to help elucidate the SARs of AMPs. sequence shuffl ing, (C) grammar-based or modular design and (D) de novo design from Their studies uncovered the modular nature amino acids to motifs and from motifs to peptides. of AMPs. A large-scale hybrid version is described below. Loose et al. (2006) derived a linguistic A large-scale operation of sequence model based on the original 525 natural shuffl ing is to construct a combinatorial AMPs collected in the APD (Wang and library, where the amino acid residues at all Wang, 2004). In the linguistic model, natural or selected positions can be varied and peptide sequences are treated as sentences optimized (Chapter 5). Monroc et al. (2006b) and the amino acids are regarded as words. demonstrated that the TIs of cyclic peptides From the 525 AMPs, the authors identifi ed against plant pathogenic bacteria were 684 regular grammars with the aid of the improved using a combinatorial library Teiresias patt ern-discovery tool. These approach. Recently, advances have also been ‘grammars’ are in essence the simple rules made in computer-aided screening and that defi ne the AMP ‘language’. Each identifi cation of short-peptide antibiotics ‘grammar’ consists of a string of ten amino (Cherkasov et al., 2009). This was made acids. The authors generated a library of feasible as a consequence of technical synthetic peptides with 20 amino acids each innovations in large-scale peptide synthesis by combining two grammars (or building using the SpotArrays, as well as activity blocks) in each case. To fi nd new candidates, evaluation based on luminescence assays. It the authors selected a subset of peptides that was necessary to build biased peptide are dissimilar to natural templates. Using libraries that were rich in residues trypto- this approach they identifi ed D28 and D51, phan, arginine and lysine and did not contain which showed antibacterial activity against glutamic acid, aspartic acid, cysteine and both Gram-positive and Gram-negative proline, because active candidates were bacteria. Although it is diffi cult to predict the absent in 200 randomly synthesized peptides. success rate of this approach, the ‘grammar’ The ANN approach and quantitative SAR approach is likely to generate AMPs that are with 44 descriptors were combined and not found in nature. trained using 1400 peptides with measured activities against Pseudomonas aeruginosa. Remarkably, the protocol successfully 4.2.4 De novo peptide design predicted 94% of the most active candidates from the 100,000 peptides in silico. It is In the reductionist method, few amino acids interesting to note that highly active peptides are utilized in peptide design: two for helical Prediction and Design of Novel AMPs 79

peptides and fi ve for β-sheet proteins (Villain peptides in the APD contain the LGK et al., 2000). A prototype helix design only sequence. When highly used motifs are involved lysine and leucine, which represent chosen, the likelihood of the peptide being positively charged and hydrophobic com- antimicrobial increases. By following the ponents, respectively. Such peptides are amphipathic patt ern, the number of peptides referred to as LK peptides. Among a series of can be further reduced. We found that peptides constructed, 14- or 15-residue GLK-19, a 19-residue peptide consisting of peptides in the amphipathic helix patt ern only glycine, leucine and lysine (Table 4.2), were found to be most active against bacteria was more active against E. coli K12 than (Blondelle and Houghten, 1992). Shorter human LL-37 (Wang et al., 2009). The peptides are inactive and longer peptides inclusion of a low glycine content may tend to be haemolytic. Wang et al. (2009) improve peptide selectivity, as peptides found that a 12-residue LK peptide was consisting only of leucine and lysine are inactive. Kang et al. (2008) were able to obtain known to be cytotoxic to human cells a highly active LK-peptide with merely 11 (Braunstein et al., 2004). Recently, Juretić et al. residues only aft er including a tryptophan (2009) arrived at an amino acid selectivity residue, an excellent membrane anchor index based on TIs and amino acid (discussed in Chapter 9). Monroc et al. occurrences in peptides (Table 4.1). (2006a) revealed that the linear form of de Adepantin-1 showed the highest TI. The seven novo-designed peptides of 4–10 residues glycine residues in adepantin-1 (sequence displayed no antimicrobial activity. However, GIGKHVGKALKGLKGLLKGLGES) could be cyclization enhanced the hydrophobicity of important for peptide selectivity. Interestingly, the peptides and rendered them antibacterial. this peptide is also rich in glycine, leucine and These results agree with the fact that the lysine residues (17 out of 23). shortest helical peptides collected in the APD In both the ‘grammar’-based (Fig. 4.2C) are of 10–12 residues. and frequently occurring amino acid-based Wang et al. (2009) found that glycine, (Fig. 4.2D) approaches, amphipathic segrega- leucine and lysine are the three most tion is along the peptide backbone (Fig. frequently occurring residues of AMPs from 4.3A). Based on the APD, Duval et al. (2009) animals (AniP in Table 4.1), including frogs. designed a two-segment amphipathic struc- This sheds light on the biological signifi cance ture (Fig. 4.3B). The peptide was found to be of the earlier choice of leucine and lysine in active against both Gram-positive and Gram- peptide design, above. Using these three negative bacteria, indicating the feasibility of residues, we designed a GLK peptide in three an alternative peptide design as previously steps (Fig. 4.2D). In brief, these three residues demonstrated by Glukhov et al. (2008). It is of can form diff erent sequence motifs such as outstanding interest to note that dermaseptin GLK and LGK, which can in turn be S9 (DRS S9 in Table 4.2), a natural AMP from com bined into multiple peptides. The number the South American hylid frog (Lequin et al., of AMPs containing a specifi c sequence motif 2006), possesses the amphipathic structure is searchable in the APD. For example, 64 depicted in Fig. 4.3B.

(A) (B)

Fig. 4.3. Amphipathic structures can be designed in (A) the classic way or (B) the two-segment mode, where + and L represent hydrophilic and hydrophobic moieties of the design, respectively. The amphipathic nature would be blurred if an end-on view is displayed for model B. 80 G. Wang

4.2.5 Database-aided enhancement of must be minimized. As noted above, AMPs peptide anti-HIV activity with toxic eff ects on mammalian cells possess the highest hydrophobic content among the Figure 4.4A shows the contents of fi ve groups fi ve AMP groups (Fig. 4.4A). This database of amino acid residues in antibacterial, fi nding concurs with the conclusion from antifungal, antiviral and anticancer peptides, SAR studies of AMPs (Tossi et al., 2000; Chen and AMPs with toxic eff ects on mammalian et al., 2005; Li et al., 2006a; Mowery et al., cells. While the average contents of the 2009) and points at the direction for im- negatively charged residues DE (3.8–4.9%), proving peptide selectivity. According to the GP group (15–16%) and the polar residues Fig. 4.4A, it is necessary to adjust the TSYQN (15–17.5%) were similar in diff erent hydrophobic content of the peptide to <50% peptide activity groups, the contents of the in order to reduce cytotoxicity to human hydrophobic residues IVLFCMAW (41–50%) cells. Ahmad et al. (2009) showed that and the positive residues HKR (17.5–20.3%) substituting large hydrophobic residues (e.g. varied by up to 10%. In particular, peptides leucine or isoleucine) with small alanine toxic to mammalian cells possessed the residues reduced the cytotoxicity of BMAP- highest hydrophobic content, whereas anti- 28 on mammalian cells. Alternatively, the fungal peptides had the lowest hydrophobic peptide hydrophobic content can be reduced content. Coincidently, the content of posi- by deleting a hydrophobic segment from the tively charged residues was highest for intact AMP sequence. Skerlavaj et al. (1996) antifungal peptides, probably complementing found that removal of the hydrophobic tail of the corresponding low hydrophobic content BMAP-27 decreased peptide cytotoxicity. By (Fig. 4.4A). A further investigation revealed removing both the N- and C-terminal that the average percentages of lysine and segments from human cathelicidin LL-37, arginine residues in the fi ve groups of Wang (2008) identifi ed KR-12, the smallest peptides diff er. On average, antiviral peptides antibacterial fragment, with 12 residues. in the APD possessed the highest arginine KR-12 displays low toxicity to human cells content (solid columns, Fig. 4.4B). We decided (Table 4.2), while its parent peptide LL-37 is to put this observation to use. When HIV-1 highly toxic. inhibitory activity was evaluated, GLK-19 Incorporation of peptoids also improves was found to be inactive. However, the cell selectivity of the peptide (Song et al., peptide (GLR-19) became HIV inhibitory 2005; Zhu et al., 2007). The side chain of (Table 4.2) aft er the lysine residues of GLK-19 peptoids is shift ed from the α-carbon were substituted with arginine. Likewise, position to its backbone nitrogen position. while frog DRS S9 showed a poor The absence of the amide proton in the HIV-inhibitory activity, an arginine mutant peptoid disrupts hydrogen bonding, leading (DRS S9r3) displayed a high TI of approxi- to a less helical structure. Therefore, peptoid- mately 26 (Table 4.2) (Wang et al., 2010). containing peptides have a reduced affi nity Currently, we are investigating which argi- for membranes of zwitt erionic lipids. Song et nine is most important and the mechanism of al. (2005) found that incorporation of Nala inhibition. Because of limited examples, it is (alanine peptoid) onto the hydrophobic too early to state that such mutations are a surface of the peptide is more eff ective than general strategy for improving the effi cacy of incorporation onto the hydrophilic face in HIV-inhibitory peptides. Other factors such improving cell selectivity of the peptide. For as peptide sequence, viral strains and a model helical peptide KLW, incorporation molecular targets could also play a role. of two Nala residues at positions 9 and 13 improved peptide selectivity. Zhu et al. 4.3 Strategies for Improving Cell (2007) utilized a diff erent approach by Selectivity of AMPs replacing proline residues in tritrpticin and indolicidin with Nlys (lysine peptoid). Both Toxic side eff ects of chemotherapeutic agents peptides became more selective aft er on healthy human cells are undesirable and engineering. Prediction and Design of Novel AMPs 81

Fig. 4.4. (A) Average contents of a group of amino acids in AMPs with different activities. Hydrophobic residues include isoleucine, valine, leucine, phenylalanine, alanine, methionine, cysteine and tryptophan; positive residues are histidine, lysine and arginine; negative residues are glutamic acid and aspartic acid; polar residues comprise serine, threonine, asparagine, glutamine and tyrosine; and the GP group contains glycine and proline residues. (B) Average content of lysine (K) and arginine (R) in the ﬁ ve groups of AMPs. AB, antibacterial; AC, anticancer; AF, antifungal; AV, antiviral; MC, toxic to mammalian cells.

Peptide hydrophobicity can also be (Table 4.2). When d-amino acids were scaled down by partial incorporation of introduced at residues 20, 24 and 28 of d-amino acids (Sharon et al., 1999; Chen et GF-17 (numbered as in LL-37), we obtained al., 2005). By combining d-amino acid a peptide that retained toxicity to E. coli incorpora tion with disulfi de-bond linked K12, but displayed no toxic eff ect to human cyclization, Oren and Shai (2000) found cells. The structural basis for peptide that the engineered LK peptide (i.e. selectivity improvement by d-amino acid leucine- and lysine-based peptide) adopted incorporation is described in Chapter 9. Li a helical structure with improved cell et al. (2006a) proposed that a decrease in selectivity. Li et al. (2006a) identifi ed a hydrophobicity is a unifi ed approach to major antimicrobial region corresponding improving cell selectivity of membrane- to residues 17–32 of human LL-37 (GF-17) targeting AMPs. 82 G. Wang

4.4 Strategies for Improving Peptide or hydrophobicity due to fl uorination has Stability to Proteases been proposed as the basis for enhanced peptide stability (Lee et al., 2004; Gott ler et Another hurdle in developing AMP-based al., 2008). therapeutics is the short lifetime of these Finally, incorporation of d-amino acids peptides in vivo, probably due to degradation provides another useful approach for by endogenous proteases from either bacteria improving peptide stability. One of the early or testing animals. The diverse structural demonstrations was the synthesis of all-d- scaff olds of natural AMPs collected in the peptide analogues by Merrifi eld et al (1995). In APD are remarkable and inspiring (see the addition, partial incorporation of d-amino face page of the APD). In general, poly- acids may improve peptide stability. Using peptides with a folded structure are more helical peptides as models, Shai and resistant to protease cleavage. For example, colleagues introduced d-amino acids at every disruption of disulfi de bonds or the key salt two to three residues to improve peptide bridge of human α-defensin 5 (HD-5) leads selectivity (Pouny and Shai, 1992; Sharon et to rapid degradation (Tanabe et al., 2007; al., 1999). Similarly, Hong et al. (1999) found Rajabi et al., 2008). In addition, N-terminal that incorporation of d-amino acids at the acetylation, C-terminal amidation, introduc- terminal regions did not disrupt the tion of d-amino acids or non-standard amino antibacterial activity of the peptide, but acids and peptide cyclization are all known improved peptide stability in serum. post-translational modifi cation strategies in Signifi cantly, Braunstein et al. (2004) demon- natural peptides (Table 1.5, Chapter 1). These strated that injection of a d,l-peptide into mice strategies can be applied to stability cured animals infected with bacteria. Of note, enhancement of AMPs (see below) as well as promising results have also been obtained other natural peptides (Craik and Adams, using peptide mimics (see Chapter 6). 2008). In analogy to circular AMPs in nature, investigators have designed diff erent 4.5 Concluding Remarks chemical strategies to connect the ends of the peptides to achieve higher stability. Dathe et It is evident that the creation of the APD with al. (2004) achieved cyclization of an arginine- a clean and well-registered dataset has and tryptophan-rich peptide RRWWRF-NH2 facilitated database-based peptide prediction. by HAPyU chemistry (Ehrlich et al., 1996). The classifi cation of AMPs into a variety of They found that the cyclized peptide had groups in the database (Wang et al., 2009) higher antimicrobial activity and cell constitutes a useful step and was used to selectivity than its linear counterpart. improve the accuracy of antibacterial peptide Another group produced cyclization by prediction (Lata et al., 2010). By grouping disulfi de bond formation. While the cyclic AMPs into more than 140 clusters, AMPer melitt in analogue displayed increased anti- (Fjell et al., 2007) has achieved a relatively bacterial activity but decreased haemolytic high accuracy in AMP prediction. In addition, activity, cyclic magainin 2 had a marked the APD provides a set of useful templates decrease in both antibacterial and haemolytic for peptide design. The choice of a starting activity (Unger et al., 2001). Therefore, the peptide template is not always trivial. When eff ect of cyclization on the properties of feasible, database screening, and preferably linear peptides is peptide dependent. virtual screening in silico followed by activity Fluorine is incorporated into 20% of evaluation, is a useful approach. Sequence pharmaceuticals on the market (Muller et al., shuffl ing, includ ing sequence reversal, 2007). Fluorinated amino acids have been generates a miniature combinatorial library introduced into AMPs to improve protease of peptides. Combining peptide building stability without disrupting the original blocks (i.e. the grammar-based approach) is peptide structure (Niemz and Tirrell, 2001; an amplifi ed version of the traditional hybrid Meng and Kumar, 2007). An increase in size approach. In addition, building novel Prediction and Design of Novel AMPs 83

peptides using a minimal number of amino References acids, such as leucine and lysine, is the classic de novo approach. Coincidentally, these Agerberth, B., Gunne, H., Odeberg, J., Kogner, P., residues overlap with part of the frequently Boman, H.G. and Gudmundsson, G.H. (1995) occurring residues identifi ed from the FALL-39, a putative human peptide antibiotic, is database analysis of hundreds of amphibian cysteine-free and expressed in bone marrow AMPs, thereby endowing biological and testis. Proceedings of the National Academy of Sciences of the USA 92, 195–199. signifi cance to these residues utilized for de Ahmad, A., Asthana, N., Azmi, S., Srivastava, R.M., novo peptide design. The desired properties Pandey, B.K., Yadav, V. and Ghosh, J.K. (2009) of AMPs can also be improved based on Structure–function study of cathelicidin-derived three-dimensional structures (Chapter 9). bovine antimicrobial peptide BMAP-28: design Continuing peptide design work not only of its cell-selective analogs by amino acid deepens our knowledge of AMPs but also substitutions in the heptad repeat sequences. enriches the peptide space, biologically and Biochimica et Biophysica Acta 1788, 2411– chemically. For future thera peutic use of 2420. AMPs, it is important to achieve cell Begley, M. Cotter, P.D., Hill, C. and Ross, R.P. (2009) selectivity and in vivo stability, and to reduce Identifi cation of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining production costs. It is noteworthy that T-20, for LanM proteins. Applied and Environmental an HIV fusion inhibitor, has been produced Microbiology 75, 5451–5460. in multi-tons by a combination of solid-phase Blondelle, S.E. and Houghten, R.A. (1992) Design and solution-phase methodologies (Fuzeon; of model amphipathic peptides having potent Roche), indicating th at chemical synthesis antimicrobial activities. Biochemistry 31, 12688– off ers a practical approach for large-scale 12694. peptide production. In addition, peptides can Braunstein, A., Papo, N., and Shai, Y. (2004) In vitro be produced by bacterial expression (see activity and potency of an intravenously injected Chapter 9). With the discovery of novel AMPs antimicrobial peptide and its DL amino acid with interesting polypeptide scaff olds as well analog in mice infected with bacteria. Anti- microbial Agents and Chemotherapy 48, 3127– as the develop ment of new tools by exploiting 3129. natural post-translational enzyme modifi - Buckheit, R.W. Jr, Watson, K.M., Morrow, K.M. and cation systems (Chapters 2–3), we have Ham, A.S. (2010) Development of topical reason to believe that the peptide production microbicides to prevent the sexual transmission issue can be resolved in the future. All of of HIV. Antiviral Research 85, 142–158. these are promising advances in overcoming Chen, Y., Mant, C.T., Farmer, S.W., Hancock, R.E., hurdles on the way to the development of Vasil, M.L. and Hodges, R.S. (2005) Rational AMP-based therapies. design of α-helical antimicrobial peptides with enhanced activities and specifi city/therapeutic index. Journal of Biological Chemistry 280, 12316–12329. Cherkasov, A., Hilpert, K., Jenssen, H., Fjell, C.D., Acknowledgements Waldbrook, M., Mullaly, S.C., Volkmer, R. and Hancock, R.E. (2009) Use of artifi cial intelligence The author appreciates the Most Promising in the design of small peptide antibiotics effective New Invention Award from UNEMED of the against a broad spectrum of highly antibiotic- University of Nebraska Medical Center, resistant superbugs. ACS Chemical Biology 4, which allowed the fi rst anti-HIV peptide 65–74. screening based on the Antimicrobial Peptide Conlon, J.M., Kolodziejek, J. and Nowotny, N. Database in collaboration with ImQuest (2004) Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a BioSciences (MD). Thanks also extend to potential source of new therapeutic agents. Alan Peterkofsky, Jim Turpin, Edward Ezell Biochimica et Biophysica Acta 1696, 1–14. and Tor Haug for critical reading of this Craik, D.J and Adams, D.J. (2008) Chemical manuscript. This work was supported by the modifi cation of conotoxins to improve stability US National Institutes of Health grants and activity. ACS Chemical Biology 2, R21AI082689 and R56AI81975. 457–468. 84 G. Wang

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William C. Wimley

Abstract

The fi eld of antimicrobial peptide (AMP) research has now spanned three decades, in which hundreds of AMPs have been discovered, designed or engineered. Yet despite a vast literature, obvious structure– function relationships are rare and this has created a bott leneck in the discovery of novel AMPs. Instead of rigorous structure–function principles, AMP activity may be best addressed using the physical chemistry concept of ‘interfacial activity’, a concept that does not help one predict or engineer AMP activity. In this chapter, I address one method of circumventing this engineering bott leneck: combinatorial chemistry and high-throughput screening. Combinatorial methods are fi rst discussed from the perspective of library synthesis techniques, for both indexed and non-indexed methods. This is followed by a discussion of available high-throughput screening techniques. Finally, I address the accomplishments to date generated using combinatorial chemistry and high-throughput screening and future directions the fi eld may take. Combinatorial chemistry and high-throughput screening are powerful and eff ective tools for discovering novel AMPs. The future of this fi eld holds great promise.

5.1 The Interfacial Activity Model of 2005; Mowery et al., 2007; Rausch et al., 2007). AMP Activity Instead it depends on ‘interfacial activity’, which has been referred to as ‘the ability of a Antimicrobial peptides (AMPs) exert their molecule to bind to a membrane, partition biological activity by fi rst acting on microbial into the membrane–water interface and to membranes (Steiner et al., 1981; White et al., alter the packing and organization of the 1995), sometimes followed by additional lipids’ (Rathinakumar and Wimley, 2008; eff ects on the microbial cell. Yet, despite Rathinakumar et al., 2009). Interfacial activity decades of intense study, compelling (Fig. 5.1) is derived from the appropriate structure–function relationships for AMPs are balance of interactions between and among rarely found and evidence for stable, well- peptides, water and membrane lipids. These defi ned transmembrane pores (Fig. 5.1) is interactions depend more on the amino acid rarely observed. Recent literature suggests composition of a peptide and on its physical that physical impact on membranes, such as chemical properties than on its exact sequence lipid domain formation (Chapter 7), is not or secondary/tertiary structure (Rathinakumar dependent on specifi c amino acid sequences and Wimley, 2008; Rathinakumar et al., 2009; or on specifi c three-dimensional peptide Chapter 7). In support of this idea, Hilpert structures (Hilpert et al., 2005, 2006; Jin et al., et al. (2006) have shown that a high

Barrel-stave pore

Toroidal pore

Interfacial activity

Fig. 5.1. Some schematic models of antimicrobial peptide activity. The literature contains numerous mechanistic models to explain the action of antimicrobial peptides on lipid bilayers. The barrel-stave and toroidal pore models, while commonly referenced, do not explain the experimentally observed actions of most antimicrobial peptides. The interfacial activity model (Rathinakumar and Wimley, 2008) can explain the actions of most AMPs. Most importantly, the interfacial activity model can be used as a basis for designing compositionally varied combinatorial peptide libraries. percentage of random versions of a potent charge, hydrophobicity; see Chapter 1). This AMP retain good activity and some even would allow the design of rational libraries have improved activ ity. from which the most active sequences can Although AMPs are known to adopt a readily be determined by high-throughput variety of three-dimensional structures in screening. In this chapter, I explore the lipid bilayers, the rational design of AMPs design, selection and screening of com- based on structure-specifi c ideas is rare binatorial peptide libraries toward the (Chapter 9). However, design based on the effi cient discovery of novel AMPs. principle of interfacial activity is not yet possible because the physical chemical basis of interfacial activity has not been 5.2 Combinatorial Chemistry Methods parameterized. In light of these considerations, the use of combinatorial chemistry and 5.2.1 Overview of library synthesis high-throughput screening is an especially att ractive approach to discovering novel Almost as soon as the Merrifi eld method of AMPs. To design a library, one can use the solid-phase peptide synthesis became widely principle of interfacial activity by requiring known, combinatorial peptide synthesis was libraries to contain peptides that adhere to envisioned as a natural extension of the common att ributes of known AMPs (e.g. chemistry. The power of combinatorial peptide length, amino acid composition, peptide synthesis is derived, in part, from Discovery of Novel AMPs 89

the fact that the chemistry required to make of peptide synthesis for deconvolution, and a library is no more complex than to perform risks the possibility that peptides in the a non-combinatorial synthesis. No novel mixture will act synergistically or reaction protocols, equipment or protection antagonistically, it is simple and eff ective, as strategies are needed. Combinatorial peptide shown by Houghton and colleagues, and can chemistry is thus directly accessible to successfully lead to the discovery of novel researchers with a moderate chemistry potent AMPs. knowledge. In this section, I describe some of Perhaps an even more powerful the more commonly used synthetic approach to non-indexed combinatorial approaches to combinatorial peptide library peptide libraries is the one-bead:one- synthesis. sequence method, also known as split and recombine libraries. In a one-bead:one- sequence library, a researcher takes advan- 5.2.2 Non-indexed methods tage of the discrete nature of solid-phase synthesis resins, which are typically in the In any high-throughput screen, one must be form of polymer microbeads, oft en made able to select for active compounds and then from polystyrene and sometimes with identify the active molecule or molecules graft ed polyethylene glycol. A synthesis exactly. Combinatorial peptide libraries can slurry of resin beads can be combined into a be separated into two broad categories: (i) single vessel when common chemistry is indexed libraries, discussed in detail below, performed; it can be split into separate which use spatial or chemical indexing that vessels for the addition of separate amino allows active sequences to be known during acids when a combinatorial site is reached. the course of the screen; and (ii) non-indexed Recombination of the resin beads into one libraries, in which indirect analytical vessel allows for randomization before the methods, such as Edman sequencing, mass next split. Each bead thus has a unique spectrometry or iterative deconvolution, history of amino acid additions, and contains must be used to identify active sequences only peptides of a single sequence. Spatial aft er they have been selected in the screen. separation of the beads is required for Here, I discuss some commonly used screening. Typical one-bead:one-sequence non-indexed combinatorial library methods. libraries have 50,000–1,000,000 beads g–1 of Houghten and colleagues were some of resin or 0.2–2.5 nmol of peptide per bead. the earliest users of combinatorial chemistry Table 5.1 shows the statistics for some and high-throughput screening to identify commonly used library beads. One-bead:one- novel AMPs (Blondelle et al., 1996). They sequence libraries are spatially segregated used several simple, partially indexed library but are usually not indexed. Screening is design methods in which the peptides performed on individual beads and the screened initially were random mixtures of positive sequences are identifi ed post facto peptides containing fi xed residues at some using chemical sequencing, mass spec- positions and random mixtures of amino trometry or deconvolution. acids at others. The relative abundance of AMP discovery requires that peptides each amino acid in the varied position was tested are free in solution, a fact that places controlled by adjusting amino acid concen- limitations on the types of combinatorial trations according to their relative reaction libraries that can be used. For example, rates. Identifi cation of active peptides was classical phage display or selectide-type accomplished by positional scanning or peptide libraries (Lam et al., 2003), designed iterative deconvolution. When necessary, to identify peptide sequences that bind to mixtures with activity were further particular targets, must be modifi ed to allow subdivided by iterative deconvolution based peptides to be released. For one-bead:one- on rounds of additional synthesis and testing sequence libraries, an orthogonal approach until single active sequences were obtained. to synthesis and release must be employed. While this approach requires a large amount Typically, researchers use a photolabile or 90 W.C. Wimley

Table 5.1. Some available synthesis microbeads for one-bead:one-sequence libraries.a

Median Typical Conc. (μM) TentaGel diameter loading Bead density Peptide per bead (measured in polystyrene with (μm) (mmol g–1) (beads g–1) (nmol per bead) 100 μl) grafted 90 0.2 1×106 0.2 1 polyethylene glycol 300 0.2 8×104 2.5 10 a TentaGel beads (Rapp-Polymere) are commonly used for one-bead:one-sequence libraries. Library size, which is inversely proportional to the amount of peptide per bead, ranges from 104 to 106 beads g–1, an easy bench-top synthesis scale. In my experience, photolabile linkers allow for release of only a portion of the peptide on the bead; the last column refl ects the actual measured release of peptide into 100 μl of buffer. other non-acid-labile linker that can be 5.3 High-throughput Screening cleaved independently from peptide chemistry and side-chain deprotection, which is In the research and development department generally acid dependent. In my work, I have of a large commercial pharmaceutical enter- used a UV-cleavable photolabile linker that prise, ‘high-throughput screening’ means allows for the peptide-bead linker to be testing millions of compounds rapidly in cleaved when the bead is dry, followed by massive parallel, automated, robotic appli- physical segregation of the beads, extraction cations. On the other hand, in a small of peptide into buff er and screening. academic laboratory, it may mean screening only a few thousand peptides by mostly manual techniques. In either case, high- 5.2.3 Indexed methods throughput screening can be defi ned by the common characteristic of performing When the individual members of a library can selection experiments on molecular libraries be identifi ed directly, either by their spatial at a pace that is orders of magnitude faster location or by a coded ‘tag’ associated with than achievable using the traditional them, the library is considered an ‘indexed’ approach of single sequence design, library. Indexing combinatorial libraries is a synthesis, purifi cation and characterization powerful method, which has the advantage of (Fig. 5.2). As discussed above, combinatorial not requiring tedious deconvolution or peptide library synthesis is not, in itself, expensive chemical sequencing. An example technically challenging. The real challenge of an indexed combinatorial library is the lies in the design of the high-throughput SPOT synthesis method (Frank, 2002) and its screen needed to effi ciently select the most variants, in which synthesis is done on active members from a library. In the fol- spatially indexed spots on a cellulose sheet, lowing sections, I explore the various followed by cutt ing of the ‘spots’ and cleavage approaches that can be used to select for AMP into individual samples. SPOT synthesis can sequences in peptide libraries. be performed with technology as simple as a pipett ing robot, as described by Hancock and 5.3.1 Biological assays colleagues (Hilpert et al., 2005). Similarly, multi-pin libraries (Maeji et al., 1990) are The most direct selection method for carried out using multiple pins as solid antimicrobial activity is to screen libraries supports with multiple reaction chambers using living microbes. Broth dilution assays controlling the chemistry. The size of SPOT (Wiegand et al., 2008) test for the ability of a libraries and pin libraries depends on a peptide to inhibit the growth of microbes in laboratory’s investment into the synthetic a rich nutrient broth. Generally a peptide technology, but libraries with thousands to and a microbe inoculum are incubated hundreds of thousands of members are overnight in media. Given the long incu- readily achievable. bation and short doubling time for most Discovery of Novel AMPs 91

(A) (B) Combinatorial High-throughput library synthesis screening

Number of Number of peptides peptides

Fig. 5.2. Combinatorial library synthesis and high-throughput screening. Library design and the high- throughput screening must be balanced. (A) Library size increases very rapidly with its complexity. The larger the library, the greater the amount of sequence space that can be explored. (B) High-throughput screening must be able to identify a small number of highly active library members in a reasonable amount of time and effort. Screens with higher throughput and better discrimination allow a researcher to design more complex libraries. microbes, expansion of the inoculum to an A second classical antimicrobial activity opaque, stationary phase will occur unless experiment that can be adapted to high completely inhibited by the peptide. In throughput is the agar diff usion assay practice, intermediate results are usually not (Wiegand et al., 2008), in which peptides are observed (Rausch et al., 2007; Rathinakumar spott ed on a thin layer of nutrient agar seeded and Wimley, 2008). Activity can be detected with bacteria. Peptides will diff use into the photometrically, but even visual inspection agar and active peptides will create a will suffi ce in this experiment because of the clearance zone where they inhibit bacterial binary nature of the result. Broth dilution growth. The clearance area is proportional to screening is readily performed in a multiwell the potency of the peptide. Examples of agar plate format and requires a minimum of diff usion experiments are shown in Fig. 5.4. automation, robotics or specialized detection An advantage of agar diff usion is that the methods. Figure 5.3 shows an example of a results are semi-quantitative. The most potent broth dilution screen in a 96-well plate members of a library can readily be identifi ed format. For more rapid screening, increased from the diameter of the spots. In one- sensitivity and automated throughput, one bead:one-sequence libraries, another possible can couple a broth dilution assay with a advantage of agar diff usion is that peptides secondary detection method using real-time do not need to be extracted from the beads growth curves or by using engineered for the assay as they do in a broth dilution enzyme activity (Hilpert et al., 2005). assay. As shown in Fig. 5.4, scatt ering beads 92 W.C. Wimley

Fig. 5.3. Broth dilution assay as a high-throughput screen. In this example of a 96-well screen, each well contains a 2.2 μM concentration of one member of a combinatorial peptide library, rich growth medium and an inoculum of 103 Escherichia coli bacteria. After overnight incubation the wells are either opaque, indicating stationary-phase growth, or they are transparent, indicating no growth (i.e. the bacteria have been sterilized by the peptide). This is a very stringent high-throughput screen, which requires no sophisticated equipment to perform. Wells C11–H11 have no added peptides and wells C12–H12 have no added bacteria.

with dry-cleaved photolabile linker on to the 5.3.2 Selection of broad-spectrum peptide agar layer is suffi cient to obtain good antibiotics antimicrobial activity because many of the peptides will spontaneously diff use from the Identifying peptides with potent anti- beads into the agar and inhibit growth. In my microbial activity against one species of experience, not all peptides diff use from the microbe is surprisingly easy. For example, I beads into the agar at a useful rate, but this and others have designed biased libraries in may actually be advantageous because the which more than a quarter of all members slowly diff using peptides are the least have potent antimicrobial activity (Rausch et soluble, and therefore also the least desirable al., 2007; Rathinakumar and Wimley, 2008; AMPs. Rathinakumar et al., 2009). A large proportion Recently, an intriguing ‘whole animal’ of random sequence variants of known high-throughput antimicrobial screen was AMPs have also been shown to have good described (Moy et al., 2009). In this screen, activity (Hilpert et al., 2006) as well as many Candida elegans nematodes in small nutrient engineered variants of natural antimicrobial chambers were used to test compounds activity. These results arise from biased simultaneously for antimicrobial activity and libraries and biased sequences, which are toxicity towards the nematode. rich in aromatics and hydrophobic and Discovery of Novel AMPs 93

(A) (B)

1 cm 1 cm

(C)

1 cm

Fig. 5.4. Agar diffusion assay for high-throughput screening of one-bead:one-sequence libraries. (A,B) For controls, the bovine AMP indolicidin was linked to synthesized resin microbeads using a photolabile linker. Indolicidin beads were placed on a thin layer of nutrient agar seeded with Escherichia coli bacteria. (A) The photolabile linker is cleaved with UV light and the released peptides create a large clearance zone where no bacteria grow. (B) The linker is not cleaved and no clearance zone is created. (C) Beads from a combinatorial peptide library described elsewhere (Rathinakumar and Wimley, 2008) are scattered on to an agar diffusion plate. This is the same library screened by broth dilution in Fig. 5.3. Some beads (black arrows) create large zones of clearance, while others (white arrows) are less active. 94 W.C. Wimley

cationic residues and, as a result, contain Tables 5.2 and 5.3 give information on these many antimicrobial members. experiments. Broad-spectrum antimicrobial activity, Biological assays have been used defi ned as activity against multiple classes of successfully to select AMPs from combina- microbes, is desirable, but is rarer and more torial libraries. However, the single-species diffi cult to select than species-specifi c nature of most experiments and the diffi culty activity. Selecting directly for broad-spectrum in performing multispecies experiments with activity using biological screens can be high throughput are problems that have not achieved if a single library member is yet been overcome. The next section de- screened in parallel against microbes from scribes lipid-vesicle-based high-throughput multiple classes. This approach requires a screens, which are simpler to perform and complex experiment that reduces the have been shown to select specifi cally for throughput of a screen. However, it has the peptides with broad-spectrum activity and advantage over other screens that broad- low toxicity. spectrum peptides are identifi ed directly and unambiguously. To test the eff ectiveness of this approach, I have performed parallel 5.3.3 Non-biological assays multispecies screening using a sample of a one-bead:one-sequence library described in Countless studies in the literature show that the literature (Rathinakumar and Wimley, AMPs interact with and perturb lipid bilayer 2008; Rathinakumar et al., 2009). We screened membranes in vitro as well as in vivo. A in parallel for activity against Escherichia coli typical in vitro experiment is performed (a Gram-negative bacterium), Staphylococcus using unilamellar lipid vesicles with an aureus (a Gram-positive bacterium) and entrapped marker for assaying membrane Crypto coccus neoformans (a fungus). We found permeability (Rausch and Wimley, 2001). species-specifi c antimicrobial activity to be Experimental lipid compositions vary widely surprisingly common, while overlapping (i.e. between laboratories, but mixtures of anionic broad-spectrum) activity was quite rare. and zwitt erionic lipids are oft en used to

Table 5.2. Abundance of broad-spectrum AMPs in a combinatorial library.a Number of peptides Percentage Total peptides screened 1040 100 Active peptides 175 16.8 Combination of microbes sterilized Only Escherichia coli 82 7.9 At least E. coli 115 11.1 Only Staphylococcus aureus 54 5.2 At least S. aureus 79 7.6 Only Cryptococcus neoformans 3 0.3 At least C. neoformans 12 1.2 Only E. coli and S. aureus 27 2.6 Only E. coli and C. neoformans 2 0.2 Only S. aureus and C. neoformans 3 0.3 All three organisms 4 0.4 a Spectrum of antimicrobial activity in a one-bead:one-sequence combinatorial peptide library described elsewhere (Rathinakumar and Wimley, 2008; Rathinakumar et al., 2009). Individual peptides were extracted from beads and the solution was divided into three 96-well plates, where the peptide concentration in each well was about 2 μM. Wells were seeded with E. coli (a Gram-negative bacterium), S. aureus (a Gram-positive bacterium) or C. neoformans (a fungus). Sterilization assays showed that there were many highly active peptides in the library (17%), but that only a small fraction (0.4%) had potent broad-spectrum activity against all three classes of microorganism. Discovery of Novel AMPs 95 cells HEK293 4 3 9 4 10 14 1 7 32 5 33 3 16 2 12 6 1 7 3 1 0 ND 0 1 2 7 1 100 4 100 3 ND Leakage (%) xed. xed. E . coli S . aureus C . neoformans (%) cells Liposome HEK293 Cytotoxicity Cytotoxicity RBC Sheep 26 8 9 55 16 89 12 4 3 50 24 97 43 6 1 79 17 84 28 5 2 69 100 72 18 6 3 39 80 76 11 6 2 25 89 71 23 6 2 93 94 47 28 8 8 65 91 52 41 4 6 67 6 65 25 7 15 72 61 22 RBC 2 4 4 ND 0 3 ND ND ND ND 0 0 Human Haemolysis (%) -leucine. D 6.5 4 10 4 1 10 ND 6.54.71.1 12 14 1 100 19 ND 93 2 93 4 24 93 98 14 24 100 23 100 ND 100 100 100 100 ND ND ND ND 10.6 6.1 6.2 5.4 2.6 2.7 4.1 3.6 8.5 9.0 >15 >15 -amino acids. D neoformans Cryptococcus 2.9 1.9 2.9 1.7 2.4 1.3 1.6 2.1 2.0 5.3 0.9 2.4 4.4 2.4 ND -leucine is replaced with a aureus L >15 Staphylo coccus Staphylo 8.82.8 >10 2.9 1.3 0.1 ND Data were selected from a 16,384-member combinatorialData were library using high-throughput screening (Rathinakumar and 3.6 2.8 2.8 2.4 2.9 3.3 1.5 1.4 1.8 2.9

>15 aeruginosa Pseudomonas Minimum sterilizing ( μ M) Minimum concentration coli 1.7 3.8 9.7 1.3 1.2 1.5 0.4 ND >15 Escherichia b ) d 10 c ) e f D a -Leu D RRGWALRLVLAY ( RRGWALRLVLAY RRGWALRLVLAY (All Indolicidin Melittin PMSD Ampicillin Triton-X WVLVLRLGY WALRLVLYY WYLTLTLGYGRR 3.1 RRGWRLVLALVY 1.4 RRGWALRLVLAY 1.9 RRGWVLRLALYAY 1.5 RRGWVLALYLRYGRR 2.1 RRGWVLALVLRYGRR 1.9 RRGWVLALVLYYGRR 4.9 RRGWVLDLVLYYGRR 1.9 randomly present or absent and the O residues can be one of the following amino acids: NDTRGAVY. The W, L and C-terminal Y residues are ﬁ L and C-terminal W, The NDTRGAVY. amino acids: present or absent and the O residues can be one of following randomly X-100). Triton dissolution with detergents (e.g. as membrane melittin had the same permeabilizing effect sequence alone is inactive and is used for a control peptide. and is used for sequence alone is inactive All novel peptides from the library are nine, 12 or 15 residues in length. The library has the form {RRG}WOLOLOLOY{RRG}-amide, where the {RRG} terminal basic cassettes are The library {RRG}WOLOLOLOY{RRG}-amide, has the form peptides from the library 12 or 15 residues in length. are nine, All novel in which the C-terminal-most The peptide RRGWALRLVLAY, neutrophil AMP indolicidin has the sequence ILPWKWPWWPWRR-amide. The bovine 5 μ M In all assays, synthetic or biological. membranes, peptide that potently permeabilizes α -helical pore-forming all lipid bilayer peptide melittin is a 22-residue, venom The honeybee The peptide RRGWALRLVLAY, in which the entire sequence is composed of The peptide RRGWALRLVLAY, the membrane-spanning However, of the whole multimeric perfringolysin to a membrane-permeabilizing PMSD is a sequence that contributes O protein toxin. ˟ -barrel in the context Control compounds Peptide Table 5.3. Table AMPs. for Example data table a b c d e f ., 2009). To assess the results of a selection process, one must have data for many aspects of biological activity, as shown here. here. as shown aspects of biological activity, many data for have one must assess the results of a selection process, To Rathinakumar et al ., 2009). 2008; Wimley, Rathinakumar et al ., 2009). 2008; Wimley, Rathinakumar and Experiments (Rausch et al ., 2005, 2007; are described in detail recent publications cell. red blood RBC, no data; ND, perfringolysin domain; membrane-spanning PMSD, cell line 293; HEK293, human embryonic kidney Abbreviations: 96 W.C. Wimley

mimic microbial membranes, while phos- chelator dipicolinic acid (DPA) added to the phat idylcholine and/or phosphatidyl choline/ external solution. The Tb3+–DPA complex, cholesterol are used to mimic mammalian which forms only when the membranes have membranes. Because cationic/hydrophobic been permeabilized, is highly ﬂ uorescent and AMPs with good interfacial activity are can be quantitated. Assays are performed in expected to perturb any lipid bilayer the 96-well plate format and ﬂ uorescence is membrane to which they bind well enough, used to rate permeabilization. Figure 5.5 the exact anionic lipid content is probably shows high-throughput screening using this not a crucial factor in vesicle-based char- method. By adjusting the concentration of acteri zation of AMPs. lipid vesicles added to each well, the We have developed a vesicle-based high- stringency of the assay can be adjusted to throughput screen to select for potent vesicle- suit the library being studied. Figure 5.6 permeabilizing peptides from combinatorial shows permeabilization data for a combina- libraries (Rausch and Wimley, 2001; Rausch torial library screened by this method. et al., 2005; Rathinakumar and Wimley, When performing high-throughput 2008; Rathinakumar et al., 2009). The high- screens for AMPs, it is important to also select throughput screen utilizes large unilamellar against peptides that have poor solubility. In vesicles with the lanthanide metal terbium the vesicle-based screen described above, we (III) (Tb3+) trapped inside and the aromatic also used a ‘premixing’ step in which library

(A) Low stringency (B) High stringency

Fig. 5.5. A vesicle-based high-throughput screen for bilayer-permeabilizing peptides. Large unilamellar vesicles containing entrapped terbium (III) (Tb3+) and external dipicolinic acid (DPA) are added to each well, which also contains about 10 μM of one peptide from a combinatorial library. Permeabilization of the vesicles results in Tb3+–DPA complex formation, which is highly ﬂ uorescent. These plates were photographed under UV light. (A) A screen with a lower lipid concentration gives high sensitivity, but lower stringency. (B) A screen conducted with a higher lipid concentration used for lower sensitivity and higher stringency screening. In practice, stringency is altered to provide the desired number of positive peptides (Rausch et al., 2005). Discovery of Novel AMPs 97

(A) 5.4 Accomplishments 1.0 Combinatorial methods have successfully led 0.8 to the discovery of novel peptides with potent antimicrobial activity. For example, 0.6 Houghten and colleagues found novel AMPs using a synthesis-intensive iterative decon- 0.4 volution method (Blondelle et al., 1996) by screening against individual organisms,

Fraction of peptides Fraction including fungi. Peptides as short as six 0.2 residues, with a classical composition of basic and aromatic amino acids, were found, 0.0 some with potent antifungal activity, others 0 20 40 60 80 100 with broad-spectrum antimicrobial activity. Contents leakage (%) Similarly, Hancock and colleagues used (B) indexed synthesis on cellulose sheets to 1.0 explore the activity of a peptide library 0.8 designed around the sequence of the AMP Bac2A (Hilpert et al., 2005). They found Extraordinary speciﬁ c sequences with improved broad- peptides spectrum activity over the parent sequence. 0.02 In recent years, we have used one- bead:one-sequence libraries and orthogonal 0.01 high-throughput lipid vesicle-based screens

Fraction of peptides Fraction to select soluble, pore-forming peptides from libraries designed to explore narrow regions 0.00 of sequence space. In these experiments, 0 20 40 60 80 100 peptides were selected based on their ability Contents leakage (%) to permeabilize lipid vesicles composed of 90% zwitt erionic phosphatidyl choline and Fig. 5.6. Example statistics for a high-throughput 10% anionic phosphatidylglycerol at screen using a one-bead:one-sequence library. peptide:lipid ratios of 1:50 to 1:200. Using Several thousand library members were screened libraries of 10,000–16,000 members, about using a high-stringency, vesicle-based screen as shown in Fig. 5.5. (A) The majority of sequences 0.1% of each library had potent membrane have little or no membrane-permeabilizing activity. permeabilizing activity and was also water (B) An altered y-axis shows that a few single soluble. Importantly, we showed that each of peptides have exceptional activity. These excep- the sequences selected in the vesicle-based tional peptides have potent, broad-spectrum screen had potent broad-spectrum activity, antimicrobial activity. being able to sterilize Gram-positive and Gram-negative bacteria as well as fungi at peptides were added to buff er and incubated peptides concentrations of <10 μM. At the for several hours before the addition of the same time, active peptides showed only vesicle test solution. Insoluble peptides will moderate haemolytic or cytotoxic eff ects on precipitate and will be inactive. By using mammalian cells. these two orthogonal screening steps, we select only for peptides that are soluble and have good activity. In one library tested, we 5.4.1 Beyond high-throughput screening found that two-thirds of all vesicle-active peptides are also insoluble. The remaining Identifi cation of potential AMPs using high- active peptides were highly soluble in water throughput screening is only the fi rst step in and thus amenable to experimentation and the pipeline towards therapeutic AMPs. First, perhaps development into therapeutics. selected peptides must be tested for broad- 98 W.C. Wimley

spectrum activity against multiple classes of may envision an orthogonal high-throughput microbes. Peptides must then be tested for pipeline strategy for the development of their lytic or toxic eff ects on mammalian cells. novel AMPs that uses high-throughput Experimentally, these latt er eff ects are quan- screening to select active peptides from titated by measuring lysis of mammalian red rationally designed libraries, followed by blood cells, lysis of mammalian nucleated focused bioactivity, formulation and toxicity cells or cytotoxicity against living mammalian assays to select the most promising cells. To exemplify these critical experiments, candidates for clinical testing. a complete example dataset is shown in Table 5.3. This gives the characteristics of peptides selected from the combinatorial library, References described elsewhere (Rathinakumar and Wimley, 2008; Rathinakumar et al., 2009), that Blondelle, S.E., Takahashi, E., Houghten, R.A. and we have used throughout this chapter as an Pérez-Payá, E. (1996) Rapid identifi cation of example. A vesicle-based high-throughput compounds with enhanced antimicrobial activity screen (Rausch et al., 2005) was used to by using conformationally defi ned combinatorial identify ten peptides from the 16,384-member libraries. Biochemical Journal 313, 141–147. Frank, R. (2002) The SPOT-synthesis technique. library. As shown in Table 5.3, these ten Synthetic peptide arrays on membrane supports peptides have potent broad-spectrum activity – principles and applications. Journal of against microbes, and only minimal lytic or Immunological Methods 267, 13–26. toxic eff ects against red blood cells and Hilpert, K., Volkmer-Engert, R., Walter, T. and cultured human cells. Hancock, R.E. (2005) High-throughput generation of small antibacterial peptides with improved activity. Nature Biotechnology 23, 1008–1012. 5.5 Future Directions Hilpert, K., Elliott, M.R., Volkmer-Engert, R., Henklein, P., Donini, O., Zhou, Q., Winkler, D.F. and Hancock, R.E. (2006) Sequence Combinatorial chemistry and high- requirements and an optimization strategy for throughput screening are powerful and short antimicrobial peptides. Chemistry & eff ective tools for discovering novel AMPs. Biology 13, 1101–1107. The future holds great promise as advances Jin, Y., Hammer, J., Pate, M., Zhang, Y., Zhu, F., in technology drive increases in throughput. Zmuda, E. and Blazyk, J. (2005) Antimicrobial However, throughput is probably not the activities and structures of two linear cationic critical factor in the discovery of thera- peptide families with various amphipathic peutically useful AMPs. Even libraries of β-sheet and α-helical potentials. Antimicrobial <1 0,000 members contain potent broad- Agents and Chemotherapy 49, 4957–4964. spectrum AMPs that can be identifi ed with Lam, K.S., Lehman, A.L., Song, A., Doan, N., Enstrom, A.M., Maxwell, J. and Liu, R. (2003) manual screening (Rausch et al., 2005; Synthesis and screening of ‘one-bead one- Rathinakumar and Wimley, 2008). Perhaps compound’ combinatorial peptide libraries. more importantly, improvements in our Methods in Enzymology 369, 298–322. understanding of AMP mechanisms of action Maeji, N.J., Bray, A.M. and Geysen, H.M. (1990) will drive improvements in library design, Multi-pin peptide synthesis strategy for T cell leading to more eff ective peptides. We can determinant analysis. Journal of Immunological also look forward in the near future to Methods 134, 23–33. orthogonal high-throughput screens that Mowery, B.P., Lee, S.E., Kissounko, D.A., Epand, select in parallel for (or against) other factors R.F., Epand, R.M., Weisblum, B., Stahl, S.S. that determine the true therapeutic and Gellman, S.H. (2007) Mimicry of antimicrobial host-defense peptides by random eff ectiveness of a potential antibiotic drug. copolymers. Journal of the American Chemical For example, screens could include selection Society 129, 15474–15476. based on membrane permeabilization, Moy, T.I., Conery, A.L., Larkins-Ford, J., Wu, G., bioactivity, solubility, bioavailability, pharma- Mazitschek, R., Casadei, G., Lewis, K., cokinetics, cytotoxicity, susceptibility to Carpenter, A.E. and Ausubel, F.M. (2009) High- acquired resistance and other factors. We throughput screen for novel antimicrobials using Discovery of Novel AMPs 99

a whole animal infection model. ACS Chemical Academy of Sciences of the USA 102, Biology 4, 527–533. 10511–10515. Rathinakumar, R. and Wimley, W.C. (2008) Rausch, J.M., Marks, J.R., Rathinakumar, R. and Biomolecular engineering by combinatorial Wimley, W.C. (2007) β-Sheet pore-forming design and high-throughput screening: small, peptides selected from a rational combinatorial soluble peptides that permeabilize membranes. library: mechanism of pore formation in lipid Journal of the American Chemical Society 130, vesicles and activity in biological membranes. 9849–9858. Biochemistry 46, 12124–12139. Rathinakumar, R., Walkenhorst, W.F. and Wimley, Steiner, H., Hultmark, D., Engstrom, A., Bennich, H. W.C. (2009) Broad-spectrum antimicrobial and Boman, H.G. (1981) Sequence and peptides by rational combinatorial design and speciﬁ city of two antibacterial proteins involved high-throughput screening: the importance of in insect immunity. Nature 292, 246–248. interfacial activity. Journal of the American White, S.H., Wimley, W.C. and Selsted, M.E. (1995) Chemical Society 131, 7609–7617. Structure, function, and membrane integration Rausch, J.M. and Wimley, W.C. (2001) A high- of defensins. Current Opinion in Structural throughput screen for identifying transmembrane Biology 5, 521–527. pore-forming peptides. Analytical Biochemistry Wiegand, I., Hilpert, K. and Hancock, R.E. (2008) 293, 258–263. Agar and broth dilution methods to determine Rausch, J.M., Marks, J.R. and Wimley, W.C. (2005) the minimal inhibitory concentration (MIC) of Rational combinatorial design of pore-forming antimicrobial substances. Nature Protocols 3, β-sheet peptides. Proceedings of the National 163–175. 6 Chemical Mimics with Systemic Efﬁ cacy

Amram Mor

Abstract

Host-derived cationic antimicrobial peptides (AMPs) and their synthetic variants are widely regarded as a potential source of future therapeutic agents against a broad range of pathogens. This is due to a remarkable set of advantageous properties, ranging from molecular simplicity that readily allows for time- and cost-effi cient establishment of structure–activity relationships, to an essentially non-specifi c multitargeted mode of action, which signifi cantly limits the capacity of pathogens to develop effi cient resistance mechanisms. Nevertheless, diffi cult challenges need to be overcome as we move towards their eventual use in therapeutics, including improved bioavailability, toxicity and production costs. To address these issues, an increasingly rich variety of strategies for improving AMP properties are currently available for designing chemical mimics that reproduce the most critical biophysical characteristics of AMPs. In this chapter, I review the main strategies for the de novo design of AMP mimics that have generated systemically active lead compounds and showed promise for therapeutic development. Conceptually, the known designs can be distinguished on the basis of backbone rigidity. Thus, aft er a brief discussion of recent advances on representative approaches used to generate relatively stiff antimicrobial structures, this chapter focuses on the acyl-lysyl approach, which allows the design of structurally bendable molecules that none the less can selectively target a variety of microbial cells.

6.1 Introduction amphipathy, supramolecular organization and conformational fl exibility in interactions Aft er over two decades of intensive with target cells. worldwide eff orts, the ubiquitous occurrence The antimicrobial properties of HDPs of host defence peptides (HDPs) and their were initially investigated for their role in critical roles as major immunity eff ectors are providing the fi rst line of defence against a now well established. It is widely believed wide range of invading pathogens (Boman that this peptide-based defence system may and Hultmark, 1987; Bevins and Zasloff , be useful in fi ghting the emergence and 1990; Hoff mann, 1995; Nicolas and Mor, spread of multidrug resistance to available 1995; Hancock and Lehrer, 1998; Bulet et al., chemotherapeutic drugs. Clearly, various fi ne 2004). Many were found to exhibit activity details of their mechanism of action are still against a broad spectrum of bacteria, fungi, poorly understood. However, structure– protozoa and enveloped viruses (Andreu activity relationship studies have helped to and Rivas, 1998; Ganz, 1999; Mor, 2009). Due reveal new strategies for the specifi c targeting to their unique mode of action, antimicrobial of pathogens and highlighted the crucial role peptides (AMPs) have demonstrated of physicochemical parameters such as excellent potential in treating very common

as well as sometimes fatal illnesses that may one or more two-component sensor/regulator not be cured by conventional antibiotics. All systems, concern was voiced that the general the evidence indicates that, in contrast to use of AMPs might provoke the evolution of common antibiotics, the antibacterial action resistance that will compromise our natural of these peptides follows a multiple-hit defences against infection. In that sense, scheme with several potential targets, AMP mimics that retain antibacterial activity including the cytoplasmic membrane, cell but lack the ability to activate PhoQ or its division and cell-wall and macromolecule orthologues would represent preferable thera- synthesis (Shai, 2002; Zasloff , 2002; Brogden, peutics. 2005), making it diffi cult for the pathogen to A rich range of strategies have been put acquire resistance. forward in the att empt to alleviate one or The therapeutic potential of AMPs more of these shortcomings. These have became evident very early aft er their included the use of peptides composed of discovery. As therapeutic agents, AMPs d-amino acids (Bessalle et al., 1990; Wade et present various a priori advantages over al., 1990; Shai and Oren, 1996), combinatorial antibiotics, including molecular simplicity, libraries (Eichler and Houghten, 1995; broad-spectrum activity (Tossi et al., 2000; Blondelle et al., 1996; Blondelle and Lohner, Radzishevsky et al., 2005; Jenssen et al., 2006), 2000) and a wealth of sequence templates or potentially low levels of induced resistance minimalistic approaches (Tossi et al., 1997, (Perron et al., 2006) and concomitant broad 2000; Epand et al., 2003; Jing et al., 2003; anti-infl ammatory activities (McInturff et al., Dartois et al., 2005; Deslouches et al., 2005). 2005; Guarna et al., 2006; Easton et al., 2009). These modifi cation strategies have yielded Nevertheless, as potential drug candidates, several peptide versions with systemic HDPs can present a variety of shortcomings antibacterial activities; to name a few that need to be addressed, particularly examples, a 24-mer peptide termed WLBU2, towards development as systemic therapeutic which is based solely on cationic (arginine) agents. Namely, antimicrobial activity of and hydrophobic (valine and tryptophan) many HDPs is signifi cantly reduced in the residues (Deslouches et al., 2005), and cyclic presence of salts and divalent cations d,l-hexapeptides composed of lysine and (Goldman et al., 1997; Bals et al., 1998; Minahk tryptophan only (Dartois et al., 2005). Most and Morero, 2003) and is susceptible to pH recently, a dimeric proline-rich peptide changes (Lee et al., 1997; Rydlo et al., 2006) (A3-APO) was designed to att ack both the and plasma components (Oh et al., 1999; bacterial membrane and the Entero- Rozek et al., 2003; Radzishevsky et al., 2005). bacteriaceae-specifi c domain of the heat- HDPs can also suff er from poor pharmaco- shock protein DnaK in order to reduce kinetic properties and systemic toxicity, as toxicity (Szabo et al., 2010). Although well as high production costs (Bradshaw, A3-APO was bactericidal to Escherichia coli at 2003; Yeaman and Yount, 2003; Gordon et al., a relatively high concentration (30 μg ml–1) in 2005; Marr et al., 2006). vitro, the designer peptide was eff ective Another concern for therapeutic uses of against systemic E. coli infections in diff erent AMPs pertains to the emergence of extreme mouse models aft er multiple dosing of 10 mg resistance phenomena to the host defence kg–1, whereas the maximal tolerated dose system. In addition to their ability to develop (MTD) was >20 mg kg–1. resistance mechanisms such as effl ux pumps, Another set of strategies has opted for secreted proteases and alterations of the cell the de novo design of peptide-like constructs surface, bacteria may also sense AMPs via a that mimic HDP structure or function variety of two-component sensor/regulator (representative structures are shown in Fig. systems (e.g. PhoPQ, PmrAB). These regulate 6.1). While diff ering with respect to their specifi c gene expression leading to greater prime philosophies, these strategies share a stability of the outer membrane and adapting common rationale, which is based on the idea bacteria for survival (Gunn, 2008; Ott o, 2009). that reproduction of the critical biophysical As some AMPs have been found to activate characteristics in unnatural oligomers should 102 A. Mor

presumably be suffi cient to endow activity template, which helps to stabilize conform- and, by the same token, circumvent the ations within the macro-cycle (reviewed in limitations associated with peptide pharma- Robinson et al., 2008). Following iterative ceuticals (Latham, 1999; Adessi and Soto, rounds of peptidomimetic library synthesis 2002; Patch and Barron, 2002). Three and screening, two variants emerged representative strategies that have generated (POL7001 and POL7080). These have specifi c systemically active compounds are briefl y antipseudomonal activity (minimum inhibi- discussed below. Additional interesting tory concentrations (MICs) in the nanomolar mimic systems have been recently reviewed range), while other Gram-negative and Gram- (Epand et al., 2008a; Lai et al., 2008; Som et al., positive bacteria are largely insensitive. 2008; Van Bambeke et al., 2008; Rotem and Unlike the parent peptide, however, these Mor, 2009; Tew et al., 2010). peptidomimetics do not act by a membrane- lytic mechanism, consistent with the nanomolar range of active concentrations. It 6.2 De Novo-designed Rigid is proposed that they interfere with outer Peptidomimetics membrane biogenesis by inhibiting lipopolysaccharide (LPS) transport. The 6.2.1 Hairpin-shaped peptides pep tidomimetics have been evaluated in a mouse septicaemia model aft er inoculation The β-hairpin fold of the HDP protegrin-1 has with Pseudomonas aeruginosa and demon- been used as a starting point for designing strated substantial activity with calculated stabilized peptidomimetics with a looped median eff ective doses in the range of sequence linked to a d-proline–l-proline 0.25–0.55 mg kg–1 (Srinivas et al., 2010).

Fig. 6.1. Chemical formulae of representative building blocks used for the de novo design of AMP mimics. The α-peptide sequence (centre) can be replaced with β-peptide (top), peptoid (bottom), arylamide (upper left), phenylene ethynylene (lower left), aminoacyl-lysyl (upper right) or lysyl-aminoacyl- lysyl (lower right) subunits. Chemical Mimics with Systemic Efﬁ cacy 103

6.2.2 Peptoids alternating 1,3-phenylene diamine units connected by an isophthalic acid. The Poly-N-substituted glycines (peptoids) are resulting conformationally restrained back- distinguished from peptides by the fact that bone oligomers displayed a typical the side chains are att ached to the backbone membrane-active mode of action (reviewed amide nitrogen instead of the α-carbon in Scott et al., 2008; Tew et al., 2010). (Zuckermann et al., 1992). Recognized Oligomeric variants using hydrophobic and att ributes of peptoids include protease hydrophilic side chains and/or switching resistance (Miller et al., 1994) and prevention benzene with pyrimidine have yielded of both backbone chirality and intrachain several compounds with potent antimicrobial hydrogen bonding. Instead, peptoids can be activities in vitro (Tew et al., 2002; Liu et al., driven to form stable helical structures via a 2004; Tang et al., 2006). Non-peptide analogous periodic incorporation of bulky α-chiral side amphiphiles with a strictly hydrocarbon chains (Armand et al., 1998; Kirshenbaum et backbone, termed phenylene ethynylene al., 1998; Wu et al., 2001, 2003; Seo et al., 2010). oligomers, have been designed to mimic Certain peptoid variants exhibit broad- structural att ributes of the HDP magainin spectrum antibacterial activity and low (Tew et al., 2006). The most active compound, mammalian cytotoxicity. For example, a meta-phenylene ethynylene (mPE), composed 12-residue compound referred to as 1-pro6 of three phenyl rings, showed potent displays potent antibacterial activity (MIC antimicrobial activity against a large panel of values of 3.1 and 1.6 μM against E. coli and pathogenic bacteria with a MIC of 0.4–14 μM Bacillus subtilis) and low haemolytic activity and 50% haemotoxicity at 153 μM.

(median lethal concentration (LC50) of >110 The mode of action of these oligomers μM against human red blood cells (RBCs)) has been investigated using various bio- (Chongsiriwatana et al., 2008). The most physical techniques, including Langmuir recent designs (binaphthyl-based peptoids 2a fi lms at the air–water interface (Arnt and Tew, and 2b) have shown promising antibacterial 2002, 2003), grazing incidence X-ray potencies against a panel of Gram-positive diff raction (Ishitsuka et al., 2006), small-angle –1 pathogens (MIC50 in the μg ml range). The X-ray scatt ering, sum frequency generation peptoids were rapidly bactericidal with a vibrational spectroscopy and dye-leakage concentration-dependent profi le, and induced experiments with model membranes (Chen et resistance was slow to develop in vitro. The al., 2006; Yang et al., 2007). Phenylene authors concluded that these compounds ethynylenes were described as effi cient hole- may act by more than one mode of action, punching membrane-active antimicrobials including inhibition of cell-wall cross-linking (Yang et al., 2008). Using lipid-knockout and cell-membrane disruption (Bremner et mutant strains of E. coli, which drastically al., 2010). A non-membrane lytic mechanism out-survived the wild-type parent strain, it was also reported in a recent study using soft was concluded that bacterial membrane X-ray tomography, showing that peptoid permeation depends on the presence of high treatment suppresses the formation of a concentrations of phosphatidylethanolamine, pathogenic hyphal phenotype of yeast cells owing to its role in inducing negative intrinsic such as Candida albicans and changes both cell curvature within bacterial membranes. As and organelle morphology (Uchida et al., oft en observed with HDPs, mPE was also able 2009). It has also been reported that in vivo to tightly bind additional anionic biomolecules potency is maintained both systemically and such as DNA and LPS, as assessed by its topically, using the mouse nasal decoloniz- ability to retard DNA migration in gel ation model (Bremner et al., 2010). electrophoresis and to inhibit LPS-mediated activation of macrophages (Beckloff et al., 2007). Moreover, using selective-pressure-type 6.2.3 Arylamides experiments under conditions that led to the Aided by molecular dynamics simulations, a emergence of resistance to conventional class of facially amphiphilic arylamide antibiotics, bacterial resistance to these oligomers has been generated based on chemical mimics was not observed. 104 A. Mor

Unfortunately, few in vivo data exist in An OAK library including >150 sequences the literature to refl ect the possible composed of either α (aminoacyl-lysyl) or β therapeutic potential of these amphiphiles. (lysyl-aminoacyl-lysyl) subunits has been While, the MTD of mPE in mice was found produced and characterized (Radzishevsky et to be 10 mg kg–1 (Tew et al., 2006), suggesting al., 2007a,b, 2008; Livne et al., 2009). Analysis a potential therapeutic window, to my of the data obtained from this library enabled knowledge no effi cacy studies have been pertinent conclusions to be drawn as to the published since. In contrast, short arylamide properties required for the potency and foldamers were recently reported to exhibit selectivity of OAKs, as detailed in the systemic effi cacy in vivo (Choi et al., 2009). following paragraphs. The most potent variants reduced staphylococcal load to various extents upon the systemic administration of 1–10 mg kg–1, 6.3.2 Structure–activity relationships while MTD was found at 20 mg kg–1. Collectively, these studies seem to imply Figure 6.2 depicts a plot of the charge (Q) that rigidifying the conformation of the and hydrophobicity (H) of the OAKs tested. peptidomimetic oligomers may lead to It also highlights relationships that exist improved antibacterial activity and selectivity, between HQ values and selective sequences and consequently suggest potential adverse – sequences that combine low haemolysis eff ects of molecular fl exibility in these (LC50 ≥ 100 μM, as assessed against human respects. erythrocytes) with high antibacterial activity (MIC ≤ 3.1 μM, as assessed against representatives of Gram-negative and Gram- 6.3 Acyl-lysyl Oligomers positive bacteria, E. coli and Staphylococcus aureus, respectively). Table 6.1 shows the 6.3.1 Design biophysical properties of the most active sequences (highlighted in Fig. 6.2). Oligomers of acylated lysines (OAKs) were One of the main trends that emerged originally designed to test the necessity for a from these data was that active sequences stable fold in AMPs (Radzishevsky et al., presented a bell-shape behaviour with an 2007a,b). To promote the lack of a stable optimal window of HQ values per tested secondary structure, acyl bridges were variable (Radzishevsky et al., 2008; Livne et intercalated between cationic amino acid al., 2009). As shown in Fig. 6.2, selective residues. The lack of hydrogen-bonding sequences also displayed a specifi c (relatively opportunities in such constructs is expected narrow) window of HQ values for each to limit the formation of stable folds because activity tested. Both the relative positions and of the rotational freedom of the carbon atoms slopes are indicative of distinct HQ within short-acyl chains (the particular case requirements for selective antibacterial of long acyls will be discussed further activities. Thus, for E. coli, hydrophobicity below). On the other hand, the OAK requirements were quite strict (i.e. H = 45–50), molecule is provided with a chance to reveal while charge levels were distributed over a antimicrobial activity as it is composed of large band of values (Q = 6–9). These values cationic and hydrophobic residues; both were strikingly diff erent for S. aureus, both in properties have long been suspected to tolerating a wider range of hydrophobicity represent essential requirements for values (H = 40–50) and in requiring lower antimicrobial activity. This method therefore charge values (Q = 3–5). As detailed further enables a fold-independent systematic tool below, selective antiplasmodial activity seems for a gradual control of molecular hydro- to require a diff erent yet distinct window of phobicity (i.e. by changing acyl chain HQ values. This trend may therefore provide lengths) and charge (by changing the nature, a useful guideline in the search for new and position or number of cationic residues). improved OAK sequences. Chemical Mimics with Systemic Effi cacy 105

40 Hydrophobicity

0 1 2 3 4 5 6 7 8 9 10 11 12 Charge

Fig. 6.2. A charge (Q) and hydrophobicity (H) map of representative oligomers of an acylated lysine (OAK) library. Plotted are the HQ values of each of >150 OAK sequences. Details are found elsewhere (Radzishevsky et al., 2008; Livne et al., 2009). The distributions of the most selective OAKs (listed in Table 6.1), deﬁ ned as sequences that combine low haemolysis (median lethal concentration ≥100 μM) with high antibacterial activity (minimum inhibitory concentration ≤3.1 μM), are highlighted. The light grey and dark grey arrows represent the active window against Staphylococcus aureus and Escherichia coli, respectively.

Table 6.1. List of the most selective oligomers of acylated lysines (OAKs; highlighted in Fig. 6.2). OAK designation a OAK sequencea Qb Hb Active against Gram-positive bacteria c *C12(ω7)K-β12 C12(ω7)K-KC12Ka 350

C8–2β12 C8-KC12K-KC12Ka 445

NC12–2β12 NC12-KC12K-KC12Ka 5 38.5 Active against Gram-negative bacteria

C12K-5α8 C12K-C8K-C8K-C8K-C8K-C8Ka 6 49.7

C12K-6α8 C12K-C8K-C8K-C8K-C8K-C8K-C8Ka 750

C12K-7α8 C12K-C8K-C8K-C8K-C8K-C8K-C8K-C8Ka 8 47.5

C12K-8α8 C12K-C8K-C8K-C8K-C8K-C8K-C8K-C8K-C8Ka 9 48.5

*C12K-3β10 C12K-KC10K-KC10K-KC10Ka 7 44.9

C12K-4β10 C12K-KC10K-KC10K-KC10K-KC10Ka 9 44.7 a For N-terminal acyls: C8, octanoyl; C10, decanoyl; C12, dodecanoyl; NC12, aminododecanoyl; C12(ω7), dodecenoyl (all intercalating residues are aminoacyl derivatives); a, amide. b Q, charge; H, estimated hydrophobicity (percentage of acetonitrile eluent) as determined by reversed-phase high-

performance liquid chromatography using a C18 column. c Asterisks indicate OAK sequences that displayed systemic antibacterial efﬁ cacy using the mouse thigh-infection model. 106 A. Mor

A number of additional sequences hydrophobic media known to induce displayed potent antimicrobial properties, secondary structures. In contrast, dodecanoyl- namely when hydrophobicity exceeded the based OAKs were oft en haemolytic, highly optimal HQ window. This oft en led to self- aggregated and displayed CD profi les of assembly in solution and consequently to stable folds reminiscent of supramolecular haemotoxicity (Radzishevsky et al., 2008; organizations. Support for this notion was Livne et al., 2009). Moreover, close inspection provided by various experiments that of the data shows that self-assembly att empted to aff ect the aggregate properties, phenomena can oft en explain why sequences either by increasing backbone charge in the neighbourhood of active compounds (Radzishevsky et al., 2008; Livne et al., 2009) (herein referred to as the ‘active HQ window’) or using analogous unsaturated (less hydro- are inactive de facto. This is not to say that all phobic) acyls (Sarig et al., 2008, 2010). This led types of aggregates are necessarily inactive, to ‘normalization’ of the CD profi le and the especially considering that an aggregated simultaneous loss of haemolytic activity. compound can be inactive against one The correlations existing between microorganism but active against another. aggregated OAKs and haemolytic activity For example, aggregated OAKs that were provide strong support to the hypothesis inactive against bacteria displayed potent underlying OAK design, proposing a link antiplasmodial properties (Radzishevsky et between stable fl ds (i.e. rigid structures) al., 2007a), highlighting the importance of and haemolytic properties of AMPs aggregate interactions with bacterial cell-wall (Radzishevsky et al., 2007b). It is stipulated components. An inverse example is rep- that rigid structures are more inclined to resented by the fact that aggregated OAKs perturb the RBC membrane architecture can be made to exhibit potent antibacterial (provided they are hydrophobic enough), activity, namely by weakening the whereas OAK interactions with RBCs are hydrophobic forces at play (Sarig et al., 2008), ineffi cient in perturbing the membrane – as further discussed below. precisely because of their high backbone The biophysical properties of OAKs are fl exibility. This logic, however, does allow very much dictated by the nature of the the very same OAKs to infl ict a range of N-terminal acyl. Thus, the presence of an damage upon bacterial membranes (Epand et N-terminal acyl essentially determined the al., 2008b, 2009; Livne et al., 2009; Zaknoon et molecular hydrophobicity of the whole series, al., 2009; Sarig et al., 2010) due to the inherent regardless of the charge or type of subunits. diff erences in membrane composition, and In contrast, in OAKs that lacked an possibly even promotes OAK internalization N-terminal acyl, the hydrophobicity value to the cytoplasm in some cases (Rotem et al., readily increased with increasing backbone 2008; Held-Kuznetsov et al., 2009). Moreover, length. In this regard, potency clearly the available data support the view that the required the presence of an N-terminal acyl, intercalated acyls can suppress the formation where dodecanoic acid most oft en of stable molecular folds as long as the represented the optimal choice (Radzishevsky acyl chains are not too long (i.e. <12 carbons). et al., 2007a,b, 2008; Livne et al., 2009). According to this view, dodecanoyl residues Comparing OAKs with short versus long are able to create amphipathic rigid acyls provided additional insight into the structures by bending or collapsing on structure–activity relationships. For instance, themselves, unlike short acyls. In that case antibacterial activity could be highly dis- only, the dodecanoyl-based OAKs gain the sociated from both stable conformation and opportunity to stabilize in aqueous solutions haemolytic activity (Radzishevsky et al., through self-assembly, whereas their 2007b, 2008). Thus, butyl- and octyl-based haemolytic activity refl ects the ability to OAKs were consistently devoid of haemolytic disassemble and reorganize within the RBC activity and displayed circular dichroism membrane, much like amphipathic con ven- (CD) profi les that refl ected random structures, tional AMPs (Feder et al., 2000; Avrahami et despite interaction with liposomes and other al., 2001; Radzishevsky et al., 2005). Chemical Mimics with Systemic Effi cacy 107

6.3.3 Antibacterial properties of action. Thus, the octamer derivative

C12K-7α8 exerts bactericidal activity through As illustrated in Fig. 6.2, the OAK library membrane disruption, as evidenced by its includes a number of OAK sequences that ability to rapidly (within minutes) induce potently aff ect bacterial viability in vitro. membrane depolarization and leakage of Thus, sequences such as the octamer cytoplasmic solutes in correlation with time– derivative C12K-7α8 (Radzishevsky et al., kill curves (Radzishevsky et al., 2007b). 2007b) and its hexamer analogue C12K-5α8 However, when compared against the very (Rotem et al., 2008) displayed signifi cant same bacteria, the hexameric version growth inhibitory activity against a large C12K-5α8 was unable to disrupt the plasma panel of bacterial species, but were most membrane and the bactericidal rates were potent against the Gram-negative species. In much slower (Rotem et al., 2008). It was contrast, NC12-2β12 (Zaknoon et al., 2009) and suggested that C12K-5α8 exerts its antibac- C12(ω7)K-β12 (Sarig et al., 2010) were rather terial eff ect aft er translocating into the active against Gram-positive species, while cytoplasm, where the OAK inhibits bio-

C16(ω7)K-β12 (Sarig et al., 2008 and synthesis of macromolecules through a direct unpublished data) and C12K-3β10 (Livne et (probably non-specifi c) interaction with al., 2009) displayed essentially non-selective DNA. With the aim of understanding the broad-spectrum cytotoxicity. Another mechanisms involved, the interaction of this non-selective broad-spectrum OAK worth pair of analogues with model phospholipid mentioning, C12K-2β12, was recently membranes was investigated by a number of evaluated for its antibacterial eff ect on complementary methodologies, including cultures of Helicobacter pylori, a gastric surface plasmon resonance, isothermal pathogen that has developed resistance to titration calorimetry, diff erential scanning virtually all current antibiotics. The OAK calorimetry and nuclear magnetic resonance exhibited MIC and minimum bactericidal (Epand et al., 2008b; Rotem et al., 2008). The concentrations of 6–26 μM and 15–90 μM, collective data indicated that only C12K-7α8 respectively, across six diff erent H. pylori exhibited a high capacity to induce clustering strains (G27, 26695, J99, 7.13, SS1 and of anionic lipids (Epand et al., 2008b). This HPAG1). H. pylori was completely killed aft er clustering eff ect is believed to lead to the 6–8 h of incubation in liquid cultures lateral segregation of domains rich in anionic containing two times the minimum and zwitt erionic lipids, producing phase- bactericidal concentration of C12K-2β12. The boundary defects that ultimately breach the OAK demonstrated low haemolytic activity permeability barrier of the cell membrane. and high stability, with effi cacy maintained The data also suggest that the hexamer’s aft er incubation at extreme temperatures aptitude to undergoing internalization is (4–95°C) and low pH, although reduced linked to its lower binding affi nities for cell- killing kinetics were observed at pH 4.5 wall components (e.g. LPS) and cytoplasmic (Makobongo et al., 2009). Thus, while these membrane phospholipids. results suggest that OAKs may be a valuable Moreover, comparing the mechanism of resource for the treatment of various bacterial action of C16K-β12 with that of C16(ω7)K-β12 or infections, they also evidenced a broad range C12(ω7)K-β12 has further accentuated the of time periods required to aff ect bacterial notion that tiny diff erences can lead to viability. These signifi cant diff erences, drastic mechanistic changes. As mentioned observed even between highly analogous above, various studies of AMPs (Avrahami et sequences, suggest the occurrence of al., 2001; Rotem et al., 2006) and OAKs mechanistic diff erences. (Radzishevsky et al., 2008) have evidenced Various mechanistic studies performed strong relationships between hydrophobicity, with the most active sequences have aggregation and haemotoxicity (Javadpour substantiated the idea that OAKs can indeed and Barkley, 1997; Radzishevsky et al., 2005). exert a number of distinct antibacterial modes Thus, excess hydrophobicity and consequent 108 A. Mor

self-assembly in aqueous media appear to be Cell death was apparently caused by the responsible for poor antibacterial per- OAK’s ability to interfere with DNA formance and potential toxicity, as refl ected functions in a similar manner to that of by haemolysis. This issue was initially C12K-7α8 (Rotem et al., 2008), as evidenced by addressed by att empting to manipulate a both the direct interaction with DNA under highly aggregated short sequence (C16K-β12) live conditions and the resulting inhibition of that exhibited both antibacterial and biosynthesis. haemolytic properties. Substitution of the There is a growing list of conventional N-terminal acyl with its unsaturated AMPs such as PR-39 (Boman et al., 1993), counterpart (C16(ω7)K-β12) signifi cantly altered tPMP-1 and HNP-1 (Xiong et al., 1999), the OAK’s supramolecular organization, even buforin II (Park et al., 2000) and pyrrhocoricin though the critical aggregation concentration (Kragol et al., 2001) that are proposed to exert (CAC) did not change (Sarig et al., 2008). antibacterial activity through interactions Thus, although antibacterial performance did with intracellular targets (Nicolas, 2009). improve, haemolytic activity remained high Combined with the OAK fi ndings, the enough to compromise its potential use in collective data show that small diff erences systemic therapy. Extending this study, the (such as a single charge, backbone length or N-terminal acyl was replaced with the less even a single double bond) are required for hydrophobic shorter acyl, dodecanoyl (Sarig an antibacterial compound to switch from et al., 2010). As a result, the C12(ω7)K-β12 CAC one mechanism to another. Further studies became signifi cantly higher than that of are, however, needed to validate this

C12K-β12 (CACs of approximately 50 and 10 hypothesis, including investigation into the μM, respectively). Reversed-phase high- mechanism of action of a compound against perform ance liquid chromatography analysis a large panel of bacterial species in order to of these compounds confi rmed that the acyl determine the possible generalization of this substitution was accompanied by reduced phenomenon. The vast majority of mech- hydrophobicity, as refl ected by the elution anistic insights published in the literature are time (50% and 54% acetonitrile eluent, based on investigations that have used a respectively). As anticipated, disassembly single strain of a single bacterial species, was responsible for reducing toxicity to which prohibits conclusions on whether the mammalian cells as assessed with erythro- compound always uses a particular cytes and primary fi broblasts. Yet C12(ω7)K-β12 mechanism, or whether it uses distinct displayed potent growth inhibitory activity mechanisms on distinct strains or species. It against Gram-positive bacteria (MIC 2.5–5 μg is also possible that an antibacterial com- ml–1), while Gram-negative bacteria were pound can use multiple or combined generally less susceptible. Furthermore, the mechanisms. This is oft en touted in the introduction of the double bond also literature, but has yet to be convincingly drastically aff ected the antibacterial mech- demonstrated experimentally. Stretching this anism of action. Thus, unlike C12K-β12, which idea further, the data support the notion that displayed classically rapid membrane simultaneous treatment of infections with a disruptive bactericidal activity, the cocktail of compounds acting by distinct unsaturated analogue C12(ω7)K-β12 exhibited mechanisms may represent a more effi cient a strict bacteriostatic mode of action (Sarig et treatment approach. Nature seems to use this al., 2010). In addition, recent unpublished strategy via the concomitant production and data indicate that the more hydrophobic delivery of multiple closely related HDPs unsaturated analogue C16(ω7)K-β12 reverted (Levy et al., 1994; Mor et al., 1994). back to the bactericidal mode of action Various preliminary in vivo investi- (causing 99% death within 2 h) as assessed gations performed so far with four OAK against multiple strains of diff erent bacterial sequences have revealed a fair ability of species. Interestingly, however, C16(ω7)K-β12 OAK-based compounds to aff ect bacterial did not disrupt the bacterial membrane(s). viability in mice and moreover highlighted Chemical Mimics with Systemic Effi cacy 109

some still ill-understood sequence depen- pharmacodynamics? We will att empt to dencies. Thus, although exerting distinct address these questions in future studies. bactericidal mechanisms of action in vitro, the analogues C12K-7α8 and C12K-5α8 have shown remarkable effi cacy in peritonitis– 6.3.4 Antiparasitic properties sepsis models, where both OAKs have increased the survival of infected mice by up A variety of in vitro and in vivo antiparasite to 100% aft er single- or multiple-dose investigations of AMPs have suggested that intraperitoneal administration (Radzishevsky these notorious antibacterial compounds et al., 2007b; Rotem et al., 2008). In another may represent a powerful tool for developing unpublished study, C12K-7α8 was examined new drugs to fi ght parasites in the host. in a P. aeruginosa pneumonia infection model Human parasites are responsible for millions when administered directly to the lungs by of deaths around the world every year. Of inhalation. At 25 μg per mouse, the OAK particular concern is the causative agent of substantially reduced the lung bacterial human malaria, Plasmodium falciparum, of population by up to 2 logs (similarly to which a large number of strains are drug tobramycin) as compared to inoculated resistant (Bruce-Chwatt , 1982). In addition to vehicle controls. MTD studies indicated that known resistance to the classical chloroquine- prolonged exposure to high dose levels of based drugs, there have been reports of fi eld

C12K-7α8 (up to 100 μg in the respiratory strains of P. falciparum with resistance to tract) did not cause overt toxicity in the mice. artemisinins, more recently introduced

This study indicates that C12K-7α8 has antimalarial drugs (Krishna et al., 2006). There potential for use as an antimicrobial agent in is a clear need for new antimalarial agents. the treatment of severe lung infections HDPs such as magainins and cecropins caused by Gram-negative pathogens. were reported to display antiparasitic However, when tested with the thigh- activities over 20 years ago (Boman et al., infection model, both C12K-7α8 and C12K-5α8 1989; Gwadz et al., 1989). Since then, failed to signifi cantly reduce bacterial load, antiparasitic activities have been reported for suggesting a poor potential for systemic numerous additional AMPs (Rivas et al., effi cacy, probably due to poor tissue- 2008). In addition to the naturally occurring penetration capacity. In contrast, C12K-3β10 antiparasite peptides and their synthetic and C12(ω7)K-β12, which exerted in vitro derivatives, recent studies suggest that potent bactericidal and bacteriostatic modes of antiparasitic properties can be generated action, respectively, were both effi cient in the from de novo-designed AMP mimics (Mor, thigh-infection model (Livne et al., 2009; 2009). Various OAK sequences have been Sarig et al., 2010). shown to inhibit the growth of diff erent Typically, OAK MTDs in mice upon plasmodial strains in culture (half the intravenous and intraperitoneal administra- maximal inhibitory concentration (IC50) of tions were approximately 5 and 10 mg kg–1, 0.08–0.14 μM). Certain sequences displayed respectively. The subcutaneous route of highly selective antiparasitic activity, where administration is much bett er tolerated (at the ratio of LC50 (haemolysis) to IC50 (parasite least up to 20 mg kg–1), although effi cacy growth inhibition) was >10,000 for the most doses are >tenfold and sometimes >100-fold selective OAK, 3α12 (Radzishevsky et al., lower (unpublished data). 2007a). The available data suggest that the Collectively, the current data raise OAK does not exert its antiplasmodial action interesting questions pertaining to the by lysis of infected RBCs, as is the case with relationship between the OAK sequence and various HDPs. Rather, it seems that it is able in vivo effi cacy. Namely, are β-OAKs more to cross the erythrocyte plasma membrane suitable for systemic development than and target internal components such as the α-OAKs? If so, why? Is it a function mitochondrion or nucleic acids of the of small-molecule pharmacokinetics? Or parasite. As described above, OAKs have 110 A. Mor

shown these abilities in other cell types unclear. None the less, these studies suggest (Rotem et al., 2008; Held-Kuznetsov et al., that effi cient antimalarial therapeutic drugs 2009). can be engineered based on the physico- The most recent att empts to generate chemical att ributes imbedded in the mol- improved antiplasmodial OAKs have re- ecular formulae of AMPs. vealed additional octanoyl-based OAKs that inhibit parasite growth at sub-micromolar concentrations (mean IC50 0.3 ± 0.1 μM) 6.4 Concluding Remarks and (unpublished data). Both the ring and Perspectives trophozoite stages of the parasite developmental cycle were sensitive to the most potent The fact that most HDPs and their synthetic OAK, which moreover exhibited no mimics essentially act by non-specifi c haemolytic activity at least up to 150 μM mechanisms has long created serious doubts (<0.4% haemolysis). Interestingly, the most as to their ability to exert suffi cient selectivity potent non-haemolytic OAKs all localized to be considered ideal candidates for drug near the S. aureus HQ window (Fig. 6.2), but development, particularly for systemic contained distinct sequences (i.e. did not therapies. In that respect, an increasing display antibacterial activity). Preliminary in number of recent studies have provided a vivo data from acute toxicity, pharmacokinetic variety of counterarguments that signifi cantly and effi cacy studies provide evidence for the contribute to dissipating this doubt. potential of OAK sequences generated during Through distinct approaches for design- this study to be considered suitable ing chemical mimics of HDPs, at least three candidates for development as systemic major achievements have been realized: antimalarials. Namely, upon daily intra- simplifying the pharmacophore composition, peritoneal administration (4 × ≥25 mg kg–1 increasing its robustness and miniaturizing day–1) to Plasmodium vinckei-infected mice, its size. These achievements should signifi - the OAK displayed high blood concentrations cantly enhance the effi ciency of future (approximately 100 μM) that were sustained structure–activity relationship studies in for at least several hours. It also demonstrated bett er defi ning the active principles, which the ability to eliminate parasitaemia, albeit will in turn drive the discovery of new and not without chronic toxicity. Nevertheless, the improved compounds. encouraging results obtained in this study Beyond their capacity to aff ect microbial regarding the selectivity and pharmacokinetic viability directly, various AMPs have been properties of some compounds justify further proposed to signifi cantly modulate host investigation, with the aim of delivering immune responses. Another unmet challenge antimalarial analogues with high safety. to be addressed in the future is therefore the Although antiparasitic properties have eff ect of these chemical mimics on the host not yet been investigated as thoroughly as immune system and its contribution to the antibacterial activities, an increasing amount observed in vivo effi cacies. of experimental evidence supports the view that the antiparasitic activity of AMPs also emanates from interactions with multiple targets. Remarkably, at least a few peptides Acknowledgement exhibit in vitro high potency and selectivity factors of several orders of magnitude, and This research was supported by the Israel appear to be endowed with the formidable Science Foundation (grant 283/08). ability to cross a number of membrane systems before reaching their target(s) within the intracellular parasite. While diff erences References in membrane composition are likely to contribute to this selectivity, the molecular Adessi, C. and Soto, C. (2002) Converting a peptide basis for these observations remains largely into a drug: strategies to improve stability and Chemical Mimics with Systemic Effi cacy 111

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Richard M. Epand* and Raquel F. Epand

Abstract

Gram-negative bacteria are more resistant to many antimicrobial agents than Gram-positive bacteria. This is because drugs must pass through an additional barrier of the outer membrane in order to access the cytoplasmic membrane or the interior of the cell. In spite of this fact, the presence of the outer membrane also provides an alternative target for the action of antimicrobial agents. In addition, the cytoplasmic membranes of Gram-negative bacteria are generally more enriched with the phospholipid phosphatidylethanolamine than those of Gram-positive bacteria. In this chapter, we outline novel strategies for using the interaction of antimicrobial agents with components of the membranes of Gram- negative bacteria in order to design agents that have a higher toxicity for these bacteria.

7.1 Differences in Chemical properties and composition. It is quite Composition and Molecular permeable to small molecules with a Organization of Gram-positive and molecular mass of ≤700 Da. This is because of Gram-negative Bacteria the presence of β-barrel proteins called porins, in which the centre of the β-barrel is Most bacteria are routinely classifi ed an aqueous pore extending through the according to the criterion of whether they are outer membrane. A simplifi ed diagram stained by Gram stain. This phenomenological depicting the diff erences in outer membrane criterion divides bacteria into two general organization between Gram-positive and groups – Gram-positive and Gram-negative – Gram-negative bacteria is shown in Fig. 7.1. that also diff er in the nature of their cell Peptide molecules covering part of the membrane(s). Gram-negative bacteria are surface of the membrane are shown as red characterized by having two membranes: an ovals. inner cytoplasmic membrane surrounding When a peptide tries to gain entry into a the cell and an outer membrane (Fig. 7.1A). Gram-negative bacterium membrane it fi rst The outer membrane is unique in its traverses the sugar chains in the O-antigen

* Corresponding author.

Fig. 7.1. Schematic diagram of the architecture of the membranes of Gram-negative and Gram-positive bacteria (not to scale). (A) Gram-negative bacteria (from left to right): O-antigen depicted as hexagons on the outer monolayer of the outer membrane; porins traversing the lipopolysaccharide (LPS) layer are depicted as cylinders; the LPS layer is followed by lipoproteins; the peptidoglycan layer is shown as dots; ovals represent peptides; the inner membrane is presented with transmembrane proteins. The complete LPS structure is shown on top and the peptidoglycan structure to the right. G, N-acetylglucosamine; M, N-acetylmuramic acid; DAP, diaminopipelic acid. (B) Gram-positive bacteria (from left to right): teichoic acid (TA) and lipoteichoic acid (LTA) as long, thin structures reaching through the peptidoglycan layer, which is depicted as dots; peptides are ovals; the membrane is shown with transmembrane proteins. The LTA structure is shown on top and the peptidoglycan structure to the right. 118 R.M. Epand and R.F. Epand

layer (Fig. 7.1A). If it has a molecular mass components that cationic antimicrobial larger than approximately 700 Da then it peptides (AMPs) are exposed to on their crosses the lipopolysaccharide (LPS) layer, journey towards the cytoplasmic membrane, which is about 8 nm thick. If it does not bind and the number of other components they completely to the phosphate moieties of the come in contact with, they emerge as truly lipid A portion in the LPS molecule then it amazing agents. will continue its journey through a number In addition to the diff erences described of lipoproteins and a peptidoglycan layer. In above, bacterial species vary greatly in the Gram-negative bacteria, the peptidoglycan phospholipid composition of their mem- layer comprises only 10% of the dry weight branes. In general, the membranes of Gram- of the cell and it is composed of a glycan negative bacteria have a far higher backbone of alternating repeating sugar phosphatidylethanolamine (PE) content than units of N-acetylglucosamine (G) and those of Gram-positive bacteria, although N-acetylmuramic acid (M). These are cross- there are some exceptions. linked directly by four amino acids (two l- and two d-amino acids) on each layer, thus forming a rigid mesh that allows facile passage of peptides. The peptide molecules 7.2 Role of Bacterial Membrane Lipid accumulate in the periplasmic space, Composition in Antimicrobial achieving high concentrations that can be Sensitivity many fold the bulk concentration of peptide. From there they are att racted electrostatically Some antimicrobial agents target specifi c to the outer leafl et of the inner membrane. At lipid components of bacterial membranes. each step the peptide molecules are exposed An example of this is the AMP nisin, which to a number of proteins that participate in binds specifi cally to lipid II (Breukink et al., specifi c biosynthetic and other functions of 1999). Duramycin and cinnamycin are two the bacteria. other AMPs of the same lantibiotic class as In trying to gain entry into a Gram- nisin that specifi cally target PE (Clejan et al., positive bacterium, a peptide encounters 1989). In addition to drugs that target specifi c teichoic acid covalently bound to a sugar lipids, less specifi c interactions of anti- backbone and lipoteichoic acid covalently microbials with membrane lipids, such as bound to the membrane (Fig. 7.1B). It gains electrostatic or hydrogen-bonding inter- direct access to the peptidoglycan layer, actions, can also infl uence the potency and which is 50% of the dry weight of the cell mechanism of action of antimicrobial agents. and contains a number of associated proteins. This can be particularly important because The peptidoglycan layer in Gram-positive the lipid composition of bacterial membranes bacteria diff ers from the one in Gram- varies widely, resulting in certain bacterial negative bacteria in the linkage between the species being more susceptible to some amino acids. This is not direct, but is rather antimicrobial agents than others. For done through an amino acid bridge, which example, the selective toxicity of two varies within species as a result of adaptation. isomeric α/β-peptides is dependent on the This makes the peptidoglycan layer wider lipid phase preference of the lipids in the and more accessible to the peptide, which bacterial membrane (Epand et al., 2005). The can easily traverse it to reach the outer leafl et toxicity of a cationic steroid compound has of the cytoplasmic membrane, in spite of the been shown to be dependent on the PE fact that this layer is 20–40 nm in length. content of the membrane (Epand et al., 2007). Gram-positive bacteria have litt le or no Additionally, the toxicity of certain oligo- periplasm; consequently, the cationic pep- acyl-lysines is most potent with bacteria that tides accumulate directly on the anionic contain both anionic and either zwitt erionic lipids by electrostatic att raction, reaching or uncharged lipid in their membrane, so threshold concentrations for membrane that these agents can promote the clustering disruption. Given the number of anionic of anionic lipid (Epand, R.M. et al., 2008). The Analysis of Membrane-targeting AMPs 119

Table 7.1. Phosphatidylethanolamine content and CSA-8 minimum inhibitory concentration for several species of bacteria. Minimum inhibitory Phosphatidylethanolamine Bacteria concentration (μg ml–1) content (%) Gram-negative bacteria Proteus mirabilis 500 80 Escherichia coli 36 80 Pseudomonas aeruginosa 20 60 Caulobacter crescentus 5 ~0 Gram-positive bacteria Bacillus polymyxa 35 60 Bacillus anthracis 20 43 Bacillus cereus 50 43 Bacillus subtilis 0.5 12 Staphylococcus aureus 2 ~0 Streptococcus pyogenes 0.5 ~0

variation in PE content for several species of 7.3 Antimicrobial Agents that Target bacteria is shown in Table 7.1. the Outer Membrane of Gram- Table 7.1 also shows some exceptions to negative Bacteria the general rule. For example, the Gram- negative bacterium Caulobacter crescentus has In addition to damage to the cytoplasmic a low PE content while three related Gram- membrane that may lead to toxicity, one must positive bacteria, Bacillus polymyxa, Bacillus also consider a role for the outer membrane anthracis and Bacillus cereus, have a higher of Gram-negative bacteria. There are several content of PE. A variable PE content can also ways in which the outer membrane can aff ect be found in the genus Clostridium. bacterial viability. The outer membrane and The relationship of PE content to the cell wall provide structural rigidity to protect antimicrobial action of the cationic sterol the cell against damage caused, for example, compound CSA-8 illustrates the importance by osmotic stress. Several classical antibiotics of membrane phospholipid content to the act by aff ecting the synthesis of cell wall or potency of this antimicrobial agent (Epand et outer membrane components. al., 2007). This conclusion was further Another role of the outer membrane can confi rmed using a mutant form of Escherichia be to act as a barrier for the passage of coli that was unable to synthesize PE (Epand antimicrobial agents to the plasma mem- et al., 2007). This study demonstrates that the brane. The interaction of negatively charged lipid composition of the bacterial membrane LPS of the outer membrane with cationic can be more important in determining antimicrobial agents can prevent the toxicity sensitivity to an antimicrobial agent than the of these agents by inhibiting access to the cell presence or absence of an outer membrane. membrane. An interesting example of this is This is an example of the more common the blockage of penetration of the small situation in which Gram-positive bacteria are cationic peptides, the temporins (Mangoni et more sensitive to the action of an al., 2008). This study showed the dependence antimicrobial agent than Gram-negative of the barrier function of the outer membrane bacteria. In the next paragraphs, we describe on the length of the polysaccharide chain of other cases in which antimicrobial agents can LPS. In addition, synergistic action between be more toxic to bacteria with a high PE pairs of temporin molecules was demon- content. strated. 120 R.M. Epand and R.F. Epand

Recently, a novel mechanism of microbial that form a homogeneous mixture, giving toxicity has been proposed in which rise to a single-component phase transition. antimicrobial agents act by blocking the This places a limit on the lipids that can be passage of polar molecules across the outer used in that they must be miscible and have membrane. In this manner, the agent can a gel to liquid crystalline-phase transition inhibit growth of the organism without temperature >0°C and below the temperature accessing or damaging the plasma membrane. of denaturation or membrane dissociation of Elucidation of this mechanism was facilitated the antimicrobial agent. For the purpose of by the availability of a strain of E. coli, ML-35p, testing for the ability of a compound to specially constructed to simultaneously cluster anionic lipid in the presence of monitor the passage of chromogenic sub- zwitt erionic lipids, we have frequently used strates across the inner and outer membranes mixtures of tetraoleoyl-cardiolipin (TOCL) (Lehrer et al., 1988). An example of an and 1-palmitoyl-2-oleoyl-PE (POPE). PE and antimicrobial agent that blocks the fl ux of cardiolipin (CL) are both major lipid small molecules across the outer membrane of components of bacterial membranes and E. coli was a fl exible sequence-random both have headgroups with hydrogen- polymer containing cationic and lipophilic bonding capabilities. PE is zwitt erionic, while subunits, which acts as a functional mimic of CL is anionic. Cationic antimicrobial agents host-defence peptides (Epand, R.F. et al., would be expected to bind preferentially to 2008). At low concentrations the polymer the anionic CL component of this mixture. If permeabilizes the outer and inner membranes; the preference for interacting with CL over at higher polymer concentrations, however, PE is great enough, then the cationic agent permeabilization of the outer membrane is will promote phase separation between the progressively diminished, while the inner two lipids. In some, but not all, cases the membrane remains unaff ected. A similar CL-rich domain can be observed at a lower mechanism has also been proposed to explain temperature since TOCL has a phase the mechanism of the action of a miniature transition below 0°C. However, there are oligo-acyl-lysine against Gram-negative cases in which the transition of the domain bacteria (Epand et al., 2009a). enriched in CL is not observed because it is shift ed or broadened as a result of binding the cationic agent. POPE in pure form has a 7.4 Antimicrobial Agents that transition at 25°C and, since a less cationic Promote Clustering of Anionic Lipids antimicrobial agent will bind to the domain enriched in this lipid, the transition 7.4.1 Evidence for lipid clustering temperature of the membrane domain enriched in POPE will be higher than that of The ability of certain antimicrobial agents to the initial pure lipid mixture and closer to cluster anionic lipids has been demonstrated that of pure POPE. In general, the transition by several methods using mixtures of lipids should be reversible on heating and cooling. corresponding to those found in bacterial However, in some cases the cationic agents membranes. The results of these analyses interact more rapidly with the lipid in the allow the prediction of which bacterial species liquid crystalline state and therefore larger will be most susceptible to the action of a temperature separations of high and low particular antimicrobial agent based on the melting components are observed in cooling lipid composition of the bacterial membrane. scans compared with heating scans. We have used diff erential scanning The evidence from DSC can be further calorimetry (DSC) to assess anionic lipid tested using spectroscopic methods. Nuclear clustering (Epand, 2007). The advantages of magnetic resonance (NMR) has provided this method are that it has broad applicability evidence for lateral phase separation. A useful and does not require the introduction of NMR method for membrane samples is magic probes that can perturb the system. The basis angle spinning NMR (MAS/NMR). This of the method is that one chooses two lipids version of solid-state NMR allows one to Analysis of Membrane-targeting AMPs 121

acquire well-resolved spectra using high one lipid from another in a mixture using molecular weight membrane samples. A Fourier transform infrared spectroscopy convenient isotope to use for the study of (Arouri et al., 2009). Deuteration is performed membranes is 31P. There are few phosphates, to separate the absorption frequencies of the usually only one, in most phospholipids. The deuterated and protonated components. The 31 paramagnetic isotope of phosphorous, P, has CH2 stretching vibrations are sensitive to the a natural abundance of almost 100% and 31P conformation of the lipid acyl chains, and can has a spin quantum number of ½, resulting in be used to follow the changes in the trans to it not being broadened by nuclear-quadrupole gauche ratio. For the protonated component, coupling. There have been two applications of the frequencies of the symmetric and 31 P MAS/NMR to test the formation of anionic antisymmetric CH2 stretching vibrations are lipid clusters with antimicrobial agents at approximately 2850 cm–1 and 2929 cm–1, (Epand, R.M. et al., 2008; Epand et al., 2009b). respectively. With this method, the Even in lipid mixtures that appear to mix antimicrobial cationic peptide RRWWRF homogeneously, 31P MAS/NMR still allows has been shown to cause segregation of the separate identifi cation of each component anionic and zwitt erionic lipids only in the gel of the mixture if they have suffi ciently phase with dimyristoyl-phosphatidylcholine diff erent chemical shift s. Although the (DMPC)/dipalmitoyl-phosphatidylglycerol chemical shift of each lipid component will be (DPPG) mixtures, while with dipalmitoyl- towards each other as a result of the presence phosphatidylethanolamine (DPEE)/DPPG – a of the other lipid in the environment, the two lipid mixture more representative of bacterial peaks are still resolvable (Epand, R.M. et al., membranes – phase separation was observed 2008; Epand et al., 2009b). If clustering of in both liquid and solid phases (Arouri et al., anionic lipid occurs, the isotropic chemical 2009). shift of the zwitt erionic component will be All of the above methods have their more towards that of the pure zwitt erionic advantages and limitations. However, they component. The line width of each peak is a are all indirect with regard to specifying the second independent parameter that can also morphological properties of the samples. be used to test for demixing. The line width of Some imaging methods have also been the anionic component increases as a result of applied that give information about the size slower motion aft er binding the cationic anti- and properties of the domains. A recent study microbial agent, while that of the zwitt erionic using atomic force microscopy in combination component changes from its value in the lipid with polarized fl uorescence microscopy mixture to that found for the pure zwitt erionic showed the formation of domains in model component. This analysis has also been membranes using a small cationic AMP, performed as a function of tem perature to PFWRIRIRR-amide (Oreopoulos et al., 2010). show the generality of the result when both This study showed the time evolution of the components are in the liquid crystalline formation of these domains. In addition, phase. freeze-fracture electron microscopy has Another NMR approach makes use of provided evidence for the formation of 2H-NMR to measure order parameters of domain structures with this same cationic deuterated lipids (Jean-Francois et al., 2008). AMP (Epand, R.M. et al., 2010). There is thus Anionic and zwitt erionic lipid components direct evidence from imaging studies, for the can be separately studied by deuteration of peptide PFWRIRIRR-amide, that membrane one of the components of the pair. The results domains are formed. suggest that the zwitt erionic component experiences two diff erent environments that are in slow exchange. One of the components resembles that of the pure zwitt erionic 7.4.2 Bacterial species specifi city of component. These results are also in accord toxicity with the clustering of anionic lipid to produce a domain enriched in zwitt erionic lipid. The ability of certain antimicrobial agents to Deuteration can also be used to distinguish cluster anionic lipids is of particular interest 122 R.M. Epand and R.F. Epand

because it explains the selective toxicity of principal one being sphingosine, bacteria can these agents towards certain bacterial have large amounts of cationic lipids. The species. If an antimicrobial agent has greater separation of cationic and anionic lipids by affi nity for one lipid species over another, cationic antimicrobial agents would not be this is suffi cient to promote segregation of very effi cient because the cationic lipid lipid components. This can happen even would prevent the cationic peptide from with a mixture of two lipids with diff erent binding to the membrane. A cationic lipid anionic headgroups, as we have shown with that is frequently found in bacterial cationic amphipathic helical peptides that membranes is lysyl-PG. It has been shown can segregate CL from phosphatidylglycerol that the presence of cationic lipids is a factor (PG) (Epand, R.F. et al., 2010); however, this in increasing the resistance of bacteria to phenomenon is not associated with the cationic antimicrobial agents (Peschel et al., specifi city of toxicity for diff erent bacterial 2001; Camargo et al., 2008). However, at least species. In contrast, when an anionic lipid is in the case of Staphylococcus aureus, most of clustered away from lipids that are this lipid is found on the cytoplasmic surface zwitt erionic or have no overall charge, the of the plasma membrane. Resistance to result is a bacteriostatic action. The reason antimicrobial agents occurs in those strains why these two diff erent kinds of lipid of S. aureus in which the lipid is translocated clustering have diff erent consequences for to the cell exterior by a mechanism facilitated bacterial toxicity is not known, but may by a membrane protein (Ernst et al., 2009; refl ect diff erences in the size of the domains Mishra et al., 2009). Although the membrane- that are formed or the nature of the phase sidedness of lysyl-PG in S. aureus has been boundary between domains. determined, in general there is litt le infor- The sequestering of anionic lipids away mation about the sidedness of membrane from ones with no overall charge will occur lipids in bacteria. only in bacterial species that contain We will now summarize the ratio of the signifi cant amounts of zwitt erionic or minimal inhibitory concentration (MIC) uncharged lipids. All bacterial species contain against S. aureus and E. coli. These two large fractions of anionic lipids, generally in bacterial species represent a species with a the form of PG and CL. However, their low content of neutral and zwitt erionic content of zwitt erionic or uncharged lipids lipids (S. aureus) and another with a high varies widely, from close to 0% to about 85%. content of the zwitt erionic lipid PE (E. coli; The most common zwitt erionic lipid is PE. In see Table 7.1). We have chosen these two addition, some bacteria have species of species as representative because they are aminoacyl-PG, most of which are zwitt erionic, commonly used and there is more as well as zwitt erionic lysyl-CL. Uncharged information about MICs with these bacteria. glycosyl-diacylglycerols may also be present. We recognize that there is a potential However, few bacterial species have a large complication with S. aureus because some amount of this lipid as a free polar lipid strains contain lysine-PG, but since this lipid component of the plasma membrane, an is generally on the cytoplasmic side of the exception being Streptococcus pyogenes in membrane, we assume it will not greatly which some of the membrane lipids are free aff ect the measured values of the MIC. Thus, glycosyl-diglycerides (Shaw, 1970; Rosch et S. aureus represents a bacteria that cannot al., 2007). It should also be pointed out that segregate anionic from zwitt erionic or glycosyl-diglycerides are also components of charged lipids, while E. coli can segregate lipoteichoic acid (LTA), a major component of these two types of lipids. An antimicrobial the cell wall of Gram-positive bacteria. agent that acts largely by the lipid clustering However, this glycosyl-diglyceride is not mechanism will thus be more toxic to E. coli usually free as a phospholipid component of than to S. aureus and hence have a high ratio the cell membrane, but rather is a covalently of S. aureus MIC to E. coli MIC. These two linked moiety of the cell wall. bacteria also diff er in that E. coli is a Gram- Unlike mammalian membranes, which negative bacterium with an outer membrane, have very low amounts of cationic lipids, the while S. aureus is a Gram-positive bacterium. Analysis of Membrane-targeting AMPs 123

Another factor lowering the ratio of S. aureus S. aureus/MIC E. coli is >1 for all of the other MIC to E. coli MIC may be a result of the MSI peptides. outer membrane of E. coli not allowing KR-12, GF-17 (Table 4.2, Chapter 4) and passage of the antimicrobial agent. GF-17 D3 are fragments of the human The MSI peptides are examples of cathelicidin peptide LL-37. KR-12 is the cationic amphipathic helical AMPs (Table shortest fragment of LL-37 possessing 7.2). They are representative of a large group antimicrobial activity (Wang, 2008). It is of AMPs. MSI-103 and MSI-469 have capable of clustering anionic lipids and identical sequences, but MSI-469 also has an shows specifi city for E. coli relative to S. N-terminal octyl group. The results indicate aureus (Table 7.2). The longer LL-37 fragment, that the octyl group does not have a major GF-17, does not have this specifi city in eff ect on antimicrobial activity. MSI-843 and microbial toxicity. This is because GF-17 is MSI-1254 are also N-octyl peptides, the more potent against S. aureus and not diff erence between them being that MSI-843 because it is less potent against E. coli. The has ornithine as its cationic residue, while lack of selective toxicity suggests that GF-17 MSI-1254 has diaminobutyric acid. We have is not acting principally by a charge cluster shown that MSI-1254 does not cross the outer mechanism. This is supported by our membrane of E. coli (Epand, R.F. et al., 2010). fi ndings that GF-17, but not KR-12, can This explains the relatively high MIC for this promote the release of entrapped dye from bacteria among the MSI peptides and also liposomes (Epand et al., 2009b). Interestingly, the low ratio of 1 for the MICs against the when three d-amino acid residues are two diff erent bacteria. The ratio of MIC introduced at diff erent locations in GF-17,

Table 7.2. Speciﬁ city of antimicrobial agents and charge. Ratio of MICb (μM) for Staphylococcus Charge/ Escherichia aureus MIC to E. Agent Chargea residue coli coli MIC Reference MSI-78 10 0.45 2.5 2 Epand, R.F. et al. (2010) MSI-103 7 0.33 0.8 15.6 Epand, R.F. et al. (2010) MSI-469 6 0.29 1.5 4 Epand, R.F. et al. (2010) MSI-843 6 0.6 2 4 Epand, R.F. et al. (2010) MSI-1254 6 0.6 7.6 1 Epand, R.F. et al. (2010) KR-12 6 0.5 66 >4 Epand et al. (2009b) GF-17 6 0.35 14 0.5 Epand et al. (2009b) GF-17 D3 6 0.35 32 >8 Epand et al. (2009b) PR-9 5 0.56 10 32c Epand, R.M. et al. (2010) RR-9 5 0.56 80 64c Epand, R.M. et al. (2010) PI-9 5 0.56 3.5 8c Epand, R.M. et al. (2010) Cateslytin 5 0.33 30 >3 Radek et al. (2008) d C12K-7α8 81 3.1 16 Rotem et al. (2008) d C12(ω7)K-˟12 31 >50 <0.1 Epand et al. (2009a) Magainin 2 3.5 0.15 40 >1 Epand, R.F. et al. (2010) a Estimated for pH 7 using + ½ for histidine. b MIC, minimum inhibitory concentration. c Ratio of Gram-positive bacteria without phosphatidylethanolamine (S. aureus) to Gram-positive bacteria with phosphatidylethanolamine (Bacillus megaterium). d Not a peptide, but an oligo-acyl-lysine. 124 R.M. Epand and R.F. Epand

the new peptide, GF-17 D3, acquires bacterial 2008) and it has a very high ratio of S. aureus species speciﬁ city of its antimicrobial action. MIC/E. coli MIC (Table 7.2). In contrast, a

This substitution destroys the amphipathic shorter oligo-acyl-lysine, C12(ω7)K-β12, does helix and GF-17 D3 no longer promotes dye not cluster anionic lipids and has a low ratio release from liposomes and consequently of S. aureus MIC/E. coli MIC because it loses its toxicity against S. aureus. We have deposits on the LPS layer of Gram-negative suggested that, in general, an increase in the bacteria (Epand et al., 2009a). conformational fl exibility of a peptide facilitates the charge cluster mechanism (Epand and Epand, 2009). This is in accord 7.4.3 Putative mechanism of action with the observation that the charge cluster mechanism is more important for GF-17 D3 It would be expected that the recruitment of than for GF-17. NMR studies also show that anionic lipids to a region of the bacterial the amphipathic helix of GF-17 D3 is largely membrane would result in the concentration destroyed (Li et al., 2006). of negative charge in a domain to which The peptides PR-9, RR-9 and PI-9 are all cationic peptides would congregate, possibly cationic nonapeptides. The three have been causing the formation of a pore. However, shown by DSC to cluster anionic lipids the pore that is formed is not very large. (Epand, R.M. et al., 2010) and the resulting Antimicrobial agents that cause clustering do domains that are formed with PR-9 have not cause leakage of dyes from membrane been imaged by freeze-fracture electron mimetic liposomes of bacterial cytoplasmic microscopy (Epand, R.M. et al., 2010) as well membranes and are not toxic to S. aureus. as by atomic force microscopy and polarized However, they may breach the membrane total internal refl ection fl uorescence barrier to a limited extent, causing slow microscopy (Oreopoulos et al., 2010). From leakage of contents and/or depolarization of circular dichroism (Epand, R.M. et al., 2010) the membrane. Evidence for this comes from and NMR (Zorko et al., 2009) measurements, studies of C12K-7α8, one of the best examples as well as the short length of the peptides, it of an antimicrobial agent causing clustering. is likely that they are conformationally It has been shown with C12K-7α8 that, in the fl exible even when bound to a membrane. presence of EDTA added to allow the dye

These peptides were shown to bind to the DiSC3(5) to reach the inner membrane, the LPS layer in Gram-negative bacteria. cytoplasmic membrane of E. coli is Therefore, the charge cluster mechanism was depolarized (Radzishevsky et al., 2007) and assessed in Gram-positive bacteria containing there is even some slow infl ux across the mostly anionic lipids in their cytoplasmic cytoplasmic membrane of E. coli ML-35 of membrane versus those containing large small organic molecules (Rotem et al., 2008). amounts of PE or uncharged lipids (Table This breach of the membrane barrier may be 7.2). The ratio of the MICs for two bacterial suffi cient to cause the bactericidal eff ect. genera within the same species was found to Furthermore, in addition to concentrat- be dramatically high, refl ecting the absence ing the cationic antimicrobial agent, the of an outer membrane. domain of anionic lipids would be sur- Cateslytin has been shown by 2H-NMR rounded by an interface with the rest of the to cluster anionic lipids (Jean-Francois et al., membrane that would be under line tension 2008) and this peptide also exhibits specifi city and be less stable. Of course, it is likely that in its antimicrobial action. there are always domains in bacterial

C12K-7α8 is an oligo-acyl-lysine, a large membranes (Matsumoto et al., 2006) that group of antimicrobial agents, many of have phase boundary defects. However, which exhibit signiﬁ cant selective toxicity those that form in the presence of lipid- towards certain bacteria (Livne et al., 2009; clustering antimicrobial agents form very Rotem and Mor, 2009; Zaknoon et al., 2009; rapidly (Oreopoulos et al., 2010) and the

Sarig et al., 2010). C12K-7α8 has been shown bacteria would not have time to compensate to cluster anionic lipids (Epand, R.M. et al., for this rearrangement. Analysis of Membrane-targeting AMPs 125

In addition to this mechanism, there can ation in the presence of anionic lipids. This also be damage to the bacteria as a result of transformation provides –0.4 kcal mol–1 the redistribution of lipids in the membrane. residue of free energy. If the process of helix This may disrupt existing natural domains in formation were to be coupled with lipid the membrane or decrease the available phase segregation, the energy provided by anionic lipid required for specifi c protein helix formation could contribute to functions. overcoming the unfavourable energy of mixing required to segregate the lipid components. In addition, insertion of 7.4.4 Properties of antimicrobial agents aromatic residues or an acyl chain of a that favour clustering lipopeptide can also supply the necessary free energy for cluster formation and Some antimicrobial agents are more eff ective stabilization. In general, the formation of any than others in promoting the clustering of kind of non-covalent bonding, either within anionic lipids. A suffi cient number and the peptide or lipid or between the lipid and density of positive charges are required. By peptide as a consequence of the interaction density of positive charges, we are referring of the peptide with lipid, could be coupled to the net number of positive charges divided with the unfavourable energy of lipid by the total number of residues. Magainin 2 clustering. Non-covalent interactions could is not eff ective in clustering anionic lipids, include an increase in the number of although a very similar peptide, MSI-78, can hydrogen bonds in addition to those induce clustering (Epand, R.F. et al., 2010). accounted for by helix formation or salt We suggest that the diff erence between these bridges, such as those between arginine two peptides is a consequence of the much residues and the phosphate groups in the greater density of positive charges on MSI-78 associated lipids. The relative importance of compared with magainin. these various forces will vary from one Another important property is the ability system to another. to pass the outer membrane of Gram-negative bacteria. This allows for a wider range of bacterial species to be tested for the correlation 7.5 Concluding Remarks and between in vitro clustering of anionic lipids Perspectives and increased toxicity to bacteria that are rich in both anionic and zwitt erionic lipids, as are The mechanism of clustering anionic lipids is most Gram-negative bacteria that have a high a component of the mechanism of action of content of PE (with some exceptions, such as many antimicrobial agents, including oligo- C. crescentus). More hydrophobic compounds, acyl-lysines, some amphipathic helical such as the oligo-acyl-lysines, appear more peptides and small, arginine and lysine-rich capable of crossing the outer membrane than peptides. This phenomenon is more short, arginine-rich peptides (Epand, R.M. important for antimicrobial agents that have et al., 2010). a high number and density of positive Conformational fl exibility is another charges, that are conformationally fl exible property associated with an increased ability and that are suffi ciently hydrophobic to to cluster anionic lipids. This has been penetrate the outer membrane of Gram- discussed above in comparing the anti- negative bacteria. Several factors contribute microbial activity of GF-17 with GF-17 D3. to the importance of this mechanism in the design of agents for clinical application. Optimizing the potency and eff ectiveness of 7.4.5 Sources of energy to account for agents that promote this phenomenon will clustering and entropy of demixing allow targeting of antimicrobial agents to specifi c strains of bacteria. In addition, it may Many cationic AMPs are not structured in provide a mechanism of bactericidal action solution, but acquire an α-helical conform- that does not involve the lysis and disruption 126 R.M. Epand and R.F. Epand

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Ju Hyun Cho and Sun Chang Kim*

Abstract

The emergence and rapid spread of multiresistant bacteria necessitates every eﬀ ort to develop new classes of antibiotics with novel targets and modes of action. One potential source of novel antibiotics is the cationic antimicrobial peptides (AMPs), which constitute an important component of the innate immune system in a variety of organisms. Most AMPs exert their activity by interacting with bacterial membranes, thus perturbing their permeability. However, an increasing number of peptides are being described that translocate across the bacterial membranes and act on intracellular targets in bacteria. These non-membrane-active AMPs have been shown to bind and inactivate intracellular biopolymers such as nucleic acids and proteins without destroying or remaining att ached to the bacterial membranes. As such, they have emerged as viable candidates for the treatment of human infections. In this chapter, we focus on the six well-characterized, non-membrane-active AMPs (buforin II, PR-39, indolicidin, apidaecin, drosocin and pyrrhocoricin) and discuss whether binding of these peptides to their intracellular targets correlates with bacterial cell death. The potential exploitation of these peptides as human therapeutics is also discussed.

8.1 Introduction through the proteolysis of precursor proteins/peptides that are encoded by the Of worldwide concern is the increasing host genome and synthesized on ribosomes. development of bacterial and fungal strains These defence peptides are short (10–50 that are resistant to currently available amino acids) and contain an overall positive antimicrobial drugs. This worsening situation charge (in general, +2 to +9) and a substantial has spurred herculean eﬀ orts to develop new proportion (>30%) of hydrophobic residues classes of antibiotics with novel targets and (Hancock and Sahl, 2006). These properties modes of action (Makovitzki et al., 2006). One permit AMPs to fold into amphipathic potential source of novel antibiotics is the α-helix and/or β-sheet structures upon cationic antimicrobial peptides (AMPs), contact with the negatively charged bacterial which are produced by all species of life and membranes. These structures can then insert play a key role in primary host defence themselves into the membranes of infectious against infection by pathogenic micro- particles and create pores by ‘barrel-stave’, organisms (Boman, 1995; Hancock and Scott , ‘carpet’ or ‘toroidal-pore’ mechanisms (see 2000; Zasloﬀ , 2002). AMPs are derived Fig. 5.1, Chapter 5), which results in the

* Corresponding author.

dissipation of transmembrane potential and peptides, representing most of the structural subsequent cell death (Oren and Shai, 1998; classes, there is no correlation between the Shai, 2002; Huang et al., 2004). concentrations leading to complete membrane The targeting of bacterial membranes by permeabili zation and the MIC. AMPs is mainly composed of two steps: the In fact, an increasing number of peptides initial binding of the peptides to the cell are being described that translocate across surface and subsequent membrane permeabil- bacterial membranes and act on intracellular ization (Huang, 2000, 2006). The mechanism targets in bacterial cells. They may inhibit by which AMPs cause bacterial cell death protein or cell-wall synthesis, interact with usually does not involve binding to specifi c DNA or RNA or inhibit some sort of receptors on the cell membrane, but is rather enzymatic activity (Cudic and Otvos, 2002; a non-specifi c interaction with membrane Markossian et al., 2004; Brogden, 2005; Otvos, phospholipids (Yeaman and Yount, 2003). 2005; Cho et al., 2009). Most of these non- The surface of bacterial cells is composed of membrane-active peptides, except for buforin negatively charged components, such as II, which contains a single proline residue in a lipopolysaccharide and teichoic acids, and an mid-chain position, over-represent proline electrostatic interaction between the cationic and adopt a non-α-helical extended structure AMPs and the negatively charged bacterial (Table 8.1). Proline is an amino acid that cell surface plays an important role in their creates a rigid bend in the peptide backbone antibacterial activity (Matsuzaki, 1999). since the carboxyl and amino groups of this Although the formation of ion channels and backbone are covalently bonded. Thus, trans membrane pores and extensive proline is generally accepted as a helix breaker membrane rupture eventually leads to the (Williamson, 1994). The infl uence of proline lysis of bacterial cells (Steiner et al., 1988; residues on the structure and activity of Wimley et al., 1994; Ludtke et al., 1995), not all α-helical peptides has been demonstrated by AMPs are thought to exert their major action a systematic structure–activity relationship on bacterial membranes. It has been shown study using peptide analogues without that the ability of various AMPs to depolarize proline, or with one or two proline residues the cytoplasmic membrane potential of (Zhang et al., 1999). The percentage helicity of Escherichia coli varies widely, with certain the peptides and their ability to permeate the peptides being unable to cause depolarization cytoplasmic membrane of bacteria decrease as at the minimal inhibitory concentration (MIC), a function of the number of proline residues while others cause maximal depolarization incorporated, suggesting the existence of below the MIC (Wu et al., 1999). For all of the intracellular targets. This does not completely

Table 8.1. Amino acid sequences of select non-membrane-active AMPs. Intracellular targets Peptide Amino acid sequencea in bacteria Mode of action Nucleic acids (DNA Inhibition of transcription/ Buforin II TRSSRAGLQFPVGRVHRLLRK and RNA) translation RRRPRPPYLPRPRPPPFFPPRLPPRIPP PR-39 Nucleic acids?b Inhibition of cell division GFPPRFPPRFP Inhibition of replication Indolicidin ILPWKWPWWPWRR-NH DNA 2 leading to ﬁ lamentation Inhibition of DnaK- Apidaecin 1a GNNRPVYIPQPRPPHRRI DnaK assisted protein folding Inhibition of DnaK- Drosocin GKPRPYSPRPT*SHPRPIRV DnaK assisted protein folding Pyrrhocoricin VDKGSYLPRPT*PPRPIYNRN DnaK Inhibition of DnaK- assisted protein folding a Proline residues are in bold and O-glycosylated threonine residues are indicated with asterisks. b Interaction of PR-39 with nucleic acids has not been directly conﬁ rmed. 130 J.H. Cho and S.C. Kim

exclude some type of interaction with the microbial action of buforin II proved to be bacterial membrane, and supports a model in diff erent from those of AMPs that function which penetration across the membrane is by membrane permeabilization. Using followed by binding to and inhibition of fl uorescein isothiocyanate-labelled buforin II functional intracellular end molecules (Otvos, and gel-retardation experiments, it was 2002). In this chapter, we focus on the six well- revealed that buforin II kills bacteria without characterized, non-membrane-active AMPs cell lysis and has a strong affi nity for DNA (buforin II, PR-39, indolicidin, apidaecin, and RNA in vitro (Park et al., 1998). Kobayashi drosocin and pyrrhocoricin) and discuss et al. investigated the interaction of buforin II whether binding of these peptides to their with phospholipid membranes and compared intracellular targets correlates with bacterial these results with those of similar experiments cell death. The potential exploitation of these with magainin 2 (Kobayashi et al., 2000). These peptides as human therapeutics is also researchers used equipotent tryptophan- reviewed. substituted peptides to fl uorometrically monitor peptide–lipid interactions. Control circular dichroism (CD) studies showed that, 8.2 Buforin II: α-Helical AMP with like magainin 2, buforin II binds selectively to Single Proline Residue Binding to liposomes composed of acidic phospholipids. Nucleic Acids However, the fl uorometric experiments revealed that, in contrast to magainin 2, Buforins are α-helical AMPs isolated from the buforin II translocates across liposome stomach tissue of the Asian toad Bufo bufo membranes effi ciently without inducing sig- gargarizans. They display a broad spectrum of nifi cant membrane permeabilization or lipid antimicrobial activity against bacteria and fl ip-fl op. Furthermore, the Pro11 residue, fungi (Park et al., 1996). Buforin I is a 39-amino which induces a kink in the buforin II α-helix, acid peptide with complete sequence identity is the key structural feature required for to the N-terminal region of histone H2A, buforin II’s unique properties. which specifi es the protein’s DNA-binding A subsequent study by Kobayashi et al. activity. Buforin II is a 21-residue peptide that (2004) revealed that buforin II crosses lipid is produced from buforin I by treatment with bilayers in a manner similar to that of the endoproteinase Lys-C. Buforin II magainin 2 – via the transient formation represents an interesting example of an of a peptide–lipid supramolecular complex α-helical AMP because it employs a unique (toroidal) pore. However, the presence of mechanism of activity that does not involve Pro11 distorts the helical structure of buforin membrane perturbation (Haney et al., 2009). II, concentrating fi ve basic amino acid The structure of buforin II was deter- residues in a limited amphipathic region mined using nuclear magnetic resonance (Arg5–Lys21); this structure destabilizes the (NMR) spectro scopy and restrained molecular pore by enhanced electrostatic repulsion and dynamics (Yi et al., 1996). Buforin II adopts a enables effi cient translocation of buforin II helix-hinge-helix structure in 50% tri- into the cell. The importance of the Pro11 fl uoroethanol; the N-terminal extended residue was also demonstrated in a systematic α-helix (residues 5–10) and the C-terminal structure–activity relationship study (Park et α-helix (residues 12–21) are separated by a al., 2000). In this study, antimicrobial proline residue at amino acid position 11. The potencies, secondary structures and mechan- four N-terminal residues are relatively isms of bacterial killing action were assessed unstructured. Interestingly, the Pro11 residue for a series of structurally altered synthetic distorts the C-terminal helix in such a way as buforin II analogues. The results revealed to maximize the amphipathic nature of the that the proline hinge (Pro11) is a key C-terminal helix. This originally led structural factor for the cell-penetrating researchers to postulate that buforin II property without permanent membrane interacts with bacterial membranes in a association, while the cell-penetrating similar fashion to other amphiphatic α-helical effi ciency, which depends on α-helical con- AMPs. However, the mechanism of anti- tent, is a critical factor for determining the Non-membrane Targets of AMPs 131

antimicrobial potency of buforin II. Indeed, from having another as yet unidentifi ed these experiments showed that only a single intracellular target. amino acid substitution at the Pro11 position With its primarily intracellular target changes buforin II into a membrane-active inhibition and ability to move between magainin-like peptide. Conversely, insertion various modes of action, buforin II derivatives of a proline hinge region (Arg5–Gly11) into are ideal candidates for antimicrobial drug the N-terminal helix of magainin 2 switches development. However, poor protease this AMP from a membrane-permeabilizing stability of short AMPs such as buforin II peptide to a cell-penetrating one. severely limits their therapeutic value. Because buforin II was shown to bind Recently, Meng and Kumar (2007) reported nucleic acids in vitro, it has been hypothesized that incorporation of hexafl uoroleucine at that buforin II kills bacteria by interacting selected sites (Leu18 and Leu19) of buforin II with their nucleic acids aft er translocation results in a simultaneous increase in potency across the cell membrane (Park et al., 1998). and resistance to protease degradation. This Although the proposed mechanism is quite observa tion suggests that fl uorination may be intriguing, many questions remain to be an important strategy for increasing the answered. The connection between nucleic metabolic stability of buforin II. In addition to acid binding and antimicrobial activity has antimicrobial activity, buforin II and buforin not been demonstrated directly, and it is IIb – a synthetic analogue of buforin II that unclear whether buforin II and nucleic acids contains a proline hinge between the two interact in a specifi c manner or whether they α-helices and a model α-helical sequence at only bind to each other because of their the C-terminus (3×RLLR) – selectively targets opposite net charges. Uyterhoeven et al. cancer cells through inter action with the cell- (2008) recently characterized the nucleic acid surface gangliosides. Buforin IIb then binding properties of buforin II using mol- traverses cancer cell membranes without ecular modelling and a fl uorescent inter- damaging them and induces mitochondria- calator displacement assay. These researchers dependent apoptosis (Lee et al., 2008). Buforin observed that, in addition to non-specifi c IIb also displays powerful cytotoxic activity electrostatic att ractions between a cationic when injected into solid tumours in p53- peptide and nucleic acids, specifi c side chains defi cient mice (Cho et al., 2009). These results (Arg2 and Arg20) of buforin II form inter- suggest that buforin IIb may be developed actions with DNA that are stronger than the into a novel therapeutic agent for the treat- non-specifi c electrostatic ones. They also ment of cancers. showed that disruption of the buforin II–DNA interactions by substituting basic residues of buforin II with alanine generally decreases the antimicrobial activity of buforin II. Moreover, 8.3 PR-39 and Indolicidin: Mammalian we recently found that buforin II dose- Proline-rich Peptides Inhibiting dependently inhibits the transcription and Macromolecule Synthesis and Cell translation of the lacZα gene of pUC19 in Division vitro (unpublished data, 2010). In addition, when E. coli harbouring pUC19 is treated PR-39 is a 39-amino acid cathelicidin-derived with buforin II at concentrations below the AMP isolated from porcine small intestine MIC, the amount of lacZ expressed, which and neutrophil azurophilic granules. It is rich is determined by β-galactosidase assay, in proline (49%) and arginine (24%) decreases as the concentration of buforin II (Agerberth et al., 1993; Shi et al., 1994). Within increases, suggesting that buforin II inhibits its primary sequence, PR-39 is characterized transcription or translation in bacteria. Taken by seven repeats of Xaa-Pro-Pro-Xaa. CD and together, these results support the assertion Fourier-transform infrared spectroscopy that buforin II kills bacteria by translocation performed with PR-39, either in aqueous into the cell and inhibition of transcription or solution or in the presence of lipid vesicles, translation through interaction with nucleic have suggested that they adopt a disordered acids, although it does not preclude buforin II left -handed polyproline II helix structure 132 J.H. Cho and S.C. Kim

because of the proline residues disposed (Gennaro et al., 2002; Lehrer and Ganz, 2002; consecutively in the linear peptide chain Zanett i, 2004; Sang and Blecha, 2009). Taken (Cabiaux et al., 1994). PR-39 has antibacterial together, it is not clear whether bacterial cell activity against several strains of both Gram- fi lamentation caused by PR-39 and PR-26 is positive and Gram-negative bacteria (Bevins, due to the blocking of DNA replication 1994). The mechanism of PR-39 bactericidal through DNA binding or the inhibition of activity has been investigated by isotope membrane proteins involved in septum incorporation experiments (Boman et al., formation. 1993). In contrast to the killing by membrane In addition to antimicrobial properties, disruption seen with most other AMPs, PR-39 exerts multiple and diverse actions on Boman and colleagues found that bacteria are mammalian cells. PR-39 has been found to killed by PR-39 through a mechanism that induce syndecan expression in mesenchymal stops protein and DNA synthesis. PR-39 cells, and to infl uence cell motility and requires a lag period of about 8 min to metastatic potential in wound repair (Gallo et penetrate the outer membrane of wild-type E. al., 1994). The peptide binds to the cytosolic coli, while subsequent killing is quite fast. component of NADPH oxidase complex Kinetic data suggest induced proteolysis in protein p47phox (Shi et al., 1996b) and a the target bacteria may be important for signalling adaptor protein p130Cas (Chan and bactericidal activity. Consistent with this Gallo, 1998), suggesting an important role for suggestion is the fi nding that actively growing this peptide in infl ammation. PR-39 also plays cells are killed more rapidly than non-growing a critical role in limiting cardiac injury cells. In addition, Shi et al. (1996a) found from through induction of angiogenesis (Li et al., a scanning electron micrographic study that 2000; Bao et al., 2001; Gaczynska et al., 2003) PR-39 and its N-terminal 1–26 fragment, and inhibition of apoptosis in hypoxic PR-26, induce fi lamentation of Salmonella endothelial cells (Ramanathan et al., 2004; Wu typhimurium without forming pores on the et al., 2004). These results underscore the bacterial outer membrane. Cells exposed to therapeutic potential of PR-39 in a number of these peptides have an extremely elongated diseases, including infl ammation, ischemia– morphology, which indicates that the peptide- reperfusion injury and heart diseases. treated cells are unable to undergo cell Indolicidin is isolated from the division. cytoplasmic granules of bovine neutrophils. It Unlike with buforin IIb and other cell- has a unique composition consisting of 39% penetrating AMPs, the translocation of PR-39 tryptophan and 23% proline, and the native into the bacterial cell and its subsequent peptide is amidated at the C-terminus (Selsted interaction with intracellular target molecules et al., 1992). Indolicidin is the smallest of the such as nucleic acids have not yet been known naturally occurring linear AMPs, confi rmed. In addition, a study on the contains the highest percentage of tryptophan secondary structure and membrane inter- of any known protein and consists of only six action of PR-39 showed that its secondary diff erent amino acids. Owing to the presence structure is not altered upon incubation of the of tryptophan residues interspersed with peptide with negatively charged vesicles and proline residues throughout the sequence, it that nearly all of the added peptide is probably assumes a structure distinct from membrane bound (Cabiaux et al., 1994). Based the well-described α-helical and β-structured on these results, Shi et al. (1996a) suggested peptides. Solution structures of indolicidin in that PR-39 and PR-26 bind to molecules diff erent environments have been determined anchored on the outer membrane of Gram- by fl uorescence, CD and NMR spectroscopy. negative bacteria and that these molecules are How ever, the proposed structures of necessary for formation of the septum. indolicidin on lipid membranes have been However, multiple cellular targets, including somewhat controv ersial. It has been suggested proteins containing PR-39-binding domains, that indolicidin adopts a polyproline helix have been identifi ed in eukaryotic cells, and structure (Falla et al., 1996) or a turn structure PR-39 has been shown to enter eukaryotic (Ladokhin et al., 1997, 1999). However, Rozek cells and regulate many biological processes et al. (2000) showed that indolicidin adopts a in which these targets are directly involved wedge-shaped structure with hydrophobic Non-membrane Targets of AMPs 133

tryptophan residues in the trough, fl anked by integrase (Marchand et al., 2006). Indolicidin two positively charged regions (see Fig. 9.6A, also inter feres with topoisomerase-I-mediated Chapter 9), which appears ideal for inter- DNA relaxation without unwinding DNA, calation between the lipid molecules of the suggesting that indolicidin may inhibit a lipid bilayer. Moreover, a study by Hsu et al. large variety of DNA processing enzymes (2005) revealed that indolicidin adopts through DNA binding. Taken together, multiple conformations in aqueous solutions indolicidin is thought to cross the mem branes and membrane-mimicking environments, into the cytoplasm at concentrations above suggesting that the structural plasticity the MIC but below the minimal lytic con- accounts for indolicidin’s eff ects. centration, and kills bacteria by multiple Indolicidin exhibits signifi cant activity actions at the level of DNA. against a wide range of targets, including The short sequence and broad spectrum bacteria, fungi, protozoa and human immuno- of activity are features that make indolicidin defi ciency virus (HIV)-1 (Zanett i et al., 2002; appealing as a putative anti-infective agent. Chan et al., 2006). Like other membrane- Although indolicidin is an eff ective AMP, permeabilizing peptides, the antimicrobial various att empts have been made to fi nd action of indolicidin was thought to increase derivatives that are more antibacterial and the permeability of the cytoplasmic mem- less cytotoxic. Omiganan pentahydrochloride, branes of target micro organisms. However, a synthetic analogue of indolicidin, is the complete mode of action of this peptide currently under clinical development for the has not been fully elucidated and is still prevention of catheter-related local and under debate. Although indolicidin binding bloodstream infections as well as for the to bacterial membranes results in membrane treatment of acne and rosacea (Melo et al., permeabilization (Falla et al., 1996; Zhao et al., 2006; Vaara, 2009). It has been reported that 2001; Schibli et al., 2002; Nan et al., 2009) or indolicidin binds abasic-site-containing DNA thinning (Shaw et al., 2006; Hsu and Yip, (Marchand et al., 2006). Abasic sites are 2007), it does not cause cell lysis at considered to be the most frequent DNA concentrations four times the MIC of the lesions in mammalian cells, with an estimated peptide (Subbalakshmi and Sitaram, 1998; frequency of 10,000 events per day per cell Wu et al., 1999). Thus, it is conceivable that (Boiteux and Guillet, 2004). There fore, further indolicidin uses its membrane-affi nity investigations on the potential interference property to enter the cytoplasm and exerts its of indolicidin with DNA repair mechanisms antibacterial activity by att acking other are needed to use this peptide in human targets, similarly to PR-39. In fact, Sub- thera peutics. balakshmi and Sitaram (1998) demonstrated that indolicidin induces fi lamentation in E. coli cells as a result of preferential inhibition of DNA synthesis, rather than RNA and 8.4 Apidaecin, Drosocin and protein synthesis. Moreover, Hsu et al. (2005) Pyrrhocoricin: Short, Insect Proline- confi rmed that indolicidin binds DNA in gel- rich Peptides Binding to Heat-shock retardation and fl uorescence quenching Protein DnaK experiments. These researchers also demonstrated that indolicidin prefers to bind duplex Apidaecin, drosocin and pyrrhocoricin are DNA rather than single-stranded DNA, using short, proline-rich AMPs isolated from insects surface plasmon resonance. It is thought that (Apis mellifera, Drosophila melanogaster and indolicidin prefers to bind strongly to Pyrrhocoris apterus, respectively). They consist phosphate groups via its positively charged of 18–20 amino acid residues (Casteels et al., amino acids, and the tryptophan residues 1989; Bulet et al., 1993; Cociancich et al., 1994). stack between the nucleotide bases or The names of these peptides refl ect their deoxyriboses in each strand of the DNA origin rather than a subdivision among the duplex. A subsequent study revealed that individual sequences. Apidaecin, drosocin indolicidin interferes with the formation of and pyrrhocoricin are remarkably similar in the catalytic HIV-1 integrase–DNA complex amino acid composition and sequence motif by directly binding to DNA and not to patt ern (Li et al., 2006). In addition to being 134 J.H. Cho and S.C. Kim

overrepresented in the sequences, proline et al., 2000a). Although the above-mentioned residues are frequently associated in doublets non-membrane-active AMPs, such as buforin and triplets with basic residues (arginine or II, PR-39 and indolicidin, act on intracellular histidine). The Pro-Arg-Pro/Pro-His-Pro components such as nucleic acids, they do motifs are either evenly distributed along the not appear to have a specifi c protein target. entire peptide sequence or are concentrated By using a biological assay to selectively in certain subdomains (Bulet and Stocklin, measure chiral interactions with bacterial 2005). Drosocin and pyrrhocoricin also targets, Castle et al. (1999) observed that all-d contain a glycosylated threonine in the enantiomers of apidaecin are rapidly mid-chain position, although the presence of associated with E. coli cells, but are then sugar is not always necessary for almost entirely recovered by exhaustive antimicrobial activity (Otvos, 2002). The washing, whereas the all-l apidaecins and presence of sugar increases the in vitro all-l and all-d magainins are not. This implies antibacterial activity of drosocin (Bulet et al., that apidaecins likely permeate and traverse 1996), but decreases that of pyrrhocoricin the outer membrane in a non-specifi c fashion, (Hoff mann et al., 1999). The solution followed by an apparently irreversible process conformations of these peptides have been due to sequentially stable binding to a determined by CD and two-dimensional periplasmic or inner-membrane component, NMR spectroscopy. Drosocin and pyrrho- or through further translocation. These coricin adopt random coil structures in researchers also showed that apidaecin uptake aqueous solution, and remain largely in E. coli is energy-driven, irreversible and can unstructured in 50% trifl uoroethanol; there be partially competed by proline in a are, however, clear indications of the presence stereospecifi c manner. Moreover, failure of of small populations with elements of local certain apidaecin mutants to kill cells aft er structure, most probably turns (McManus et entering the cytosol implies the existence of al., 1999; Otvos et al., 2000a). In addition, another downstream target. Based on these apidaecin appears to form an ordered results, they proposed a permease/transporter- oligomer in a membrane-mimicking environ- mediated mechanism for the mode of action ment (Dutt a et al., 2008). of apidaecin on E. coli. The proposed Apidaecin, drosocin and pyrrhocoricin mechanism involves an initial, non-specifi c are generally active against Gram-negative binding of the peptide with an outer bacteria, while only a few Gram-positive membrane component and self-promoted species are aff ected (Bulet and Stocklin, uptake in the periplasmic space, followed by 2005). The most unique feature of their mode interaction with an inner membrane-bound of action is a complete lack of membrane transporter/receptor protein allowing trans- permeabilization activity. Casteels and location into the cytosol. In the fi nal step, the Tempst (1994) showed that the addition of peptide in the cytosol meets one or more apidaecin does not cause any unmasking of macromolecular targets, the inhibition of the β-galactosidase activity of E. coli ML-35, a which results in bacterial cell death. strain with a phenotype allowing an easy Putative target proteins of apidaecin, calorimetric evaluation of the kinetics of drosocin and pyrrhocoricin in the bacterial inner-membrane permeabilization, and the cell have been identifi ed by mass spectroscopy, sensitivity of apidaecin-resistant mutant Western blott ing and fl uorescence polarization strains to pore-forming AMPs is undimin- (Otvos et al., 2000b). Otvos et al. found that ished. Moreover, unlike most AMPs that kill these peptides specifi cally bind to the E. coli bacteria in a non-stereospecifi c fashion heat-shock protein DnaK, which functions (Oren and Shai, 1998), the all-d enantiomers co-translationally and post-translationally to of apidaecin, drosocin and pyrrhocoricin promote protein folding and inhibit the are totally inactive against strains susceptible formation of toxic protein aggregates of to the all-l isomers, suggesting that the misfolded proteins (Bukau and Horwich, antibacterial eff ects of these peptides could 1998; Hartl and Hayer-Hartl, 2002; Slepenkov involve stereoselective recognition of a chiral and Witt , 2002), but not to the homologous cellular target, most likely a protein (Casteels DnaK fragment of Staphylococcus aureus (a and Tempst, 1994; Bulet et al., 1996; Otvos species not susceptible to the peptides) or the Non-membrane Targets of AMPs 135

human equivalent Hsp70. These peptides activity spectrum of drosocin can be 100% also interact with lipopolysaccharide, which correlated with the homology to E. coli DnaK could be responsible for their initial binding at the D-E helix region in the 31 studied to the bacterial surface, and in a non-specifi c bacterial species. However, there is some manner with the bacterial chaperonin GroEL. disagreement as to the specifi c binding site of In addition, an inactive pyrrhocoricin DnaK. Chesnokova et al. (2004) studied the analogue, made of all-d amino acids, does chemistry of pyrrhocoricin binding to DnaK not interact with DnaK. This suggests that using complementary pre-state kinetic and inhibition of DnaK’s function by these single-turnover ATPase assays, and showed peptides might be responsible for bacterial that pyrrhocoricin binds to and stimulates the killing. ATPase activity of both wild-type and lidless A subsequent study by Kragol et al. variants of DnaK. In addition, a study of CD (2001) revealed that pyrrhocoricin and curves by Zhou et al. (2008) showed that drosocin aff ect DnaK’s two major functions: obvious spectral alteration is detected when ATPase activity and refolding of misfolded apidaecin is added to lidless DnaK, while no proteins. In this study, they found that alteration is observed when it is added to biologically active l-pyrrhocoricin diminishes DnaK D-E helix fragments. Thus, these the ATPase activity of recombinant DnaK, researchers suggested that pyrrhocoricin while the inactive d-pyrrhocoricin analogue binds primarily to the conventional substrate- and membrane-active AMPs such as cecropin binding site of DnaK, much like other A or magainin 2 fail to inhibit ATPase activity substrates, and eff ectively decreases the of DnaK. In addition, both alkaline cellular concentration of DnaK by competing phosphatase and β-galactosidase activities with natural substrates. (refl ecting DnaK’s function of refolding Ideally, a protein present only in bacteria misfolded proteins) of live bacteria are that carriers a signifi cant function, but is reduced upon incubation with l-pyrrhocoricin absent or non-homologous in human cells, or drosocin. In contrast, d-pyrrhocoricin, may form the basis for the rational design of magainin 2 or buforin II have only a species-specifi c antibacterial peptides and negligible eff ect. According to fl uorescence peptidomimetics (Casteels and Tempst, 1994). polarization and dot-blot analysis of synthetic DnaK shares only 50% sequence homology DnaK fragments and labelled pyrrhocoricin with eukaryotic Hsp70 (Bardwell and Craig, analogues, the peptide binds to the hinge 1984), with some well-conserved N-terminal region around the C-terminal helices D and E regions and some less conserved C-terminal of DnaK, located just above the conventional ones (Karlin and Brocchieri, 1998). Therefore, substrate-binding site. Structure–activity the variable sequence domain of DnaK relationship studies of pyrrhocoricin identi- appears to off er a sensible target for fi ed that the N-terminal half (residues 2–10) is antimicrobial drug development, and responsible for inhibition of the ATPase apidaecin, drosocin and pyrrhocoricin are an activity of DnaK, while the C-terminal half excellent starting point for these species- (residues 11–20) is responsible for membrane specifi c drug development eff orts. It is penetration (Kragol et al., 2002). Overall, these conceivable that drugs that act on a specifi c data suggest that the binding of pyrrhocoricin target protein likely have the disadvantage of and drosocin to DnaK prevents the frequent an easier selection of drug-resistant strains opening and closing of the multihelical lid through mutations at the target level, over the peptide-binding pocket of DnaK, similarly to conventional antibiotics, which permanently closes the cavity and inhibits generally act by binding to a specifi c target chaperone-assisted protein folding. The (Gennaro et al., 2002). However, Cassone et al. sequence of the D-E helix is remarkably (2009) recently showed that induced dissimilar in various bacterial and resistance to the designer proline-rich peptide mammalian DnaK proteins. This may explain dimer A3-APO does not involve changes in the selectivity and relatively restricted the intracellular target DnaK, thus dispelling spectrum of activity of these peptides and the concern of target modifi cation and their lack of toxicity to eukaryotic cells. In arguing for focused research for novel fact, Bikker et al. (2006) demonstrated that the bacterial targets. 136 J.H. Cho and S.C. Kim

8.5 Concluding Remarks and Despite the fact that non-membrane- Perspectives active peptides show great potential as novel antibiotics, a number of issues must be solved The emergence and rapid horizontal spread of before these peptides can be developed as antibiotic-resistant traits in bacteria of human human therapeutics. Although these peptides and veterinary clinical signifi cance has been a exhibit signifi cant in vitro activity against driving force in the search for new classes of bacteria, they are cleaved in vivo by endogen- antibiotics (Coates et al., 2002). AMPs have ous mammalian proteases, severely reduc- been regarded as a potential solution to ing their therapeutic value. In particular, serious worldwide problems caused by infec- chymotrypsin-like enzymes att ack proteins at tious diseases (Mookherjee and Hancock, basic residues, which are an obligate feature 2007). The potential value of AMPs for clinical of AMPs. In this regard, there are strategies purposes includes their use as single antimi- for protecting peptides from proteases, includ- crobial agents, synergistic agents to existing ing liposomal incorporation and chemical antibiotics, immunostimulatory agents and modifi cation (McPhee et al., 2005). Advances endotoxin-neutralizing agents (Gordon et al., in peptide engineering, including physical 2005). The non-membrane-active AMPs dis- (structural), chemical or composition modifi - cussed in this review display many of the cations of AMPs, will likely contribute to the desirable features of a novel antibiotic. As development of peptide antibiotics and with any new class of antimicrobial thera- improve their effi cacy. In fact, the designer peutics, a central issue is whether resistance proline-rich peptide dimer A3-APO kills can be provoked. Unlike most AMPs, which β-lactam- and fl uoroquinolone-resistant kill bacteria via subsequent cytoplasmic clinical isolates even in the presence of serum membrane disruption, non-membrane-active proteases and is eff ective against systemic AMPs translocate across the bacterial mem- models of infection in vivo, showing clear branes and act on intracellular targets in potential for therapeutic utility (Szabo et al., bacteria. The fact that these peptides have 2010). The next step in the development of multiple targets, as well as their fundamental new antibiotics is the large-scale production interaction with the bacterial membrane, of pharmacologically pure AMPs in a cost- means the chances of resistance by target eff ective manner. Novozymes Inc. has modifi cation are slim, as this would require reported, using a proprietary fungal-based the complete alteration of the membrane or system, recombinant production of the pep- bypassing of several biochemical pathways tide plectasin at the scale and purity required (Marr et al., 2006). Buforin II, PR-39 and indoli- for therapeutic administration (Mygind et al., cidin, which target nucleic acids, have the 2005). Jang et al. (2009) recently developed a potential to be developed into more effi cient, novel translationally coupled, two-cistron broad-spectrum antimicrobials; apidaecin, expression system for the production of drosocin and pyrrhocoricin, which target the recombinant AMPs in their natural forms. variable sequence domain of DnaK, are an Using this system, they produced approxi- excellent starting point for species-specifi c mately 100 mg of several potent AMPs from 1 drug development eff orts. In addition to their l of E. coli culture. These eff orts may lead to a direct antibacterial eff ects, some of these non- universal cost-eff ective solution for the mass membrane-active AMPs, such as buforin II production of AMPs, and AMPs may soon and PR-39, exert multiple and diverse actions fulfi l their promise as alternatives to conven- on mammalian cells, suggesting their thera- tional antibiotics for the treatment of human peutic potential in a number of human infections. diseases, including cancer and infl ammation. Moreover, peptides such as buforin II and indolicidin, which at the same time cross the Acknowledgements eukaryotic membrane and bind DNA, may be used as a gene-delivery vehicle in the treat- This work was supported in part by ment of a variety of genetic and acquired grants from the Research Program of New diseases. Drug Target Discovery (M10748000314- Non-membrane Targets of AMPs 137

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Guangshun Wang

Abstract

This chapter reviews structural studies of antimicrobial peptides (AMPs), with a focus on human defensins and cathelicidins. Also discussed are the major steps for structural determination of AMPs by nuclear magnetic resonance spectroscopy, including bacterial expression and puriﬁ cation, sample preparations, data collection, sequential signal assignments, structure calculations, validation and coordinate deposition. A variety of three-dimensional structures (α-helices, β-strands, αβ-fold and non-αβ structures) have been discovered for AMPs, which can induce similar biophysical consequences in membranes such as positive curvature or lipid domain formation. Therefore, it is the amphipathic surface, not polypeptide backbone scaﬀ olds, that is essential for the antimicrobial activity of AMPs. A proper presentation of side chains (e.g. cationic and hydrophobic) on the surface of AMPs determines not only membrane perturbation potential, but also other biological functions. Reduction in hydrophobicity is a fundamental strategy to improve peptide selectivity. Structures of AMPs in complex with membranes or non-membrane targets also form the foundation for engineering a new generation of antimicrobials that will supplement or replace traditional resistant antibiotics.

9.1 Human Defensins and additional aspartic acid residue at the Cathelicidins N-terminus, is inactive (Raj et al., 2000). The antimicrobial and chemotactic activities of In mammals, defensins and cathelicidins are HNP-4 are somehow comparable to other the two major families of antimicrobial neutrophil defensins. While HD-5 is highly peptides (AMPs) (Zasloff , 2002; Zanett i, 2005). bactericidal, the antibacterial activity of HD-6 Six α-defensins have been found in humans: has yet to be observed (Szyk et al., 2006). four from neutrophils (HNP-1, -2, -3 and -4) Table 9.1 also contains four well-known and two from intestinal Paneth cells (HD-5 human β-defensins (hBD-1, -2, -3 and -4). and -6) (Lehrer et al., 2009). Note that HNP-1, Salts appear to aff ect the antimicrobial -2 and -3 have essentially identical sequences activities of defensins, indicating the import- and only diff er at the N-terminal residue ance of electrostatic interactions in bacterial (Table 9.1). Even such a small sequence membrane binding (Pazgier et al., 2007). A diff erence infl uences the antifungal activity of third family of defensins is small (18-residue these peptides. While HNP-1 and -2 are active minidefensins) and in circular form against Candida albicans, HNP-3, with an (θ-defensins). They are expressed in non-

‡ Chapter editor: Richard M. Epand.

Table 9.1. Primary structures of human cathelicidins and defensins. APD No. AA Net Hydrophobic IDa Name AA sequence residues charge % (PBP)b 176 Human α-defensin-1 ACYCRIPACIAGERRYGTCIYQG 30 +3 53 (1.07) (HNP-1) RLWAFCC 177 Human α-defensin-2 CYCRIPACIAGERRYGTCIYQGR 29 +3 51 (1.17) (HNP-2) LWAFCC 178 Human α-defensin-3 DCYCRIPACIAGERRYGTCIYQG 30 +2 50 (1.42) (HNP-3) RLWAFCC 179 Human α-defensin-4 VCSCRLVFCRRTELRVGNCLIGG 33 +4 51 (1.40) (HNP-4) VSFTYCCTRV 180 Human α-defensin-5 ATCYCRTGRCATRESLSGVCEIS 32 +4 40 (2.60) (HD-5) GRLYRLCCR 181 Human α-defensin-6 AFTCHCRRSCYSTEYSYGTCTV 32 +4 40 (1.71) (HD-6) MGINHRFCCL 451 Human β-defensin-1 DHYNCVSSGGQCLYSACPIFTKI 36 +5 36 (1.30) (hBD-1) QGTCYRGKAKCCK 524 Human β-defensin-2 GIGDPVTCLKSGAICHPVFCPRR 41 +7 36 (0.90) (hBD-2) YKQIGTCGLPGTKCCKKP 283 Human β-defensin-3 GIINTLQKYYCRVRGGRCAVLSCL 45 +11 33 (2.87) (hBD-3) PKEEQIGKCSTRGRKCCRRKK 675 Human β-defensin-4 FELDRICGYGTARCRKKCRSQEY 49 +7 32 (3.35) (hBD-4) RIGRCPNTYACCLRKWDESLL NRTKP 310 Cathelicidin LL-37 LLGDFFRKSKEKIGKEFKRIVQRIK 37 +6 35 (2.99) DFLRNLVPRTES 624 Cathelicidin ALL-38 ALLGDFFRKSKEKIGKEFKRIVQRI 38 +6 36 (2.87) KDFLRNLVPRTES a Additional information for each peptide can be found in the APD (http://aps.unmc.edu/AP/main.html) by entering the APD ID number in the search interface (Wang et al., 2009). b Hydrophobic % is calculated as the ratio between the numbers of hydrophobic residues and total residues. Abbreviations: AA, amino acid; APD, Antimicrobial Peptide Database; HNP, human α-defensin; PBP, protein-binding potential (Boman, 2003).

human primates, but not in humans due to activity, human defensins possess other the stop codon in the pseudogenes (Tran et functional roles in host defence such as anti- al., 2002). Although the exact disulﬁ de bond human inﬂ ammatory virus (HIV), antitoxin, (S–S) patt ern varies, all defensins possess chemotaxis and immune-modulation proper- multiple such bonds. In human α-defensins ties (Lehrer et al., 2009). (29–33 residues), the three S–S bonds are The precursor proteins of the cathelicidin CysI–CysVI, CysII–CysIV and CysIII–CV; in family share a well-conserved N-terminal human β-defensins (36–49 residues), they are ‘cathelin’ domain while the sequences at the CysI–CysV, CysII–CysIV and CysIII–CysVI. The C-terminus are highly variable, encoding a S–S bond connections in rhesus monkey plethora of AMPs (Zanett i, 2005). Several θ-defensins are CysI–CysVI, CysII–CysV and cathelicidins exist in animals such as sheep, CysIII–CysIV. The cysteines in these peptides cows and pigs. For example, PMAP-23, are labelled from CysI to CysVI according to protegrin-1 and PR-39 are all pig cathelicidins their order in the sequence. The S–S bridges, that form α-helix, β-hairpin and extended as well as the circular polypeptide chain in structures in membrane environments, the case of θ-defensins, confer stability to the respectively. In contrast, only one cathelicidin three-dimensional (3D) structure of defensins. gene has been found in humans. The In addition to the known antibacterial precursor of human cathelicidin is an 18 kDa Structural Studies of AMPs 143

human cationic antimicrobial protein defensins, LL-37 also possesses other bio- (hCAP-18). Depending on the expression logical functions such as immune modulation, sites, hCAP-18 can be cleaved into diff erent angiogenesis, apoptosis and wound healing forms of active peptides. In neutrophils, the (Nĳ nik and Hancock, 2009). peptide, released by proteinase 3, contains 37 To elucidate the functional roles of residues and starts with two leucines, and is defensins and cathelicidins, 3D structures are thereby referred to as LL-37 (Gudmundsson essential. While circular dichroism (CD) and et al., 1995). In human production systems, Fourier transform infrared spectroscopy are the released peptide is ALL-38. LL-37 and low-resolution techniques that give an ALL-38 (Table 9.1) have similar antimicrobial estimation of secondary structures of proteins activities (Sørensen et al., 2003). In addition, in various states, X-ray crystallography and LL-37 can be cleaved into short active nuclear magnetic resonance (NMR) peptides in human skin or sweat, indicating a spectroscopy are able to determine the regulatory role of proteases in host defence structure of proteins to the atomic resolution. (Yamasaki et al., 2006). Of note, these LL-37 By February 2010, the Antimicrobial Peptide fragments display greater antimicrobial Database (APD) (Wang et al., 2009) contained activity in the presence of physiological salts 195 NMR structures and ten crystal such as carbonate (Dorschner et al., 2006). structures, indicating that NMR is the major Therefore, it is important to perform player (Wang, 2006; Haney et al., 2009). As a antibacterial assays under physiological consequence, this chapter focuses on conditions. Of these protease products of structural studies of AMPs by NMR. An hCAP-18, LL-37 has been best studied. This outline of the procedure for NMR structural molecule protects humans from infectious determination of AMPs is provided in Fig. diseases (Nizet et al., 2001; Pütsep et al., 2002), 9.1. For structural analysis, highly purifi ed cystic fi brosis (Bucki et al., 2007) and sepsis by AMPs are required. Because cationic AMPs neutralizing lipopolysaccharides (LPS or usually associate with anionic bacterial endotoxin) (Cirioni et al., 2006). LL-37 and its membranes, NMR studies are oft en con- fragments all inhibit HIV-1 infection ducted in membrane-mimetic systems. Also (Bergman et al., 2007; Wang et al., 2008), but discussed are NMR methods, major structural they may also perform diff erent biological types (α-helices, β-sheets and extended functions. While LL-37 promotes cancer structures), peptide–membrane interactions metastasis, an N-terminal fragment (LL-25) and structure-based peptide design. inhibits the process (Weber et al., 2009). In addition, a C-terminal fragment has been shown to have anticancer activity in vitro (Li et al., 2006a). It is proposed that overexpressed 9.2 Bacterial Expression and LL-37 att racts mesenchymal stem cells to Purifi cation of Antimicrobial Peptides tumour microenvironments by directly interacting with formyl peptide receptor-like Most AMPs are relatively short and can 1 (FPRL-1). The chemotactic activity is readily be synthesized by the solid-phase att ributed to the N-terminal region of LL-37, method (Merrifi eld et al., 1995). In particular, and the antibacterial region is mapped to the this method enables the incorporation of C-terminus of the peptide (Braff et al., 2005). isotope-labelled amino acids at specifi c sites. A recent review lists >100 LL-37 fragments Such peptides are important for solid-state discovered by various laboratories (Burton NMR studies. Advances have also been made and Steel, 2009). KR-12, the smallest anti- in the chemical synthesis of defensins (Raj et microbial fragment with merely 12 residues al., 2000; Wu et al., 2004; Klüver et al., 2006). (Table 4.2, Chapter 4), has been mapped to For longer peptides or those with residues 18–29 of LL-37 (Wang, 2008c). multiple disulfi de bonds, chemical synthesis Evidently, polypeptide fragmentation is less effi cient and bacterial expression shows provides a useful approach for elucidating advantages. In addition, bacterial expression the functional regions of AMPs. Like human enables uniform labelling of polypeptides 144 G. Wang

the expression host and to protect the peptide from being degraded. For a particular case, AMP synthesis NMR data NOESY and the carrier may be optimized by comparing collection 3D fold peptide expression levels. Other favourable properties of the carriers are increased solubility and easy separation from the Peptide Signal Structure purification assignments refinement recombinant peptide. Some tested carrier and validation proteins are glutathione S-transferase, maltose-binding protein, thioredoxin, Staphy- NMR sample Secondary Structure lococcus aureus protein A, immunoglobulin G structures deposition binding protein, OprF, RepA, baculoviral and publication polyhedron, PaP3.30, small ubiquitin-related modifi er (SUMO), PurF and TAF12 (Zhang et Fig. 9.1. A fl ow chart for the structural al., 1998; Kim et al., 2008). In the case of LL-37, determination of AMPs by NMR spectroscopy. The carriers such as thioredoxin, glutathione peptide may be synthesized chemically using the S-transferase and a family III carbohydrate- well-established solid-phase method or biologically using bacterial expression systems. In many binding module (CBM3) have been used cases, it is diffi cult to purify a suffi cient amount of for peptide expression in Escherichia coli peptide from natural organisms for structural (Table 9.2). The recombinant LL-37 was studies. NOESY, nuclear Overhauser effect not separated from thioredoxin by high- spectroscopy. performance liquid chromatography (Li et al., 2006c), but was separated from CBM3 (Ramos at an acceptable cost, facilitating 3D double- et al., 2010). The introduction of the CBM3 and triple-resonance NMR studies of carrier simplifi es the original protocol (Li et AMPs (Fig. 9.1), especially when two- al., 2006c) developed for the expression and dimensional (2D) homonuclear NMR experi- purifi cation of recombinant LL-37. There are ments do not provide suffi cient spectral also other LL-37 expression and purifi cation resolution (e.g. LL-37; see Wang, 2008c). By protocols (Yang et al., 2004; Moon et al., 2006). using recombinant DNA technology, the AMP The bacterial expression systems in Table 9.2 gene can be inserted into an expression vector gave peptide yields ranging from 0.3 to 2.6 (Fig. 9.2) and expressed in bacteria, yeasts or mg l–1 of bacterial culture. By fusing histonin cell-free expression systems (Wang, 2008a). to part of PurF protein and adopting a The AMP gene can be chemically synthesized multimeric expression strategy, Kim et al. or amplifi ed via polymerase chain reactions (2008) obtained 167 mg of the recombinant using natural DNA as a template. Both the peptide from 1 l of bacterial culture. It codons in the synthetic AMP gene and DNA appears that the expression yields of oligomers for polymerase chain reaction recombin ant AMPs vary substantially amplifi cation can be optimized with the aid depending on the peptide and expression of computer programs (Puigbo et al., 2007; systems and purifi cation methods. Welch et al., 2009). A carrier protein is chosen Since a purifi cation tag (Fig. 9.2) is to att enuate the potential toxicity of AMPs to att ached to the fusion protein, it can be

Table 9.2. Select bacterial expression systems for human LL-37. Carrier Molecular form protein Cleaving agent Peptide yield (mg l–1) Reference GS-LL-37 Trx Thrombin 1.1 Yang et al. (2004) P-LL-37 Trx Formic acid 1.7 Li et al. (2006c) LL-37 GST Factor Xa 0.3 Moon et al. (2006) P-LL-37 Trx Formic acid 2.6 Li et al. (2007) P-LL-37 CBM3 Formic acid 1.0 Ramos et al. (2010) Abbreviations: CBM3, family III carbohydrate-binding module; GST, glutathione S-transferase; Trx, thioredoxin. Structural Studies of AMPs 145

and effi ciently cleaves the carrier protein from the peptide; the peptide released possesses no additional residues from cloning. Only polypeptides starting with a proline residue are diffi cult to cleave (Malakhov et al., 2004). Although chemical cleavage is not as specifi c as enzyme cleavage, it off ers some advantages. Chemical cleavage is cost- eff ective and suitable for industrial production. The commonly used chemical cleaving agents are cyanogen bromide (Park et al., 2009; Seo et al., 2009), hydroxylamine Fig. 9.2. A putative expression vector for AMPs. (Hu et al., 2010) and formic acid (Li et al., Several purifying tags and carrier proteins applied 2006c; Ramos et al., 2010). The cleavage sites to the expression of human LL-37 are listed in for these agents are given in Table 9.3. Due to Table 9.2. Some commonly used cleavage methods and cleavage site sequences their small size, chemical agents are able to (represented by ABCDE in the fi gure) are listed in access the cleavage sites of fusion proteins Table 9.3. and release the expressed AMPs. In contrast, poor or no cleavage is sometimes reported when proteases are used for cleavage, rapidly separated from other unwanted probably owing to inaccessibility of the molecules by affi nity chromatography. The cleavage sites by a bulky protease. In purifi ed fusion protein is next cleaved by a particular in the case of LL-37, its aggregation protease or chemical agent to release the may hinder the access of the proteases and recombinant peptide. Select cleaving agents cause poor cleavage at the site adjacent to the and peptide cleaving sites are listed in Table peptide by enzymes. Cleavage may be 9.3. Factor Xa (Moon et al., 2006), enterokinase successful aft er a formic acid cleavage site is (Bang et al., 2010) and thrombin (Li et al., introduced (Table 9.3). Recombinant LL-37 2007) are frequently used proteases. Recently, obtained in this manner retains an extra tobacco etch virus protease (TEV) and SUMO proline residue at the N-terminus of the have become popular. A mutant form peptide. It is important to validate that extra (Ser219Val) of TEV is a bett er choice, since it residues of recombinant proteins from is 100-fold more stable than the wild type expression constructs do not substantially (Kapust et al., 2001). Another att ractive alter the biological properties of the feature of TEV is that it appears to be active expressed polypeptide. We found that the even in the presence of detergents. This recombinant LL-37 displayed similar property is useful for removing purifi cation antibacterial activity to the native peptide. tags from membrane proteins in detergent- Therefore, this form of recombinant LL-37 is containing buff ers (Lundbäck et al., 2008). suitable for structural studies. For NMR SUMO recognizes the entire protease fold studies, isotope-labelled peptides were

Table 9.3. Select enzymes and chemical agents and cleavage sequences. Protease Cleavage sequence Chemical agent Cleavage site Thrombin Arg↓Gly, Lys↓Leu or Lys↓Ala CNBr Met↓Xxx Enterokinase AspAspAspAsp↓Lys Formic acid Asp↓Pro Factor Xa IleGluGlyArg↓Xxx Hydroxylamine Asp↓Gly SUMO protease 1 SUMO↓Xxxa TEV GluGlnLeuTyrPheGln↓Gly/Ser a Xxx should not be proline. Abbreviations: CNBr, cyanogen bromide; SUMO, small ubiquitin-related modiﬁ er; TEV, tobacco etch virus protease. 146 G. Wang

obtained by using a minimal labelling in complex with seven diff erent micelles. medium, where glucose and ammonium Due to the small size of the peptide, micelles chloride are replaced with 13C- and determined the diff usion rate of the complex. 15N-labelled molecules (Li et al., 2006c; Seo et The longer the acyl (alkyl) chain of lipids al., 2009). Similarly, defensins have also been (detergents), the slower the diff usion of the expressed and purifi ed in E. coli for complex. The rapid tumbling of smaller structure–activity relationship studies (Chen micelles led to a narrower spectral line- et al., 2006; Pazgier and Lubkowski, 2006; Seo width. Evidently, small micelles are desirable et al., 2009). It is worth noting that no for high-resolution NMR studies as long as chemical or enzyme cleavage was needed in the native structure of the peptide is a recently reported cistron expression reserved. Sodium dodecylsulfate (SDS) and system, where the termination codon of the dodecylphosphocholine (DPC) are the two fi rst cistron (encoding an anionic poly- most popular micelles (Wüthrich, 1986; peptide) overlaps with the initiation codon of Opella and Marassi, 2004), deuterated forms the second cistron (encoding a cationic of which can be purchased from commer- AMP). This interesting system may be of cial sources. Deuteration makes the proton universal use as a cost-eff ective AMP signals of micelles invisible, thereby sim- expression system (Jang et al., 2009). plifying the 1H-NMR spectra of the peptide–detergent complex. The two membrane-mimetic agents have identical 9.3 Structural Studies of Membrane- C12 carbon chains that diff er only in their targeting Antimicrobial Peptides head groups (for chemical structures, see Wang 2008b). While SDS has an anionic 9.3.1 Membrane-mimetic models sulfate head group, DPC has a zwitt erionic phosphatidylcholine head group. These two Many AMPs function by targeting biological micelles are used to mimic bacterial mem- membranes. Due to the complex nature of branes and human cell membranes, respect- bacterial membranes, high-resolution NMR ively (Haney and Vogel, 2009). studies of membrane proteins are usually Because the major anionic lipids in performed in membrane-mimetic models. bacterial membranes are phosphatidyl- Lipid bilayers are regarded as the closest glycerols (PGs), high-resolution structures of membrane-mimetic model (Fig. 9.3A). This AMPs in such an environment are of great model is suitable for solid-state NMR studies interest. We have demonstrated structural that provide insight into peptide orientation determination of AMPs in PGs with an acyl as well as aggregation state in lipid bilayers chain length ranging from six to ten (Wang et (Opella and Marassi, 2004; Mani et al., 2006; al., 2003; Wang, 2008c). We chose dioctanoyl Resende et al., 2009). As the name implies, PG (D8PG) because it can stabilize the bicelles (Fig. 9.3B) are a hybrid of bilayers membrane-bound structures of AMPs at a and micelles. The size of a bicelle is much lower concentration than dihexanoyl determined by the molar ratio between the PG. A low concentration of protonated D8PG long- and short-chain lipids. While large reduces the intense micelle proton signals, bicelles are good for solid-state NMR, smaller thereby improving the quality of NMR bicelles (molar ratio <0.25) are useful for spectra. It is also important to control the pH solution NMR studies. Structural determin- of the sample to about 5–6. A lower pH can ation in this interesting model is gaining reduce the solubility of the AMP–D8PG momentum (Losonczi and Prestegard, 1998; complex and a high pH can cause the loss of Opella and Marassi, 2004; Prosser et al., 2006; important amide proton signals (Wang, Wu et al., 2010). Detergent micelles (Fig. 2007). For NMR analysis, high temperatures 9.3C), nevertheless, are most commonly used are helpful owing to faster tumbling of the in solution NMR studies. Keifer et al. (2004) peptide–micelle complexes in solution. compared the translational diff usion Indeed, improved NMR spectra have been coeffi cients of a membrane-targeting peptide reported at 50°C (Park et al., 2007; Wang, Structural Studies of AMPs 147

(A) Lipid bilayer

(B) Bicelle (C) Micelle

Fig. 9.3. Three membrane-mimetic models for NMR studies. (A) A lipid bilayer is composed of long-chain lipids; (B) a bicelle comprises a mixture of short- and long-chain lipids; and (C) a micelle consists of short-chain lipids (see the text for more details).

2008c). We also suggest the use of the NMR dimensional NMR methods are well suited chemical shift standard externally, since for structural studies of AMPs because of anionic compounds such as sodium their relatively small size (usually <50 2,2-dimethyl-silapentane-5-sulfonate (DSS) residues). The standard set of 2D (1H, 1H) may interact with cationic AMPs (Wang et al., NMR experiments includes total correlation 2003). Note that DSS is unable to form a spectroscopy (TOCSY), nuclear Overhauser micelle by itself based on translational eff ect spectroscopy (NOESY) and double- diff usion measurements by NMR (Keifer et quantum-fi ltered correlation spectroscopy al., 2004). (DQF-COSY). These NMR experiments allow for sequential signal assignments in two steps (Wüthrich, 1986). First, the amino 9.3.2 NMR methods acid spin systems are identifi ed in the TOCSY spectrum and verifi ed in DQF- Two-dimensional NMR methods COSY. Secondly, the relationships of these NMR spectroscopy was established as a spin systems are established based on structural tool by Kurt Wüthrich (2002 NOESY spectra. In principle, nuclear Nobel Laureate), whose group determined Overhauser eff ect (NOE) cross peaks can be the fi rst protein structure during 1984–1985. detected when two protons are within 5 Å. The earlier 2D homonuclear NMR work of Signal assignments constitute a critical step peptides and proteins is summarized in a towards structural determination of AMPs classic book by Wüthrich (1986). Two- (Fig. 9.1). 148 G. Wang

Natural abundance NMR spectroscopy and shift s can be employed to provide dynamic its applications information. The basis of this application is the observed correlation between 15N The birth of cryogenic NMR probes increased heteronuclear NOE values and secondary the signal to noise ratio by several-fold, 13Cα shift s (Wang, 2010). A structured region allowing the recording of 2D heteronuclear is rigid with positive 15N NOE values and correlated spectra at the natural abundance large 13Cα secondary shift s. In contrast, (0.36% for 15N and 1.11% for 13C). High residues in a disordered region display concentrations of peptides, if possible, are negative 15N NOE and small 13Cα secondary always recommended to further improve the shift s. Therefore, a plot of the secondary 13Cα signal to noise ratio. I routinely collect (1H, shift s versus residue number of AMPs can be 15N) or (1H, 13C) heteronuclear single- used to locate rigid or fl exible regions. quantum coherence (HSQC) spectra for Fourthly, the secondary 13Cα plot may also be micelle-bound peptides. The heteronuclear applied for structural validation. A poorly chemical shift s can be assigned based on the defi ned region may result from insuffi cient known proton resonances achieved from 2D NMR restraints or peptide motion. The NMR. The 15N and 13C chemical shift s have poorly defi ned region may be regarded as multiple applications. First, they can be truly fl exible if the 13Cα secondary shift s are employed to verify 1H chemical shift also small in the corresponding region. Lastly, assignments (Wang, 2006). Secondly, the plot of (1H, 13C) cross-peak intensity as a heteronuclear chemical shift s also contain function of residue number displays a wave structural information. Statistical analysis of patt ern. Interestingly, residues with lower protein NMR chemical shift s has revealed peak intensities tend to be hydrophobic. This that 13Cα are up- and downfi eld shift ed in fi nding may off er an approach for identifying β-sheet and α-helical structures, respectively. micelle-binding residues (Wang, 2010). This means that the 13Cα secondary shift s (diff erences between the measured and random shift s) are positive in helical regions Heteronuclear 3D NMR studies of isotope- β and negative in -stranded regions (Wishart labelled AMPs and Sykes, 1994). Such empirical relationships enable the identifi cation of secondary For helical peptides or those with diffi cult structures (α-helices or β-strands) in poly- sequences, 2D NMR may not provide peptides (Fig. 9.1). Since 13C chemical shift s suffi cient spectral resolution (see Fig. 1 in are related to protein dihedral angles, Ad Bax Wang, 2008c). Under these circumstances, 3D and colleagues have established and heteronuclear NMR methods are needed. improved the TALOS program, which The power of 3D NMR results from its high predicts backbone dihedral angles of resolution by separating the signals in a polypeptides based on 1Hα, 13Cα, 13Cβ, 13C crowded 2D spectral region onto multiple 2D carbonyl and 15N chemical shift s (Shen et al., planes along the third dimension, allowing 2009). For unlabelled AMPs, fewer than fi ve structural studies of proteins in the 20–30 chemical shift s have been found to be useful kDa range (Bax and Grzesiek, 1993). For in predicting backbone angles (Wang, 2007). small AMPs, an 15N-labelled peptide might Therefore, the measurement of natural be suffi cient to resolve the overlapped cross abundance chemical shift s off ers a practical peaks by recording 3D-edited TOCSY and approach to obtaining dihedral angles, since NOESY (e.g. Park et al., 2007). In this case, it is diffi cult to measure scalar couplings for sequential signal assignments can still be micelle-bound peptides. The inclusion of achieved using the classic NOE-based natural abundance chemical-shift -derived method (Wüthrich, 1986). If spectral overlap backbone angles into structural calculations persists, triple-resonance NMR experiments has improved the structural quality of can be applied to a 15N,13C-labelled peptide. micelle-bound peptides (Wang et al., 2005). In this method, sequential signal assignments Thirdly, natural abundance 13C chemical are obtained by utilizing the simultaneous Structural Studies of AMPs 149

i and i – 1 types of through-bond connectiv- such as spin-spin coupling constants and 15 13 13 3 ities, typically from N to Ci and Ci – 1 in hydrogen bonds. The JHNHαvalues are >8 Hz pairs of experiments such as HNCACB in a β-stranded protein structure and <6 Hz and CBCA(CO)NH. While the HNCACB in an α-helical structure (Wüthrich, 1986). experiment provides through-bond connect- Doreleĳ ers et al. (2009) showed that more ivity information from the backbone amide recent soft ware could give bett er-quality proton to nitrogen as well as to α and β structures. An ensemble of structures is carbon nuclei from both residues i and i – 1, chosen, usually based on the lowest energy. CBCA(CO)NH only provides connectivities The root mean square deviation (RMSD) is from 1H and 15N to α and β carbon nuclei of calculated to refl ect structural precision. Low residue i – 1. The 1H, 13C and 15N chemical RMSD values indicate that the peptide region shift s of 15N,13C-labelled LL-37 in complex is well defi ned by a suffi cient number of with SDS or D8PG micelles were assigned in NMR restraints in that region; high RMSD this manner (Wang, 2008c). For more diffi cult values suggest motion. High RMSD values problems, four-dimensional NMR spectra may also result from insuffi cient NMR may be recorded as well. With 15N labelling, restraints due to poor spectral quality or heteronuclear NOEs and longitudinal and overlap. This issue can be resolved by transverse relaxation times (T1 and T2) of performing additional measurements, such backbone nitrogen nuclei can be measured to as of 15N NOE values (Wang, 2008c). Prior to provide site-specifi c information on peptide coordinate deposition into the Protein Data dynamics in solution (Kay et al., 1989). Bank (Fig. 9.1), the structural quality of the AMP should be validated by viewing the violated distance and angle restraints and NMR structures structural geometry as represented by a Ramachandran plot, and by using other tools The next critical step in structural such as PROCHECK. Structural coordinates determination (Fig. 9.1) is to convert NMR of shorter peptides (<23 residues) are now measurements into coordinates that allow us required to be deposited with the to view the folding of a polypeptide chain in BioMagResBank (Markley et al., 2008). space. Since NOE is inversely proportional to distance to the power of six (r–6), NOESY spectra yield a set of distance restraints for structural calculations (assuming the same 9.3.3 Three-dimensional structures of correlation time for the entire molecule). The antimicrobial peptides current practice is to classify the cross peaks into strong, medium, weak and very weak Based on 3D structures, AMPs are classifi ed and then convert them to distance ranges into four major groups: α-helices, β-sheets, (1.8–2.8, 1.8–3.8, 1.8–5.0 and 1.8–6.0 Å). The αβ structures and non-αβ structures (i.e. common lower boundary (1.8 Å) is the extended or loops) (Chapter 1). In the shortest distance for two protons to approach following discussion, β-sheet-containing in space during distance geometry or AMPs (i.e. β-sheets and αβ structures) are simulated annealing calculations. Thus, combined under the same umbrella of diff erent distance sets can be sampled within ‘β-sheet structures’. the distance ranges to generate multiple conformations that all satisfy the boundary conditions. This is why NMR structures AMPs with α-helical structures determined in this manner are usually reported as an ensemble of structures (10– More than 100 AMPs have been annotated as 50). However, the distance restraints obtained having helical structures in the APD (Wang from NOESY spectra are incomplete and et al., 2009). When multiple coordinates are structural calculations are normally available for a particular AMP in the Protein complemented with other NMR restraints Data Bank, only the high-quality structure is 150 G. Wang

linked to the APD and discussed herein. A pleurocidin (Syvitski et al., 2005), insect few AMPs are helical even in aqueous spinigerin (Landon et al., 2006) and human solutions, primarily due to the formation of LL-37 (Wang, 2008c) all contain a single helix-bundle structures. Saposin-like proteins helical region with one or both ends (approximately 80 residues) form pores in disordered. In the high-resolution structure bacterial membranes. Such polypeptides of micelle-bound LL-37 determined by 3D occur widely in nature, ranging from NMR, residues 2–31 form a continuous protozoan parasites (e.g. Entamoeba histo- helical structure, whereas the C-terminal lytica) to worms (e.g. Caenorhabditis elegans) residues 32–37 are disordered (Fig. 9.4C). and mammals (e.g. Sus scrofa), including Such a structure is fully consistent with humans. The helix-bundle structure of these 15N heteronuclear NOE measurements, sup- antimicrobial proteins is further stabilized by porting that only the C-terminus is fl exible. three disulfi de bonds. In the structure of The high-resolution LL-37 structure deter- caenopore-5 from C. elegans (Pro81 cis mined by 3D NMR methods is interesting in conformer in Fig. 9.4A), the fi ve helices are several aspects. First, it is the longest located between residues 6–16, 24–36, 45–50, continuous helix (with 30 residues) found for 58–66 and 70–78 (Mysliwy et al., 2010). In AMPs to my knowledge. Secondly, the addition, there are two S–S bonds (Cys6- hydrophobic surface of the amphipathic Cys80 and Cys9-Cys74) between helices I helix is punctuated by a hydrophilic residue and V at the N- and C-termini of the protein; Ser9, leading to segregation of the hydro- the third S–S bond (Cys35-Cys49) is formed phobic surface into two regions. Thirdly, between helices II and III. This AMP plays an there is a helical bend between residues essential role in the survival of the worm by 14–16, and all hydrophobic side chains are eliminating any E. coli ingested. Upon located on the concave surface of the curved association with bacterial membranes, the structure. Interestingly, the corresponding helix bundle structure may open at a site that helical-bend residues in homologous primate has several exposed hydrophobic side chains cathelicidins (Zelezetsky et al., 2006) are (arrow in Fig. 9.4A). Despite a similar protein usually glycines (Wang, 2008c). These data fold, the antibacterial activity of tick suggest that the LL-37 structure determined microplusin is att ributed to Cu2+ binding, in SDS micelles serves as a useful model another antimicrobial mechanism diff erent to understand the activity of homologous from pore formation in bacterial membranes cathelicidins. Fourthly, the aromatic– (Silva et al., 2009). The structures of both aromatic packing at the N-terminal region of caenopore-5 and microplusin were deter- LL-37 is proposed to play a role in peptide mined by 3D heteronuclear NMR techniques. aggregation, as well as to confer chemotactic Another unique AMP that forms a helix and cytotoxic properties to the peptide. bundle structure in water (determined by 2D Some AMPs have been found to adopt a solution NMR) is distinctin. This amphibian helix-hinge-helix motif (also referred to as a peptide comprises two chains linked by one ‘helix-break-helix’ or ‘helix-turn-helix’ motif). S–S bond. This S–S bond is critical for Examples are insect cecropin A (Holak et al., structure stability to proteases (Fig. 9.4B) 1988), fi sh pardaxin 4 (Porcelli et al., 2004), rather than antimicrobial activity (Dalla Serra spider latarcin 2a (Dubovskii et al., 2006), et al., 2008). Recent solid-state NMR studies amphibian gaegurin 4 (Chi et al., 2007) and have revealed that both helices are located on dermaseptin B2 (Galanth et al., 2009). The the membrane surface of lipid bilayers, linker region of these peptides was proposed excluding the possibility of pore formation to be important for antimicrobial activity, (Resende et al., 2009). allowing optimal binding of both helices to In the membrane-bound state, a variety bacterial membranes (Park et al., 2007; of helical structures have been determined Galanth et al., 2009). Normally, these AMPs for linear AMPs with <40 residues. induce positive membrane curvature, leading Amphibian magainin 2 (Gesell et al., 1997), to toroidal pore formation or micellization of dermadistinctin K (Verly et al., 2009), fi sh membranes (Haney et al., 2010). However, Structural Studies of AMPs 151

(A) (B)

S9 F17 F15 F27 F5/F6

(E) (F)

F3 F13 F17 F27 Fig. 9.4. Three-dimensional structures of AMPs from the α-helical family. Depicted are: (A) caenopore-5 from Caenorhabditis elegans (Protein Data Bank (PDB) ID: 2JS9) (arrow, exposed hydrophobic side chains); (B) distinctin, a two-chain AMP from frogs (PDB ID: 1XKM); (C) LL-37, a cathelicidin from humans (PDB ID: 2K6O); (D) pardaxin 4 from ﬁ sh (PDB ID: 1XC0); (E) FK-13, an LL-37 antimicrobial core peptide discovered by NMR (PDB ID: 2FBS); and (F) aurein 1.2 from Australian bell frogs (PDB ID: 1VM5). In the short peptides (panels C–F), phenylalanine residues (labelled) are common and are important for anchoring these AMPs into bacterial membrane. To clearly view the polypeptide fold, the disulﬁ de bonds in S–S-containing peptides are not displayed here, but are described in the text.

pardaxin 4 was found to induce negative vesicles, but vesicle lysis in the presence of curvature in lipid bilayers. The aromatic side anionic PG (Vad et al., 2010). chains are not located on the same side in the In the helical family, AMPs with longer 3D structure (Fig. 9.4D). In fact, they might polypeptide chains tend to display toxicity to not be needed or able to, because the mammalian cells (Ciornei et al., 2005; N-terminal short helix is rather hydrophobic Dubovskii et al., 2006). This observation laid and can insert into the membranes. The the foundation for truncating such AMPs to C-terminal helix is amphipathic and its improve peptide selectivity. By using NMR, membrane orientation and mode of action we have identiﬁ ed a 13-residue antibacterial depend on lipid chain length and com- core peptide (FK-13) corresponding to position (Porcelli et al., 2004). This peptide residues 17–29 of human LL-37 (Li et al., induces pore formation in phosphocholine 2006a). FK-13 showed a similar antibacterial 152 G. Wang

activity to LL-37 and adopted a helical backbone dihedral angles of a d-amino acid. structure upon association with micelles (Fig. Indeed, the protein fold was retained when 9.4E). Subsequently, Phe17 of FK-13 was Gly16 was changed to a d-amino acid found to be critical for cytotoxicity to human (alanine, glutamic acid, phenylalanine, argin- cells, because removal of this residue led to ine, threonine, valine or tyrosine), but the KR-12 that displayed selective toxicity fold was disrupted when Gly16 was changed against E. coli (Wang, 2008c). FK-13 is com- to l-alanine (Xie et al., 2005). parable in size to aureins and temporins, the Likewise, structures of hBD-1, hBD-2 shortest helical peptides from amphibians in and hBD-3 have been determined by X-ray the APD. For comparison, the high-quality diff raction and NMR (Hoover et al., 2000, structure of micelle-bound aurein 1.2 is 2001; Bauer et al., 2001; Sawai et al., 2001; presented in Fig. 9.4F (Wang et al., 2005). Schibli et al., 2002). Human β-defensins Interestingly, we obtained a peptide that contain one N-terminal helix packed on the shows sequence homology to aurein 1.2 aft er three β-strands (Fig. 9.5, panels F–H). Using reversing the sequence of FK-13 (Li et al., hBD-1 as a model, Pazgier et al. (2007) 2006b). Retro-FK13 (sequence in Table 4.2) determined the structure of ten mutants by showed a higher antibacterial activity than X-ray diff raction. These mutants have a aurein 1.2 owing to additional cationic protein fold that is identical to the wild type. residues (fi ve versus two). In contrast, aurein It is proposed that the charged residues 1.2 and the non-toxic bacterial membrane Arg29, Lys31, Lys33 and Lys36 are critical for anchor both have two positively charged antibacterial activity, whereas the N-terminal residues in their membrane-binding region. helical region (residues 1–8), including The antibacterial activity of aurein 1.2 was adjacent residues such as Lys22, Arg29 and att ributed to a longer and broader hydro- Lys33, form the surface for chemotaxis to phobic surface than that of the membrane CCR6-transfected HEK-293 cells. It is more anchor. These structure–activity relationship convenient to map the binding sites by NMR. studies have revealed and emphasize the This is because membrane binding to an importance of both the hydrophobic surface isotope-labelled protein can cause selective and cationic side chains of the amphipathic peak shift s of those residues that form the helix in binding to bacterial membranes. The binding surface (this is also the principle for combined consideration of charge and a screening potential drug molecules by NMR) hydrophobic surface is important, because (Shuker et al., 1996). As an example, the we have found it diffi cult to correlate a single NMR-identifi ed membrane-binding sites on structural parameter (e.g. helicity, net charge, the structure of plant defensin Psd1 are hydrophobic surface area, transfer free indicated by an arrow in Fig. 9.5I (De energy) with antibacterial activity in a series Medeiros et al., 2010). of helical peptides (Wang et al., 2005). X-ray crystallography and NMR provide complementary information under diff erent conditions. While the structural coordinates AMPs with β-sheet structures determined in crystals are normally more Human defensins have a folded structure in accurate, NMR can be applied to the study of water, enabling crystallization and structural peptide dynamics in solution (Skalicky et al., determination by X-ray crystallography (Hill 1994), as well as the interaction with bacterial et al., 1991; Szyk et al., 2006). Human membranes (see below). In addition, NMR α-defensins share a similar three antiparallel also provides insight into the oligomerization β-strand fold stabilized by three S–S bonds of defensins in solution. While a dimer for (Fig. 9.5, panels A–E). The disulfi de bonds, as hBD-1 and octamer for hBD-2 have been well as salt bridges, are critical to maintaining found under crystal conditions (due to the defensin fold. Using HNP-2 as a model, molecular packing), they are monomers in the structural basis for the Gly-Xaa-Cys motif solution. However, hBD-3 is a dimer in (Chapter 4) was elucidated. The conserved solution. The dimeric structure of hBD-3 is glycine in the classic β-bulge has the proposed to be important for its greater Structural Studies of AMPs 153

(A) (B) of the peptide enhances antimicrobial activity (Klüver et al., 2006). Further increases in hydrophobicity make the peptide cytotoxic to human cells. The hydrophobic residue percentages of defensins are given in Table 9.1. Because segments of defensins have been (C) (D) shown to be antibacterial, the entire sequence is not a must. Furthermore, the cysteine residues are not required for antibacterial activity (Hoover et al., 2003). However, the (E) (F) fi ft h cysteine residue is critical for chemotaxis activity (Taylor et al., 2008). Recent NMR studies have revealed that, without cysteine residues, the hBD-3 mutant adopts a two- domain helical structure in micelles (G) (H) (Chandrababu et al., 2009), which diff ers entirely from the structure of native hBD-3 (Fig. 9.5H). Therefore, the cysteine residues are essential for stabilizing the defensin folds (Fig. 9.5), which are essential for specifi c (I) (J) interactions with chiral molecular targets such as proteins. Indeed, disruption of the S–S bonds of HNP1 and HD5 is detrimental to their binding to Zn2+-dependent metal lo- protease (bacterial lethal factor) or HIV gp-120 (Wei et al., 2009). Fig. 9.5. Three-dimensional structures of AMPs Circular θ-defensins such as RTD-1 from the β-sheet family. Depicted are: (A) HNP-1 (Trabi et al., 2001) contain two antiparallel (Protein Data Bank (PDB) ID: 3GNY); (B) HNP-3 β-sheets stabilized by three S–S bonds (Fig. (PDB ID: 1DFN); (C) HNP-4 (PDB ID: 1ZMM); (D) 9.5J). Several linear AMPs adopt a similar HD-5 (PDB ID: 1ZMP); (E) HD-6 (PDB ID: 1ZMQ); β-hairpin structure. These include thanatin (F) hBD-1 (PDB ID: 1IJV); (G) hBD-2 (PDB ID: (Mandard et al., 1998), protegrin-1 (Aumelas 1FD3); (H) hBD-3 (PDB ID: 1KJ5); (I) Psd1 (PDB et al., 1996), tachyplesins (Laederach et al., ID: 1JKZ) (arrow, membrane-binding site); and (J) 2002), polyphemusin I (Powers et al., 2004), RTD-1 (PDB ID: 1HVZ). Structures in panels A–G were determined by X-ray crystallography, while gomesin (Mandard et al., 2002) and arenicin-2 those in panels H–J were determined by NMR (Ovchinnikova et al., 2007). Thanatin consists spectroscopy. For simplicity, the disulfi de bonds of only one S–S bond. This bridge is essential are not displayed but are described in the text. for specifi c interaction of the peptide with E. coli, but unimportant for non-specifi c binding to membranes of the Gram-positive bacter- antimicrobial activity than hBD-1 or hBD-2 ium Micrococcus luteus (Imamura et al., 2008). (Schibli et al., 2002). It is not clear whether Other AMPs listed above contain two S–S protein-binding potential (Table 9.1) is related bonds. The S–S bond distant to the β-turn to peptide oligomerization. Another factor region is more important than the one close that plays a role could be net charge, since to the turn. Similar to the situation with hBD-3 has a net charge of +11, higher than defensins (Landon et al., 2008), the diff erent that of other human defensins (Table 9.1). cationic and hydrophobic side chains present Bacterial membrane composition is on the same backbone fold (Fig. 9.5J) another important factor that determines the determine their antimicrobial activity antimicrobial activity and selectivity of spectrum (Rodziewicz-Motowidło et al., hBD-3 (Böhling et al., 2006). Indeed, an 2010). In addition, peptide oligomerization increase in positive charge or hydrophobicity plays a role. While solid-state NMR data 154 G. Wang

indicated the existence of diff erent oligomers adopted a non-αβ structure with a distorted for protegrin-1 in lipid bilayers (Mani et al., backbone. In these structures (Fig. 9.6), the 2006), molecular simulation led to an octamer six-membered aromatic rings of tryptophan model as the active conformation in bacterial residues tend to point towards the membranes (Langham et al., 2008). membrane. In the case of indolicidin and lactoferrin, this is supported by fl uorescence spectroscopy as well as by 5-doxyl stearic AMPs with non-αβ structures acid spin label experiments. The lactoferrin Several tryptophan-rich peptides have been peptide could be further shortened to a found to adopt non-αβ structures (extended, hexamer fragment with a similar anti- turns or loops). Rozek et al. (2000) found that microbial activity to the long fragment (Chan the CD spectra of indolicidin in the presence et al., 2006). All of these NMR structures of membrane-mimicking vesicles or micelles indicate a unique role for tryptophan in did not resemble those of either α-helical or micelle binding. Tryptophan residues have β-sheet proteins. In DPC micelles, NMR long been recognized as an important structural determination revealed a unique membrane anchor, preferring the membrane– amphipathic structure, where the middle water interface (Wang et al., 1996; Yau et al., tryptophan-rich region plays the major role 1998; Schibli et al., 1999). Arginine residues to anchor the peptide into the micelle, while provide positive charges for recognition of the peptide termini are exposed and negatively charged bacterial membranes. The sampling a wide conformational space (Fig. importance of these two residues is further 9.6A). In the structure, the aromatic rings of verifi ed by the identifi cation of hexapeptides Trp6 and Trp9 pack against Pro7 and Pro10, based on com binatorial library screening respectively. It is worth noting that the (Chapter 5). These hexamers are rich in both structure of indolicidin in SDS micelles arginine and tryptophan residues and adopt somewhat diff ers from that in DPC. Such a extended structures in micelles (Rezansoff et diff erence could be real, as CD spectra of the al., 2005). peptide in lipid vesicles or in micelles also Then what is the minimal length for a diff er (Rozek et al., 2000). Likewise, Vogel tryptophan/arginine-rich peptide to be active and colleagues found that tritrpticin adopted against bacteria? Strøm et al. (2003) answered an amphipathic turn structure in SDS this question. While several hexapeptides micelles (Fig. 9.6B), where the three with 50% of each residue were eff ective tryptophan residues are clustered, but against both E. coli and S. aureus, only one terminal cationic residues occupy a broader pentamer with three tryptophan residues conformational space, implying motions (WRWRW-amidated) showed good activity (Schibli et al., 1999). Tinoco et al. (2002) found against both bacteria. Synthetic tetramers a WW+ motif in micelle-bound PW2 (Fig. were inactive. It appears that fi ve residues are 9.6C), an AMP (sequence: HPLKQYWWRPSI) required for a tryptophan/arginine-rich obtained from phage display libraries. The peptide to be active. (Coincidently, the WW+ motif was also found in other peptides. shortest AMPs collected in the APD also have It is remarkable that the side chain of Arg9 is fi ve residues (sequences: ACSAG, AMVSS well defi ned in this case and packs against and AMVGT). It is not clear whether these the aromatic ring of Trp8, providing an earthworm peptides also act by binding to example for the cation–π interaction. The bacterial membranes.) However, the inactive aromatic ring has π electron clouds on the tryptophan/arginine peptides are useful surface, whereas the arginine side chain templates to design selective AMPs (for other donates positive charge (Burghardt et al., strategies, see Chapter 4). By att aching three 2002; Chan et al., 2006). As a fourth example, hydrophobic tert-butyl moieties to the the structure of a tryptophan-rich segment tryptophan ring, Haug et al. (2008) made an corresponding to residues 4–14 of lactoferrin inactive tripeptide RWR bactericidal. B2 is presented in Fig. 9.6D (Nguyen et al., Importantly, the tripeptide analogue only 2005). In SDS micelles, this peptide also associated with anionic membranes, but not Structural Studies of AMPs 155

Fig. 9.6. Structural ensembles of AMPs from the non-αβ family. Presented are: (A) 16 structures of bovine indolicidin in dodecylphosphocholine micelles (Protein Data Bank (PDB) ID: 1G89) with the backbone atoms of residues 6–11 superimposed; (B) 19 structures of tritrpticin in sodium dodecylsulfate (SDS) micelles (PDB ID: 1D6X) with the backbone atoms of residues 4–11 superimposed; (C) 19 structures of PW2 in SDS micelles (PDB ID: 1M02) with the backbone atoms of residues 5–9 superimposed; and (D) 20 structures of lactoferrin B2(4–14) in SDS micelles (PDB ID: 1Y5C) with the backbone atoms of residues 3–5 superimposed. Key residues are labelled. All structures were determined by two-dimensional NMR.

with lipid vesicles consisting of phospho- AMP is determined by a proper display of cholines. This example indicates the feasibility hydrophobic and cationic side chains on the of engineering peptides. peptide surface, rather than the structural In summary, membrane-targeting AMPs scaff olds underneath the peptide backbone. can adopt various 3D structures, ranging from α-helices to β-sheet, αβ-fold and non-αβ structures (Figs 9.4–9.6). Furthermore, pep- 9.3.4 Interactions of antimicrobial tides with diff erent 3D structures can induce peptides with bacterial membranes by similar eff ects, such as positive curvature in NMR spectroscopy membranes (Haney et al., 2010) or lipid domain formation (Epand et al., 2010). The interactions of human LL-37 with Therefore, it is not the specifi c type of 3D bacterial inner and outer membrane structure of the peptide, but the amphipathic components nature that is critical for membrane targeting. Because of this, we have proposed a model HSQC spectra are useful to follow the that unifi es membrane-targeting AMPs conformations of human LL-37 in diff erent (Wang et al., 2005). In this universal model, states. In an HSQC spectrum, the backbone the membrane perturbation potential of an amide proton of each amino acid gives only 156 G. Wang

one cross peak with a covalently bonded model, since it is a PG with shorter acyl nitrogen nucleus. In water and at an acidic chains. The NMR T1/T2 ratio-derived pH (Fig. 9.7A), the (1H, 15N) cross peaks of correlation times suggest that the LL-37– LL-37 are sharp and clustered in a narrow D8PG complex (6.9 ns at 50°C) tumbles spectral region of 8.0–8.6 ppm, indicating a slightly slower than the LL-37/SDS complex randomly coiled structure as previously (7.0 ns at 25°C). This may explain why the suggested by CD (Johansson et al., 1998). At NMR spectra of LL-37 in D8PG were poor at approximately pH 7, only a few cross peaks lower temperatures, but became well remain (Fig. 9.7B), probably due to the dispersed at 50°C (Fig. 9.7D). However, all formation of elongated aggregates (Li et al., the NMR data collected for LL-37 in the two 2007) and perhaps structural heterogeneity. membrane models are remarkably similar Since CD spectra indicate a helical structure and indicative of similar structures (Wang, at this pH (Johansson et al., 1998) and size- 2008c). In addition, we found similar con- exclusion chromatography suggests a formations for several other peptides in SDS tetramer for synthetic LL-37 (Li et al., 2007), and D8PG micelles (Wang et al., 2003, 2004, we propose that LL-37 adopts a four-helix 2005; Li et al., 2006a). Therefore, the LL-37 bundle structure at the physiological pH. As structure determined in SDS (Fig. 9.4C) can only Ser37 is clearly visible, Fig. 9.7B also be applied to the PG case as well. In implies that residues 2–36 of LL-37 par- summary, the high-quality structure of LL-37 ticipate in tetramer formation. Such a helix provides a basis for understanding its bundle could dis sociate into monomers interactions with bacterial outer and inner when bound to anionic membranes (Oren et membranes. While nearly the entire LL-37 is al., 1999). required for peptide–peptide interactions (i.e. LPS is the major outer membrane oligomerization), only the long amphipathic component of Gram-negative bacteria. For helix is involved in peptide–membrane some AMPs, binding to LPS is the fi rst step in interactions (Fig. 9.7). interaction with such bacteria. Furthermore, LPS can cause sepsis. In this sense, the Atomic insight into peptide–membrane binding of AMPs to LPS is a desired property. interactions In the presence of LPS (Fig. 9.7C), the cross peaks for the N-terminal region of To provide additional insight into the 15N-labelled LL-37 disappeared, but the mechanism of action of LL-37, we also signals for a few C-terminal residues were explored the possibility of detecting peptide– detected. This NMR spectrum implies that lipid interactions directly by NMR. We noted the N-terminal portion of LL-37 is bound to that the amide proton signals of arginine side LPS and forms large complexes, leading to chains of LL-37-derived fragments tend to line broadening. The observation of intense overlap with aromatic protons of phenyl- signals for C-terminal residues 35–37 at alanine residues in SDS. In DPC micelles, nearly the same positions in the spectrum as they can overlap with the backbone amide observed for them in SDS or D8PG micelles protons. However, they are well resolved in indicates that the tail of LL-37 is also fl exible D8PG micelles (see Wang, 2008b). This in complex with LPS. In the presence of lipid unique spectral window for arginine side A (portion of LPS, see Fig. 7.1), CD spectra chains enabled the detection of arginine–PG indicated a helical structure (Turner et al., interactions by solution NMR for the fi rst 1998). Therefore, the ordered–disordered time (Wang, 2007). A useful strategy is to structural model of LL-37 determined in SDS draw one-dimensional NMR slices at micelles (Fig. 9.4C) can be applied to the LPS resolved lipid signals. The well-resolved case. Consistent with this model, deletion of a D8PG protons at 2.34 ppm (C2-H) and 5.25 few N-terminal residues from LL-37 reduced ppm (Hβ) (lipid head group) enabled LPS binding activity (Kirikae et al., 1998). through space 1H–1H dipolar interactions Next, LL-37 moves towards the inner with the arginine side chain amide protons membrane of bacteria. We utilized D8PG as a at 7.3–7.5 ppm (summarized in Fig. 9.8). As Structural Studies of AMPs 157

Fig. 9.7. Heteronuclear single-quantum coherence spectra of 15N-labelled recombinant LL-37 in water at: (A) pH 3.6 and (B) pH 6.9; (C) in the presence of lipopolysaccharide (LPS to peptide molar ratio 1:1, pH 5.4, 30°C); and (D) in the presence of D8PG (peptide to D8PG ratio 1:30, pH 5.4, 50°C). The arginine side chain peaks are boxed and asparagine/glutamine side chain signals are connected by lines. Figure adapted from Li et al. (2007) and Wang (2008c).

these intermolecular NOE cross peaks cationic AMPs can have a global impact on showed normal build-up curves with the the bacterium, in particular those biological increase in mixing time (Wang, 2007), they processes that require PGs. Examples are resulted from direct dipolar interactions bacterial signal-transduction pathways (Wang rather than spin diﬀ usions (Wüthrich, 1986). et al., 2003) and voltage-dependent potassium Thus, such NMR measurements provide channels (Schmidt et al., 2006). Therefore, direct evidence for the association of cationic lipid domain formation will perturb the peptides with anionic lipids. The arginine–PG bacterial membrane physiology, leading to interactions observed herein provide the bacterial death (Wang, 2008c). driving force for lipid domain formation in In addition, intermolecular NOE lipid bilayers induced by cationic peptides. observations provide evidence for the For KR-12, the smallest bactericidal fragment approximation of hydrophobic side chains of of LL-37, the lipid domain formation is peptides to acyl chains of lipid micelles (Fig. supported by solid-state NMR, diﬀ erential 9.8). For the bacterial membrane anchor, scanning calorimetry and freeze-fracture aurein 1.2 and LL-37 fragments (e.g. KR-12 electron microscopy (Epand et al., 2009, and GI-20, sequences in Table 4.2), 2010). The clustering of anionic PGs around intermolecular NOEs were detected from the 158 G. Wang

Fig. 9.8. Schematic representation of peptide–lipid interactions according to intermolecular nuclear Overhauser effect cross peaks measured for GI-20 in complex with dioctanoyl phosphatidylglycerol (D8PG) (peptide to D8PG molar ratio 1:5, pH 5.4, 25°C). GI-20 is a synthetic peptide corresponding to residues 13–32 of human LL-37, with the positions between residues Ile13 and Gly14 swapped

(sequence: GIKEFKRIVQRIKDFLRNLV-NH2). NH2 indicates C-terminal amidation. Note that Phe17 and Phe27 are likely to interact with different lipid molecules due to the excess of D8PG. For a detailed description, as well as the raw NMR data, please refer to Wang (2007a).

peptide aromatic rings to the protons of the Interactions of AMPs with specifi c membrane lipid head group as well as acyl chains (Wang components et al., 2003, 2005; Wang, 2007). Similar results were observed for the aromatic rings of Phe5, Bacterial AMPs also target bacterial mem- Phe6, Phe17 and Phe27 on the hydrophobic branes. Hsu et al. (2002) have investigated the surface of intact LL-37 in complex with D8PG interactions of nisin Z, a bacteriocin, with micelles (Wang, 2008c). We conclude that cell-wall precursor lipid II by NMR. Using an these peptides associated with the membrane 15N-labelled peptide, they found selective surface. In the case of amphibian aurein 1.2 chemical shift perturbation at the N-terminal and human LL-37, our conclusions are in line region of this lantibiotic, indicating that the with solid-state NMR measurements in lipid N-terminal region, including rings A, B and bilayers (Henzler Wildman et al., 2003; Balla et C, is responsible for nisin recognition. The al., 2004). Solution NMR, however, provides C-terminal region (rings D and E) appeared the long-desired evidence that these amphi- not to be involved in the recognition. pathic AMPs indeed associate with bacterial However, the hinge between these two membranes via both electrostatic and hydro- domains is critical for biological activity. phobic interactions. These results for animal Such an interaction patt ern is consistent with AMPs indicate that solution and solid-state mutagenesis studies as well as other NMR NMR studies provide complementary insight measurements such as temperature coef- into peptide–membrane interactions in the fi cients. The insertion of the C-terminus into diff erent membrane-mimetic systems depicted the membrane completes the pore formation in Fig. 9.3. process. Structural Studies of AMPs 159

In response to pathogenic invasion, successful example is the identifi cation of plants produce defence proteins that selective KR-12 from human LL-37 (Wang, recognize chitin, a polysaccharide in the 2008c). Secondly, peptide hydrophobicity can cell wall of fungi as well as in the also be diminished by altering the peptide exoskeleton of insects. Aboitiz et al. (2004) backbone structure. Shai and colleagues have established a structural model for a plant conducted extensive studies of the eff ect of hevein in complex with chitin based on selective d-amino acid incorporation on NMR data. The extended binding site of the peptide structure and activity. Their scheme protein can accommodate at least fi ve of incorporating d-amino acids at every two N-acetylglucosamine units. Plant and insect to three residues along the peptide chain has defensins also recognize sphingolipids in eff ectively disrupted helical structures of fungal membranes (Thevissen et al., 2004). In peptides based on CD and NMR studies the case of plant Psd1, De Medeiros et al. (Papo and Shai, 2004, and references cited (2010) identifi ed the binding sites for therein). Using this incorporation scheme, glucosylceramide (indicated by an arrow in we found a non-classical amphipathic struc- Fig. 9.5I) by NMR based on both chemical ture when residues 20, 24 and 28 of GF-17, an shift mapping and relaxation measurements. LL-37-derived fragment (sequence in Table The binding involves hydro phobic 4.2), were replaced by d-amino acids (Li et al., interactions with residues Val13, Phe15, 2006a). Backbone distortion caused an out- Ala18 and Trp38, as well as hydrogen of-phase packing of hydrophobic side chains bonding with Thr16 and Asn17. The recog- in GF-17D3 compared to the in-phase nition of unique fungal sphingolipids is hydrophobic packing in a regular amphi- essential for plant and insect defensins to pathic helix (see Fig. 9 in Li et al., 2006a). We induce subsequent events, release of reactive propose that the out-of-phase packing of oxygen species or cell-cycle inhibition that hydrophobic side chains provides a leads to cell death (Aerts et al., 2007; Lobo et structural basis for the decrease in peptide al., 2007). hydrophobicity, as refl ected by a short In summary, AMPs from bacteria, plants retention time on the reversed-phase high- and animals can recognize diff erent bacterial performance liquid chroma tography column. membrane components by adopting a variety of 3D structures (Figs 9.4–9.6). These structural data will be invaluable for the future engineering of diff erent molecular devices 9.4 Structure-based Peptide that specifi cally target certain microbes based Engineering on diff erences in membrane composition. The ultimate goal of peptide engineering is to improve peptide selectivity, effi cacy, stability, 9.3.5 Structural basis of peptide formulation and delivery. Structure-based selectivity design deals with one or more aspects of these properties to improve AMP druggability. For Due to cytotoxicity to human cells, many example, CP-11, an indolicidin derivative AMPs cannot be utilized directly. Select (sequence ILKKWPWWPWRRK-NH2), has strategies for improving peptide selectivity been determined by 2D NMR to have a (or therapeutic index) to reduce peptide U-shaped backbone with the N and C termini hydrophobicity have been discussed in close to each other when in complex with Chapter 4. These strategies can be grouped micelles. Based on this conformation, Rozek et into two types based on whether there is al. (2003) introduced a disulfi de bond between conformational change in the peptide the termini of CP-11. While CP-11 and backbone. First, the peptide can be partially cycloCP-11 showed similar antimicrobial truncated or mutated residue by residue to activity, the cyclized form was more stable to reduce hydrophobicity without changing the trypsin than its parent molecule. Micelle- structure of the peptide backbone. A bound structures of AMPs also guide the 160 G. Wang

design of non-peptidic compounds. By mim- have illustrated by NMR that the long icking the 3D structure of cyclo(RRWWRF), amphipathic helix of LL-37 is responsible for Appelt et al. (2007) success fully designed association with bacterial outer- and inner- peptide analogues without using the peptide membrane components (LPS and PGs) (Fig. backbone. Based on the novel amphipathic 9.7). In addition, we have observed direct structure determined by Li et al. (2006a), phenylalanine–PG and arginine–PG inter- Gellman and colleagues succeeded in actions by solution NMR. These data depict synthesizing random nylon co-polymers as a picture that the positively charged peptide peptide mimetics (Mowery et al., 2009). These is indeed located on the negatively charged studies, along with the synthesis of other membrane surface via both electrostatic and antimicrobial peptidomimetics (Chapter 6), hydrophobic interactions. Such interactions illustrates that the peptide backbone is not provide the driving forces for the formation absolutely required. Rather, a proper of non-lamellar phases or lipid domains. It is arrangement of functional groups into an useful to restate the membrane perturbation amphipathic scaff old is important. Structure- potential model (Wang et al., 2005). This based engineering is not limited to membrane- model emphasizes the importance of a bound AMPs. When a unique target molecule proper spatial presentation of cationic and is verifi ed within pathogenic microbes, novel hydrophobic side chains on AMP surfaces peptides can be rationally designed based on that determine its ability to perturb the the 3D structure of the AMPs in complex with bacterial membranes, as well as the outcome non-membrane targets such as proteins or of peptide–membrane inter actions that lead DNA. Future structural determination of such to bacterial death. Our investigations of complexes can borrow the NMR strategies peptide–membrane inter actions have already established and demonstrated for deepened and broadened our view on AMPs. protein–protein or protein–nucleic complexes In addition to lipid bilayers, AMPs also (Wang et al., 2000; Marintchev et al., 2007). target specifi c molecules on the cell surface. While lantibiotics such as nisins are known to bind to cell-wall precursor lipid II, insect 9.5 Concluding Remarks and and plant defensins specifi cally recognize Perspectives unique fungal sphingolipids. In other situations, AMPs also recognize carbo- AMPs can interact with membranes and non- hydrates. It is evident that NMR has played membrane targets. Much research has been an important role in providing atomic focused on the membrane-binding property insights into a variety of molecular inter- of AMPs (Chapter 7). In the membrane- actions involving cationic AMPs. bound state, they can adopt a variety of Not all AMPs target bacterial membranes backbone scaff olds (α-helices, β-strands and and interactions of AMPs with other targets extended structures). Typical examples in the have emerged (for examples, see Chapter 8). helical family are magainins, aureins, A hallmark for AMPs to act on non-chiral dermaseptins, temporins and human bacterial membranes is that the d- and cathelicidin LL-37. Human defensins are l-enantiomers with symmetric ‘mirror images’ typical members of the β-sheet family. have essentially identical antimicrobial Indolicidin and tritrpticin are representative activity (Wade et al., 1990; Wei et al., 2009). members of the extended-structure (non-αβ) This does not apply when AMPs target chiral family. Although these polypeptide back- molecules such as proteins. The interactions bones fold in diff erent manners, their of AMPs with non-membrane targets are surfaces share a common amphipathic nature important for many biological functions of (i.e. a clear segregation of hydrophobic and human cathelicidin and defensins. Under cationic side chains). Evidently, such a what conditions is there a correlation between feature is essential for a positively charged peptide aggregation and biological activity? amphipathic peptide to associate with How does an AMP traverse bacterial negatively charged bacterial membranes. We membranes and bind to intracellular targets Structural Studies of AMPs 161

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Paul A. Janmey* and Robert Bucki

Abstract

Under disease conditions, human LL-37 and other cationic antimicrobial factors are expressed but inactive due to their binding to anionic polyelectrolytes such as DNA, F-actin or acidic polysaccharides. This chapter discusses the basis for this electrostatic interaction and alternative strategies that liberate LL-37 and other amphipathic cations from the bound state so that they are available to ﬁ ght invading pathogenic bacteria. The potential of this approach for therapeutic use is discussed.

10.1 Introduction in the inner membrane, together with the requirement for appropriate hydrophobic 10.1.1 Electrostatic properties of LL-37 surfaces that disrupt bacterial lipid bilayers, can be satisfi ed by many peptide and non- Antimicrobial agents are generally, or perhaps peptide structures, and the guiding principles universally, multivalent cationic amphiphiles. for this polycationic and hydrophobic The fact that similar bactericidal eff ects can be combination are beginning to be elucidated achieved against Gram-negative and Gram- (Yang et al., 2007). The generality of this positive bacteria by natural peptides such as physico-chemical mechanism is perhaps the LL-37 expressed in humans (Burton and Steel, main reason why bacteria cannot easily 2009; Bucki et al., 2010), steroid derivatives develop resistance to these agents by such as squalamine expressed in dogfi sh mutations of single or even multiple genes, sharks and a host of synthetic peptide and but it also places restraints on the environ- non-peptide compounds strongly suggests ments in which antibacterial functions of that general physical chemical features, rather cationic amphiphiles can be maintained. In than stereospecifi c biochemical interactions particular, the antibacterial eff ects of LL-37, with a unique bacterial target, defi ne the which has a net charge of +6 at pH 7 (for the mechanisms by which these agents exert their micelle-bound three-dimensional structure, biological eff ect. The necessity for a poly- see Fig. 9.4C, Chapter 9), are lost in purulent cationic surface to direct the antimicrobial fl uids such as cystic fi brosis (CF) sputum. CF agent fi rst to the highly anionic bacterial cell sputum is highly enriched in the linear wall and then to negatively charged surfaces polyelectrolytes DNA and fi lamentous

* Corresponding author.

(F)-actin, glycosaminoglycans and mucins with the square of the distance between 2 produced by the host, as well as in anionic charges as: Epoint = q/4πεr . Here, q is the polyelectrolytes secreted by bacteria such as charge on the ion (in this case the cationic Pseudomonas (Weiner et al., 2003; Baranska- antimicrobial peptide (AMP)), ε is the Rybak et al., 2006; Bergsson et al., 2009). permitt ivity of water and r is the distance between the two charges. In contrast, the

ﬁ eld from a line with linear charge density λl 10.1.2 Interaction of polyvalent cations (such as F-actin, DNA or alginate) decays

with linear polyelectrolytes only linearly with r as: Eline = λl/2πεr. The fi eld from an infi nitely large charged surface Charged lines and surfaces interact with (here, the anionic surface of the bacterium) is soluble counterions diff erently from the a constant with respect to r for distances that interactions of two point charges, and when are small relative to the size of the cell. The the linear charge density of a rod-like fi lament simplest consequence of these laws is that for is suffi ciently high, more multivalent than a given number of anionic charges, arranging univalent counterions can condense on the them as distributed points, lines or surfaces fi lament surface. If a suffi cient number of has a large eff ect on their att raction for polyvalent counterions condense, the electro- counterions, with the greatest att raction for static repulsion between like-charged linear the charged surface. Another result of polyanions (DNA or F-actin in the case of CF) polyelectrolyte theory is that multivalent is overcome by att ractive interactions, and counterions are drawn to the fi lament much multivalent cations, such as LL-37, induce the more avidly than ions of smaller valence, formation of bundled fi lament arrays with largely because the entropy loss from counterions trapped inside the bundle. sequestering a smaller number of high- Experimental and theoretical work on valence ions to lower the surface charge of polyelectrolyte condensation, developed the fi laments is less than the same degree of initially to model the condensation of DNA charge neutralization due to condensation of (Perdue et al., 1976; Overman et al., 1998; a larger number of lower valence counterions. Fuller et al., 2007; Devkota et al., 2009), shows In addition, because the fi xed charges on that multivalent counterions do not bind actin, DNA and other biopolymers are tightly to the surface of the polyelectrolyte, univalent (phosphodiesters or carboxyls), but maintain mobility along the fi lament sequestration of multivalent counterions surface. When suffi cient counterions con dense produces regions of positive charge that can on the fi lament, the electrostatic repulsions lead to fi lament–fi lament interactions due to between fi laments that keep them separated the formation of structures analogous to become weaker than att ractive interactions Wigner latt ices (Lenac and Sunjic, 1991). and the fi laments collapse into bundles. These features of polyelectrolytes have Previous studies of F-actin (Tang and Janmey, recently been shown to account for unusual 1996; Tang et al., 1997) and alginate (Bu et al., structures formed in rings of F-actin bundled 2004; Donati et al., 2006) confi rm that the by divalent metal ions (Tang et al., 2001; polyelectrolyte behaviour fi rst observed with Cebers et al., 2006), and for the fact that DNA also applies to these fi laments, which fi lament bundles stabilized by counterions, also have fi xed charge spacing smaller than such as F-actin bundled by lysozyme the Bjerrum length and are therefore classifi ed (Guaqueta et al., 2006), cannot be dissociated as strong poly electrolytes. by increased monovalent ions. The mechanism by which polyelectrolyte There are many assumptions – including fi laments can trap multivalent cations such that the lines and surfaces are infi nite and as LL-37 depends on the strength of the uniform – but at the crudest level, this electric fi eld att racting the counterion to the hierarchy of electrostatic att ractions ration- fi lament. The electric fi eld, and therefore the alizes why cationic antimicrobial agents force on an ion, emanating from a point selectively att ack bacterial membranes charge (such as a small soluble ion) decays without an apparent unique bacterial binding Design of Alternative Antimicrobial Agents 171

partner, and why polyelectrolyte fi laments monomeric actin, which can polymerize and such as actin, alginate and DNA can inhibit form a mixed network of DNA and actin their functions. Of course, AMPs are not fi laments. F-actin bundles (Fig. 10.1) have simply cations and hydrophobic moieties. been identifi ed as an additional abnormal The juxtaposition of cationic charges with biopolymer that can aff ect the viscous spacing suitable for binding lipopoly- properties of lung airway fl uid in respiratory saccharide (LPS), lipoteichoic acid (LTA) or disease (Vasconcellos et al., 1994; Sheils et al., other anionic targets, and other modifi cations 1996). that optimize lethal eff ects on bacteria, are Bacteria also produce anionic poly- essential for the biological function of AMPs. saccharides, in part as extensions from the lipid A moiety of LPS and oft en as much longer polysaccharides extruded into the 10.2 Production of Anionic space surrounding the bacterium. During Polyelectrolytes by Host and infection, bacteria can begin to produce Microbial Sources exopolysaccharides – alginate in the case of Pseudomonas aeruginosa – and form biofi lms. During physiological cell turnover, anionic These biofi lms contain bacterial DNA polyelectrolytes located inside the cell, such (Whitchurch et al., 2002) along with other as DNA and F-actin, are not accumulated in polymers that create a hydrated and charged extracellular compartments due to suffi cient environment around the bacterial surface, phagocytic elimination of cell fragments preventing access by cationic antimicrobial generated during apoptosis. Bacterial agents such as LL-37. Other polyanionic infection rapidly leads to infi ltration of white blood cells, mainly neutrophils, which are (A) highly enriched in cytoskeletal proteins. During the course of necrosis, both the anionic cytoskeletal fi laments and DNA of the neutrophils that crawled into the extracellular fl uid, which contains bacteria, F-actin are released. Considering that DNA was fi rst isolated from pus due to its high concentration of neutrophils, prolonged infection may generally lead to neutrophil

accumulation and necrosis as well as other DNA host cell death that would release polyelectrolytes from the cytoskeleton and the nucleus into the extracellular space. Here, they can inhibit cationic antibacterial (B) peptides (CAPs) and potentially contribute to biofi lm production. This concentration of DNA and F-actin is most evident in sputum collected from CF patients with chronic bacterial infection of the lungs. Large DNA- containing fi bres, hundreds of microns long, have been documented in CF sputum (Fig. F-actin DNA 10.1A) since at least the 1960s (Chernick and Fig. 10.1. F-actin and DNA bundles in (A) cystic Barbero, 1959; Shak et al., 1990). They are also fi brosis sputum and (B) pus samples. F-actin and present in other infl ammatory sites char- DNA were visualized by Alexa Fluor phalloidin and acterized by pus formation (Fig. 10.1B). YOYO-1 labelling, respectively. The morphology of The necrotic death of neutrophils and corresponding cystic fi brosis samples is shown by epithelial cells that releases DNA into the phase contrast microscopy (black and white extracellular space also releases F-actin and images). Scale bars: 200 microns. 172 P.A. Janmey and R. Bucki

bacterial factors that can inhibit LL-37 and other AMPs are bacterial outer-wall con- stituents, LPS from Gram-negative bacteria such as P. aeruginosa and LTA from Gram- 5 positive bacteria, which both appear to be the targets of antimicrobial agents on the 4 bacterial cell wall (Gutsmann et al., 2005), but P. aeruginosa PAO1 ) P.

which once released from bacteria are potent –1 inhibitors of AMPs (Bucki and Pastore, 2006). 3 Complex formation by LPS with antibacterial peptides such as cathelicidins has been 2 studied to characterize how the peptides log (cfu ml 0 2 4 6 8 10 inhibit the endotoxic eff ects of bacterial LPS LL-37 (µM) (Nagaoka et al., 2001). Although systemic Fig. 10.2. Inhibition of LL-37 function by F-actin concentrations of LPS (endotoxin) are much and DNA. Growth of Pseudomonas aeruginosa lower than those of LL-37, in localized PAO1 in a microbroth dilution assay, after incuba- chronic infections LPS accumulation may tion with various concentrations of LL-37 alone lessen the eff ect of AMPs on the intact (open circles) or with the addition of DNA (tri- bacteria. Anionic glycosaminoglycans such angles) or F-actin (fi lled circles). cfu, colony as chondroitin sulfate, hyaluronic acid and forming units. various mucins that function at the surface of epithelial cells as part of a mucosal barrier 10.4 Releasing LL-37 from may also reduce the antibacterial activity of Polyelectrolyte Bundles CAPs. However, in the case of mucins and CAPs, which from a functional point of view In the absence of multivalent counterions, are both antibacterial, partial inactivation of the linear polyelectrolytes at sites of infection CAPs in the presence of mucins is – including DNA, F-actin, mucin, alginate compensated for by an increased local CAP and the micelles or other aggregates of LPS concentration at the epithelial surface due to and LTA from lysed bacteria – would be sequestration by these anionic polymers mutually repulsive and so tend not to (Felgentreff et al., 2006). aggregate. However, DNA and F-actin, the stiff est polyelectrolytes and therefore the easiest to visualize by light microscopy, are arranged in bundles that are many times 10.3 Sequestration and Inactivation of larger than the cells from which they come. LL-37 by DNA and F-actin These aggregates of like-charged polyelectrolytes must be stabilized by strong Both bactericidal and bacteriostatic functions forces that overcome their electrostatic of LL-37 are strongly inhibited by DNA and repulsions, and the multivalent elements of F-actin (Fig. 10.2). These two polymers are the innate immune system, including LL-37, highly concentrated at infl ammatory sites defensins and lysozyme, are the most such as in CF lung airway fl uid, where their abundant such molecules in airway fl uid and concentrations can reach up to approximately extracellular sites of infection. 20 and 2 mg ml–1, respectively. In the highly Immunohistochemical assays show that purulent sputum characteristic of chronic LL-37 is co-localized in bundles of actin and bacterial infection in the lungs, the expression DNA in CF sputum (Bucki et al., 2007a). In of antimicrobial factors is oft en upregulated, this scenario LL-37, even when upregulated but they are not active, presumably due to by the lung epithelium and released from their sequestration within polyelectrolyte neutrophils, would be trapped within bundles (Weiner et al., 2003). fi lament bundles and therefore unavailable Design of Alternative Antimicrobial Agents 173

to att ack bacteria. This hypothesis suggests combination of DNase I and gelsolin was that strategies that destabilize polyelectrolyte more eff ective than either treatment alone. bundles might release LL-37 and thereby Even high concentrations of LL-37 were not enhance endogenous antibacterial function able by themselves to eradicate bacterial even in the absence of increased LL-37 growth, but addition of gelsolin signifi cantly expression. Polyelectrolyte bundles can be increased the function of LL-37. However, dissociated by several mechanisms. If the upon action of DNase and gelsolin, which counterions are dynamic within the bundles, disaggregates CAPs–DNA–F-actin bundles, LL-37 might be released by competitive the effi ciency of CAP release may still be binding of a stronger polyvalent cation compromised if CAPs form precipitates with (Purdy Drew et al., 2009). short DNA or F-actin fragments.

10.4.1 Severing polymers with DNase, gelsolin and alginase

Because the electrostatic eff ects that sequester multivalent cations to the surface of a highly charged linear polyanion require that the linear polyelectrolyte is suffi ciently long as to be approximated as an infi nite line, decreasing the fi lament length to some critical value should abrogate counterion condensation. Experimental tests show that, for actin fi laments or DNA, this critical Fig. 10.3. Quantitative immunoblot analysis of length is in the order of a few hundred LL-37 released from cystic fi brosis sputum as a nanometres (Tang and Janmey, 1996; Tang et percentage of the total LL-37 concentration in the al., 1996). This degree of fi lament shortening whole volume of individual cystic fi brosis sputa. can be achieved by depolymerizing DNA Data represent the means of six different patient with DNase or severing actin fi laments with samples. Error bars represent standard deviations. gelsolin, a protein present in both cytoplasm *P<0.05 versus the respective values in super- and extracellular fl uids and capable of natant (unpaired t-test). GSN, gelsolin; poly-Asp, breaking the non-covalent bonds that hold polyaspartate. F-actin together (Yin et al., 1984). Indeed, release of LL-37 from a pellet and into a supernatant fraction has been observed when CF sputum samples were treated with DNase I, gelsolin, polyaspartate or their combination (Fig. 10.3). These data confi rm the hypothesis that LL-37 and other cationic antimicrobial factors are trapped in actin/DNA bundles in CF sputum and can be released by mucolytic agents directed at polyanionic fi laments. Additionally, in vitro studies show that, by depolymerizing actin with gelsolin or DNA with DNase, antibacterial function is partially restored in CF sputum. Figure 10.4 compares Fig. 10.4. Bacterial load of cystic fi brosis sputa the degree to which addition of gelsolin, before (control) and after treatment with 0.5 μM DNase and exogenous LL-37 lessen bacterial gelsolin (GSN) or 100 μg ml–1 recombinant human colony growth from CF sputum. Each of DNase, alone or in combination with GSN, and these agents could decrease bacterial exogenous LL-37 peptide, alone (3 μM) or in outgrowth by approximately 50%, and the combination with gelsolin (0.5 μM). 174 P.A. Janmey and R. Bucki

Polyelectrolyte networks at the site of are stabilized by polyvalent counterions they infection can also directly aff ect invasive will be dissociated by the addition of soluble bacteria. It was recently shown that P. co-ions. This prediction has been verifi ed by aeruginosa growth, in sett ings that included experiments in vitro with purifi ed DNA or neutrophils, enhanced formation of biofi lms F-actin (Tang and Janmey, 1996). In the case of (a patt ern of bacterial growth associated with actin/DNA bundles in CF sputum, high resistance to antibiotic treatment) by polycationic ligands such as LL-37, histones stimulating the expression of alginate by P. and lysozyme trapped in the bundles will be aeruginosa (Walker et al., 2005). Neutrophils released when the polyelectrolyte bundles are likely to have multiple eff ects on P. dissolve, even if the fi laments do not aeruginosa biofi lm production, some involv- depolymerize. Soluble multivalent anions ing the eff ects of neutrophil-derived such as polyaspartate and polyglutamate have hydrogen peroxide on P. aeruginosa mucoid been shown to both reduce the viscosity of CF conversion. In vitro, however, extracts of sputum (Tang et al., 2005) and release LL-37 neutrophils are 94% as potent as intact into the soluble fraction (Fig. 10.3). neutrophils in enhancing biofi lm production The most signifi cant potential of anionic by P. aeruginosa. Of three fractions tested, polyaspartate is likely to be its ability to granular proteins and DNA had litt le to no restore, at least partially, the antimicrobial eff ect on biofi lm production, whereas activity of endogenous CAPs within purulent purifi ed F-actin had a strong stimulating fl uids such as CF sputum. This antimicrobial eff ect. Depleting actin from whole neutrophil activity is inactivated by a number of factors, lysates diminished their eff ect. Therefore, including polyelectrolyte fi laments (Tang et dissolution of bundles with polyanions and al., 2005). It has been shown that, consistent depolymerization of actin with gelsolin or with the electrostatic hypothesis (Tolker- DNase might reverse the strong eff ect of Nielsen and Hoiby, 2009), the addition of actin and possibly other cellular debris to polyaspartate to CF sputum lowered the stimulate P. aeruginosa biofi lm production. outgrowth of bacteria from these samples by In addition to its eff ect on actin, gelsolin 30%, while polyaspartate had no direct may have another benefi cial eff ect in infected antibacterial eff ect when added directly to P. fl uids. Gelsolin is a strong ligand for LPS and aeruginosa PAO1 (Fig. 10.3) (Tang et al., 2005). LTA (Bucki et al., 2005, 2008a) and can inhibit Increased doses of DNase or gelsolin together LPS and LTA cell immunostimulatory eff ects with polyaspartate may further improve the through inhibition of their binding to Toll-like release of LL-37. These data demonstrate the receptors, aft er their release from the bacterial potential for polyaspartate to lessen bacterial cell wall. Bacterial exopolysaccharides such as burden through a mechanism that liberates those produced by mucoid strains of P. antimicrobial functions already residing aeruginosa have been shown to interfere with within CF sputa. Additionally, polyaspartate the antibacterial activity of amino glyco- eff ectively prevents and disrupts P. aeruginosa sides and granulocyte-mediated non-opsonic biofi lm formation. This potentially thera- phagocytosis. However, these eff ects of peutic eff ect of polyaspartate is exopolysaccharides were reversed by pre- synergistically enhanced by DNase (Parks et treatment of the mucoid cells with alginase al., 2009). (Bayer et al., 1991; Hatch and Schiller, 1998; Alipour et al., 2009), again suggesting that decreasing polyelectrolyte length lessens the 10.5 Designing Antimicrobial Agents inhibition of antibacterial factors. Resistant to Inactivation by Polyelectrolytes

10.4.2 Destabilizing bundles with small In addition to treatments directed at making multivalent anions linear polyelectrolytes less inhibitory to the native antimicrobial agents resident within Theoretical models of polyelectrolyte solutions purulent sputum, alternative antibacterial predict that if bundles of charged ﬁ laments factors that are more resistant to inactivation Design of Alternative Antimicrobial Agents 175

by DNA or F-actin have potential for lytic eff ect (a measure of peptide toxicity for practical use. host cells) of CAPs has been based on rational modifi cations of existing peptide sequences. Since minor diff erences in amino acid 10.5.1 Cationic steroids: minimizing and sequence can produce signifi cant diff erences redistributing charge in antimicrobial activity (Raj et al., 2000), the possibility of strategically varying key amino AMP activity can be mimicked by cationic acids has been explored by many steroid antibiotics (CSAs), which were fi rst investigators (Travis et al., 2000; Hancock and developed with the intent of reproducing or Patrzykat, 2002; Jenssen et al., 2006; Saugar et improving the antibacterial activities of al., 2006; Zelezetsky and Tossi, 2006). polymyxin B (Ding et al., 2004). These Sequence modifi cations off er an enormous antibacterial agents can have potency similar number of combinatorial possibilities, includ- to that of LL-37, but compared to natural ing deleting, adding or replacing one or more antibacterial peptides their net positive charge residues, as well as truncating the peptide or is usually lower and chemical synthesis assembling chimeric peptides based on provides the opportunity to control their sequences from the same or diff erent species. charge distribution. One such synthetic com- In addition, conjugating peptides with pound, CSA-13, is signifi cantly less sensitive lipophilic acid (Avrahami and Shai, 2002) or to inactivation by DNA or F-actin compared rhodamine B (Bucki et al., 2004), incorporating to LL-37 and shows bett er effi cacy in CF carbonate bond(s) (Lee and Oh, 2000) or sputum (Bucki et al., 2007b). Synthetic CSAs peptoid residues (Song et al., 2005) and share some structural and functional α-amino acid epimerization (Mangoni et al., properties with squalamine, a membrane- 2006) have been found to change peptide active CSA (Moore et al., 1993) that was fi rst physico-chemical properties and oft en isolated from tissues of the dogfi sh shark. translate to bett er biological activity. Enhanc- Their bactericidal properties are due to ing antibacterial potency by manipulating membrane disruption and they display a hydrophobicity proves the potential to reduce moderate degree of selectivity for prokaryotic or reorganize cationic moieties, thereby over eukaryotic membranes (Salunke et al., weakening interactions with anionic poly- 2006). Several cholic acid derivates (Bellini et electrolytes. However, modifi cations that al., 1976) such as cholic acid with basic amino increase hydrophobicity are also likely to acids (Bellini et al., 1979; Li et al., 1999), result in increasing peptide toxicity towards guanidine (Savage and Li, 2000) and host cells. spermidine (Chen et al., 2006) have been characterized as potent antimicrobial agents that are eff ective against multidrug-resistant bacteria (Schmidt et al., 2001) and fungi (Hazra 10.6 Possible Therapeutic Use et al., 2004). In addition to these steroidal conjugates (Salunke et al., 2006), a conjugate of 10.6.1 Selective use in some body fl uids spermine with two dexamethasone moieties and not others displays strong antibacterial activity with low Bacterial infection takes place in various lytic eff ect on eukaryotic plasma membranes areas of the body and CAP activity may vary (Fein et al., 2009). This molecule also inhibits depending on the local environment factors. the infl ammatory response induced by Understanding the physiological homeostasis bacterial wall products in vitro. of CAPs in these sett ings is important to design eff ective antimicrobial and anti- 10.5.2 Increasing the hydrophobicity of infl ammatory therapies. Generally, CAPs, antimicrobial agents including human cathelicidin LL-37, have been shown to be active in body fl uids with The approach to enhancing the desired low protein concentrations such as airway bactericidal activity and reducing the haemo- surface fl uid (Bucki et al., 2007a), tears 176 P.A. Janmey and R. Bucki

(Huang et al., 2006), sweat (Rieg et al., 2006), phils (Barlow et al., 2006), the diff erentiation gastric juice (Leszczynska et al., 2009) and of dendritic cells that bridge innate with saliva (Gutner et al., 2009). However, several adaptive immune responses (Kandler et al., body fl uid components have been identifi ed 2006; Skokos and Nussenzweig, 2007) and as potent inhibitors of CAP functions. In the chemotactic activities of monocytes, blood, most natural and synthetic cationic eosinophils and T cells (De et al., 2000; antibacterial molecules lose their antibacterial Koczulla and Bals, 2003; Khine et al., 2006). activity due to their insertion into plasma LL-37 directly activates endothelial cells, lipoproteins and interaction with divalent which results in the increased proliferation cations. Accordingly, LL-37 is inactive in the and formation of vessel-like structures in cell presence of human serum, and its lack of culture. Decreased vascularization during antibacterial activity cannot be explained by wound repair in mice defi cient for CRAMP, proteolytic degradation. LL-37 activity the murine homologue of LL-37/hCAP-18, inhibition in human serum is mediated shows that cathelicidin-mediated angio- mostly by binding to human apolipoprotein genesis is important for cutaneous wound A-I (Wang et al., 1998, 2004). Interestingly, the neovascularization in vivo (Koczulla et al., de novo-engineered AMP WLBU2 resists the 2003). Additionally, LL-37 induces wound inhibitory action of blood and physiological healing, proliferation and migration of 2+ 2+ concentrations of Mg and Ca (Deslouches airway epithelial cells, suggesting that this et al., 2005), suggesting that it is possible to peptide might be involved in the regulation develop antibacterial peptides that will of tissue homeostasis in the airways maintain activity in serum. Mucin is another (Shaykhiev et al., 2005). The regenerative factor, widely present at the surfaces of activities of LL-37 support its therapeutic airway, digestive or urogenital tracts, that potential to promote wound healing can compromise the antibacterial function of (Tokumaru et al., 2005; Carretero et al., 2008). LL-37 (Bucki et al., 2008b). On the other hand, interaction of CAPs with mucin can increase the concentration of CAPs in the area most subject to microorganism att ack, and mucin 10.7 Conclusion has been shown to be involved in protecting peptides from proteolysis (Gutner et al., LL-37 and other cationic antimicrobial factors 2009). Antimicrobial activity at mucosal sites essential to the innate immune response and ocular surfaces is aff ected by interactions appear to function largely by physico- between diff erent classes of antimicrobial chemical interactions with bacteria in which factors (Nagaoka et al., 2000; McIntosh et al., electrostatic att raction is a fundamental 2005). feature. This mechanism also limits the chemical environments in which these cationic amphiphiles can perform as eff ective 10.6.2 Stimulation of host cells bactericidal agents. Release of polyanionic fi laments and other aggregates into the Considering that bacterial killing by LL-37 extracellular space in which infection occurs occurs primarily by depolarizing and can inactivate the antimicrobial eff ects of permeabilizing bacterial membranes or by LL-37, even under conditions where it is induced autolysis (Ginsburg, 2004), it has upregulated and protected against degrad- been suggested that a similar mechanism of ation. Multiple strategies directed at action can be used to target disruption of disrupting the complex of cationic AMPs cancer cells by C-terminal LL-37 fragments with anionic DNA, F-actin, alginate or other (Li et al., 2006). In addition to its bacteria- polyelectrolytes, or designing novel anti- killing ability, LL-37 can also aff ect host microbial agents that have less affi nity for leukocyte functions that are associated with polyelectrolytes, have the potential to host immune defence. LL-37 aff ects the life improve antibacterial therapies in respiratory span and infl ammatory functions of neutro- infections and other diseases. Design of Alternative Antimicrobial Agents 177

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(2005) Induction of template to guide structure–activity relationship keratinocyte migration via transactivation of the studies. Biochimica et Biophysica Acta 1758, epidermal growth factor receptor by the 1436–1449. 11 Role of Vitamin D in the Enhancement of Antimicrobial Peptide Gene Expression

John H. White* and Ari J. Bitton

Abstract

1,25-Dihydroxyvitamin D (1,25D), the hormonally active form of vitamin D, was initially identifi ed as a key regulator of calcium homeostasis. 1,25D signals through the nuclear vitamin D receptor, a ligand- regulated transcription factor. More recent studies have revealed that 1,25D regulates a number of physiological processes, including innate immunity. The innate immune system is responsible for rapid, non-specifi c host responses to pathogenic infection. Unlike adaptive immunity, innate immune responses do not confer lasting protection on the host. 1,25D induces the expression of LL-37 and hBD-2, antimicrobial peptides that serve as vanguards of innate immune responses. Antimicrobial peptides are small proteins with antibiotic properties that can also acts as signalling molecules to modulate specifi c responses such as wound healing and cytokine induction. This chapter focuses on the gene regulatory activity of 1,25D, with an emphasis on its role in the potentiation of innate immune responses and the promotion of antimicrobial peptide expression.

11.1 Vitamin D lished roles for vitamin D in controlling nervous system function and cell growth and Vitamin D is somewhat of a misnomer in that diff erentiation, and regulating immune sys- it is not a true vitamin by classical defi nition tem responses. At the molecular level, much (Crowle et al., 1987). A vitamin is an essential of the recent emphasis has refi ned the focus substance that is required for proper on vitamin D and its role as a potent physiological function, but is not produced in modulator of gene expression. suffi cient quantity by the body (Ziegler and Filer, 1996). As detailed below, we can generate physiologically adequate levels of 11.2 Early History of Vitamin D vitamin D from the action of solar ultraviolet (UV) light on the skin. Vitamin D has been Vitamin D therapy can be traced back to best known for its role in calcium homeostasis Hippocrates, the father of medicine, who and bone maintenance. However, there are used heliotherapy, or exposure to sunlight, to threads of evidence for a broader spectrum of treat phthisis (tuberculosis (TB)) (Masten, actions of vitamin D that extend back over 1935). Nutritional vitamin D therapy arose millennia. More recently, research has estab- from the early pharmacology of cod-liver oil

* Corresponding author.

use. Cod-liver oil was fi rst described as a temperate climates, this phenomenon is medicinal agent for the treatment of chronic commonly referred to as ‘vitamin D winter’. rheumatism in 1789. Throughout the next For example, one survey found widespread century, medical literature documented its vitamin D defi ciency among female eff ectiveness for treating a number of populations across Northern Europe prevalent conditions such as gout and (Andersen et al., 2005). Another study found scrofula, a form of TB that infects the lymph that a signifi cant portion of African-American nodes (Guy, 1923). Beginning in the 1820s, women living in the USA are seriously studies showed that administering doses of vitamin D defi cient (Chapuy et al., 1997). cod-liver oil to affl icted children could cure While correlations between vitamin D rickets (Guy, 1923), a nutritional disease defi ciency and disease go back for well over characterized by a lack of vitamin D or a century, more recent studies have estab- calcium leading to bone soft ening and lished a direct connection between the two deformity. However, it was not until several conditions. Since the association between TB decades later that the active compound in and vitamin D defi ciency was described over cod-liver oil was identifi ed as vitamin D. By 20 years ago (Davies et al., 1985), numerous 1849, the list of conditions treatable with cod- epidemiological studies have linked vitamin liver oil had grown to include TB (Grad, D defi ciency to increased rates of cancer, 2004; Martineau et al., 2007). multiple sclerosis, type I diabetes, Crohn’s Several independent observations in the disease and infection (Schwalfenberg, 2007; 19th and early 20th centuries fostered further White, 2008). links between sunlight and cutaneous vitamin D synthesis. This was highlighted by reports such as the following (Guy, 1923; 11.4 Vitamin D Signalling and Crowle et al., 1987; Grad, 2004; Lin and Mechanisms of Action White, 2004; Martineau et al., 2007): • A relationship was found between the Vitamin D is generated primarily by the prevalence of rickets and lack of exposure photochemical action of UVB radiation (295– to sunlight. 320 nm) in the skin (Fig. 11.1), but is also • UV light therapy was found to cure obtained from limited dietary sources such rickets. as fi sh oils and fortifi ed dairy products. • Feeding UV-irradiated skin to rats was Vitamin D refers collectively to closely found to have the same protective eff ects related molecules (Fig. 11.2) known as against rickets as did cod-liver oil. vitamin D2 (ergocalciferol) and vitamin D3 • UV light was also found to be useful in (cholecalciferol) (D’Aldebert et al., 2009). treating cutaneous TB (lupus vulgaris). Dietary vitamin D2 is derived from the fungal steroid ergosterol. Vitamin D3 is These studies support associations estab- found in animal sources such as cod-liver oil lished between vitamin D defi ciency and the and is generated by cutaneous photochemical prevalence of certain diseases. conversion of the cholesterol metabolite 7-dehydrocholesterol. Vitamin D undergoes a series of 11.3 Vitamin D Defi ciency hydroxylation reactions, which are required for both biological activation and deactiva- Today, vitamin D defi ciency is regarded as a tion of the molecule. First, 25-hydroxylation widespread and completely preventable is catalysed by the enzymes cytochrome health concern. Although there is no strict P (CYP)2R1 or CYP27A1, producing defi nition, vitamin D defi ciency is char- 25-hydroxyvitamin D (25D), the major acterized by low circulating vitamin D circulating vitamin D metabolite (Jones et al., metabolite levels, caused by a combination of 1998; Cheng et al., 2004; Prosser and Jones, seasonal variations in sunlight and 2004; Shinkyo et al., 2004; Holick, 2007). This inadequate dietary consumption. In more occurs primarily in the liver, but also in the Vitamin D and AMP Gene Expression 183

Vitamin D3 Skin Provitamin D3 Liver (skin) 25-OHase (CYP27A1 and others)

Kidney and peripheral tissues (incl. skin) 1α-OHase (CYP27B1) 1,25-Dihydroxy- 25-Hydroxy- vitamin D3 vitamin D3

Fig. 11.1. Biosynthesis of 1,25-dihydroxyvitamin D3. CYP, cytochrome P; VDR, vitamin D receptor. skin. Then, 1α-hydroxylation of 25D is of vitamin A, among others. Nuclear receptors catalysed by the enzyme CYP27B1, are composed of highly conserved DNA and producing hormonally active vitamin D, ligand-binding domains (Rochel et al., 2000; 1,25-dihydroxyvitamin D (1,25D) (Cheng et Chawla et al., 2001; Cheng et al., 2004). Upon al., 2004; Prosser and Jones, 2004; Shinkyo et ligand binding, the VDR undergoes a al., 2004). Sites of 1α-hydroxylation include conformational change triggering dimeriz- the kidneys and peripheral tissues, including ation with a related nuclear retinoid X several cell types of the immune system. receptor (RXR). The VDR–RXR heterodimer Hormonal 1,25D serves as the molecular complex is essential for high-aﬃ nity DNA ligand of the vitamin D receptor (VDR). binding with speciﬁ c DNA sequence motifs Production of 1,25D triggers expression of known as vitamin D response elements CYP24, the enzyme that initiates catabolism of 25D or 1,25D via a 24-hydroxylation reaction to generate 24,25-dihydroxyvitamin D or 1,24,25-trihydroxyvitamin D, in a physio logical negative-feedback loop. The 24-hydroxylated metabolites undergo further catabolism to calcitroic acid, which is then excreted in urine (Cheng et al., 2004; Prosser and Jones, 2004; Shinkyo et al., 2004). Regulation of gene expression by 1,25D occurs via binding to the VDR. The VDR is a nuclear receptor and transcription factor that is part of a family of ligand-regulated transcription factors, which includes receptors Fig. 11.2. Chemical structures of vitamin D2 for steroid hormones and the hormonal form (ergocalciferol) and vitamin D3 (cholecalciferol). 184 J.H. White and A.J. Bitton

(VDREs), located in the regulatory regions of gene encoding RANK ligand, a tumour specifi c target genes. VDREs are direct repeats necrosis factor-like ligand secreted by (DR) or everted (inverted) repeats (ER) of mesenchymal cells (Kim et al., 2006). hexameric nucleotide motifs composed of PuG(G/T)TCA consensus sequences, characterized by their spacing of either three 11.5 Overview of Vitamin D and (DR3), six (ER6) or eight (ER8) nucleotides Immune System Regulation (Lin and White, 2004; Tavera-Mendoza et al., 2006). Two copies of cognate DNA sequence A substantial body of evidence exists linking motifs refl ect the binding of heterodimeric vitamin D signalling with regulation of the VDR–RXRs in ‘heel-to-toe’ or ‘toes out’ innate and adaptive arms of the immune orientations (Fig. 11.3) (Rochel et al., 2000; system. The VDR is present in most cells of Chawla et al., 2001; Holick, 2007). the immune system, including T lympho- Aft er DNA binding, VDR–RXRs recruit cytes, neutrophils and antigen-presenting a series of co-regulatory proteins required for cells such as macrophages and dendritic cells the regulation of transcription of adjacent (DCs) (Provvedini et al., 1983; Bhalla et al., target genes. Regulation of transcription of 1984; Brennan et al., 1987; Adorini, 2005; protein-coding genes in eukaryotes is a bio- Norman, 2006). 1,25D polarizes T-helper (Th) chemically complex process that culminates responses towards a more regulatory Th2 in recruitment of RNA polymerase II and phenotype, which is considered to be a key initiation of transcription. For further component in its capacity to suppress Th1- information, readers are referred to a series driven autoimmune responses. It inhibits DC of excellent reviews on the subject (Glass and maturation and T-cell proliferation (van Ett en Rosenfeld, 2000; Dilworth and Chambon, and Mathieu, 2005). Gene expression studies 2001; McKenna and O’Malley, 2002). Recent have shown that 1,25D represses the studies suggest that the VDR can regulate transcription of genes encoding key Th1 target gene transcription from a considerable cytokines, including interferon-γ and inter- distance. For example, work has suggested leukin (IL)-2 (Alroy et al., 1995; van Ett en and that a VDRE located as far as 75 kb from the Mathieu, 2005). The net eff ect of 1,25D start site can regulate transcription of the signalling leads to a suppression of antigen

RXR VDR RXR VDR

PuG(G/T)TCAN3PuG(G/T)TCA TGA(A/C)CPyN6PuG(G/T)TCA

DR3 ER6

Fig. 11.3. Vitamin D receptor (VDR)–retinoid X receptor (RXR) dimeric binding to vitamin D response elements of the direct repeat (DR; left) or everted repeat (ER; right) type. DBD, DNA-binding domain; LBD, ligand-binding domain. Vitamin D and AMP Gene Expression 185

presentation and activation and recruitment regulation of DEFB2 transcription was found of Th1 cells. to be modest (Wang et al., 2004). However, it Innate immune responses are found in a is now clear that 1,25D can enhance DEFB2 wide variety of plant and animal life and expression with stimulation by other represent the front-line defences to patho- regulators (see below). Remarkably, several genic challenge. The innate immune system studies showed that 1,25D strongly stimu- defends the host from infection in a non- lated LL-37 expression in a number of human specifi c manner but, unlike adaptive cells and tissues (Wang et al., 2004; Gombart immunity, does not confer long-lasting or et al., 2005). These fi ndings are highly protective immunity to the host. Molecular signifi cant because AMPs represent the evidence for the regulation of innate immune vanguards of innate immune responses to responses by 1,25D has been accumulating infection. for some time. However, it is only in the last 5 years or so that we have begun to fi ll in many of the details of the central role of 11.7 Antimicrobial Peptides vitamin D signalling in innate immune responses in humans. AMPs are a group of small proteins with intrinsic antibiotic properties that are synthesized and secreted in cells and tissues 11.6 Vitamin D as a Regulator of exposed to environmental pathogens. Much Antimicrobial Peptide Gene of the eff ectiveness of AMPs stems from their Expression ability to eradicate a broad spectrum of pathogens, including bacteria, fungi and Modern genomics techniques have shift ed viruses, without triggering antibiotic resist- the focus of vitamin D into fi elds other than ance (Patil et al., 2004; Hancock and Sahl, regulation of calcium homeostasis, and have 2006; Jenssen et al., 2006). Additionally, being begun to reveal the molecular mechanisms a natural constituent of host immune underlying the ancient use of heliotherapy to systems, AMPs have been discovered to have treat infectious diseases. This new focus has additional immunoregulatory properties, its roots in studies of how 1,25D modulates leading to the term ‘immune effector mol- gene expression. As detailed above, the VDR e cules’. These regulatory properties include is a direct regulator of gene transcription alteration of gene expression, cytokine with a well-defi ned binding site (Lin and induction and modulation of DC responses White, 2004). Therefore, initial large-scale (Patil et al., 2004; Hancock and Sahl, 2006; identifi cation of 1,25D target genes began by Jenssen et al., 2006; Giuliani et al., 2007). As utilizing a combination of microarrays and pathogens continue evolving into increas- genome-wide screens for VDREs, yielding ingly antibiotic-resistant strains, AMPs hold signifi cant results (Akutsu et al., 2001; Lin et promise as a potent method in the control of al., 2002; White, 2004; Wang et al., 2005). Of microbial infection. particular interest, these screens identifi ed Cathelicidins are a family of anti- consensus DR3 VDREs located in the microbial polypeptides, the precursors of promoter region of two genes encoding the which are characterized by a highly con- antimicrobial peptides (AMPs) defensin β2 served N-terminal cathelin region with a (DEFB2/hBD-2) and LL-37 (Wang et al., 2004). variable C-terminal antimicrobial domain. As The 1,25D-dependent binding of the VDR to a subset of a larger group, the cathelin these elements was confi rmed by chromatin domain shares sequence homology and immunoprecipitation assays. These assays of function with the cystatin family of cysteine VDR binding entail immunoprecipitation of proteinase inhibitors (Zanett i, 2004). Cath- chemically cross-linked complexes of the elicidins serve as a critical component of the VDR bound to target DNA sequences, innate immune response, providing rapid followed by detection of specifi c DNA defence against infection. While genes sequences by PCR amplifi cation. 1,25D encoding for cathelicidins are widely 186 J.H. White and A.J. Bitton

conserved among vertebrates (Zanett i, 2004; immune function (Lehrer, 2004; Klotman and Chang et al., 2005; van Dĳ k et al., 2005), the Chang, 2006). Structurally, defensins are human gene remains the subject of focus. characterized by a cysteine-rich backbone In humans, a single gene encodes the forming β-pleated sheets stabilized by 18-kDa cathelicidin precursor protein (hCAP- disulfi de bonds (Klotman and Chang, 2006). 18), widely expressed throughout cells of the Classifi ed by their orientation of disulfi de immune system. Proteolytic cleavage of the bonds to cysteine residues, two subfamilies proprotein releases LL-37, also known as of defensins exist in humans: α-defensins, human cathelicidin AMP (CAMP) (Fig. 11.4). which are commonly produced by LL-37 is a linear-chain polypeptide with a net neutrophils and Paneth cells, and positive charge of +6 due to an excess of basic β-defensins, which are secreted by epithelial amino acids (Hancock and Diamond, 2000). tissues (for primary and tertiary structures, The basis of cathelicidin’s antimicrobial see Table 9.1 and Fig. 9.5, respectively). activity is due to the cationic nature of the peptide. Bacterial cell membranes contain high lipid compositions with a negatively 11.8 Regulation of DEFB2/hBD-2 charged surface, thereby creating a selective Expression by 1,25D preference for the positively charged AMP. Upon binding to bacterial cell surfaces, 1,25D induction of DEFB2 has been linked human cathelicidin LL-37 folds into an with cytokine production during the immune amphipathic α-helical structure (see Fig. response. As mentioned above, Wang et al. 9.4C, Chapter 9), leading to insertion of the (2004) found the induction of DEFB2 protein and interference with cell membrane expression by 1,25D alone to be relatively integrity. Increasingly, in vivo studies have modest (maximum twofold). However, this documented a more complete role for human initial study also showed that fold expression cathelicidin beyond the direct eradication of of DEFB2 could be elevated by the addition microbes. Cathelicidins have been shown to of IL-1β to the incubation media. IL-1β is a mediate a number of immune system cytokine and key regulator of infl ammatory responses, including chemotaxis, altering immune responses. The combined eff ects of transcription, directing the infl ammatory 1,25D and IL-1β on DEFB2 transcription response and promoting wound healing hinted at crosstalk between the two (Hancock and Diamond, 2000; Zanett i, 2004; signalling pathways. Expanding upon these Chang et al., 2005; van Dĳ k et al., 2005). results, Liu et al. (2009b) demonstrated the Defensins represent another family of synergistic activity of IL-1β with 1,25D in the 1,25D-induced AMPs that contribute to potentiation of antimicrobial responses innate immune defence. Similar to cath- induced by Toll-like receptors (TLRs). TLRs elicidins, defensins are small poly peptides are patt ern-recognition receptors that stimu- with cationic properties that are integral to late innate immune responses upon their antimicrobial activity. Found in both recognition of molecular motifs characteristic vertebrates and invertebrates, defensins are of pathogens. More recent research has also well recognized for their dual capacity found that 1,25D stimulates the expression of to both eradicate microbes and modulate another patt ern-recognition receptor called

Fig. 11.4. A schematic representation of the precursor protein hCAP-18 and the cleavage product LL-37. Basic amino acids in the LL-37 peptide are noted in bold. Vitamin D and AMP Gene Expression 187

NOD2/CARD15 (Wang et al., 2010). NOD2/ a number of fi ndings: (i) the VDR is found in CARD15 is an intracellular receptor that most immune cells, including T lymphocytes, recognizes lysosomal breakdown products of neutrophils, DCs and macrophages (Prov- bacterial peptidoglycan in the form of vedini et al., 1983; Bhalla et al., 1984; Brennan muramyl dipeptide (MDP). Notably, others et al., 1987; Adorini, 2005; Norman, 2006); (ii) have shown that signalling by MDP through studies have linked VDR induction of AMP NOD2/CARD15 induces expression of DEFB4 production with TLR signalling in innate (formerly DEFB2/hBD-2) (Liu, P.T. et al., immune responses (Stead et al., 1990; 2009b). The combination of MDP and 1,25D Brightbill et al., 1999; Thoma-Uszynski et al., synergistically induced DEFB4 expression. 2001; Liu et al., 2006, 2007); and (iii) VDR These results are of clinical signifi cance signalling is important for both the process because att enuated or defective expression of of pathogen recognition and antimicrobial NOD2/CARD15 or DEFB2/hBD-2 has been response during tissue injury (Schauber et al., associated with the pathogenesis of Crohn’s 2007). disease (Wang et al., 2010). Crohn’s disease is TLR patt ern-recognition receptors are a a chronic infl ammatory bowel disease that family of cell-surface and intracellular recep- arises from a defect in intestinal innate tors. TLRs have long been studied for their immune responses to bacterial fl ora. roles in responses to microbial infection, and have recently been linked to VDR immunoregulation. TLR2 receptors are cell- 11.9 1,25D Regulation of LL-37 is surface proteins that recognize bacterial Human and Primate Specifi c lipopeptides. By 2006, it was established that TLR2 activation of human or murine The induction of antimicrobial gene macrophages directly stimulates anti- expression by 1,25D does not appear to be microbial activity against TB infection (Liu et widely conserved among mammals. The al., 2006). However, the intermediate VDRE in the human hBD-2/DEFB2 gene is signalling pathways leading to AMP not conserved in the mouse, for example. production are believed to be diff erent Moreover, work by Gombart et al. (2005) between the two species. In mice, studies showed that the regulation of LL-37 showed that antimicrobial activity is expression seen in humans is not conserved dependent upon the induction of nitric oxide in mice. Indeed, a comparison of several synthase (iNOS) and the production of nitric mammalian genomes showed that the oxide (NO) in infected tissue (Liu et al., 2006, mechanism in 1,25D induction of LL-37 is 2007). Further work showed that NO-induced conserved specifi cally in humans and non- antimicrobial activity is mediated through human primates. Conservation of the VDRE TLR signalling in mice, and that iNOS in the LL-37 promoter can be att ributed to the inhibitors are able to block this induction of presence of the element within a short antimicrobial activity (Liu et al., 2006). interspersed element of the Alu subfamily. Additionally, these studies demonstrated Alu repeats are a family of transposable that human macrophages do not depend DNA elements that are primate and human upon the production of NO for their anti- specifi c. This specifi c conservation of the Alu microbial activity. However, one intriguing repeat containing the LL-37 VDRE can be study has cast doubts on these initial results traced to an insertion event dating back to and shown that iNOS activity does indeed 55–60 million years ago (Gombart et al., play a role in host defences to TB infection in 2009), prior to the divergence of the primate human macrophages (Lee et al., 2009). lineage leading to humans, apes and Old and Research has confi rmed the induction of New World monkeys. AMPs as the basis for TLR-induced Regulation of the LL-37 gene by 1,25D is antimicrobial activity in human macrophages believed to hold a selective advantage given and linked VDR signalling to AMP its functional signifi cance in innate immune production (Liu et al., 2007). Using micro- responses. This signifi cance is highlighted by arrays, studies found that TLR stimulation 188 J.H. White and A.J. Bitton

upregulated expression of the VDR as well the importance of regulation of non-renal as CYP27B1 in human monocytes (Liu et al., CYP27B1 expression in immunoregulation 2006, 2007). These fi ndings are highly by vitamin D. signifi cant as they reveal that macrophages react to pathogen detection by acquiring the capacity to respond to circulation levels of 25D and convert it into hormonal 1,25D. The 11.10 Broader Physiological and functional signifi cance of this discovery was Pathophysiological Implications of demonstrated by the observation that the Regulation of LL-37 Expression reduced Mycobacterium tuberculosis viability by 1,25D in infected macrophages is dependent on 1,25D-induced production of LL-37 (Liu et al., The skin occupies a key position in vitamin D 2007). Furthermore, LL-37 expression has physiology. Epidermal keratinocytes express recently been linked to the induction of the hydroxylase enzymes CYP27A1 and autophagy and the degradation of M. CYP27B1 and can thus produce 1,25D tuberculosis in infected human macrophages (Norman, 1998) in the presence of suffi cient (Yuk et al., 2009). Autophagy plays an UVB irradiation. Additionally, keratinocytes important role in the degradation of express the VDR, making them responsive to damaged cellular components and misfolded vitamin D inputs. Under conditions of tissue proteins, as well as in organellar turnover damage, 1,25D regulates a variety of responses (Baehrecke, 2005). Importantly, a critical role in keratinocytes, including changes in cell has recently emerged for autophagy as a proliferation, diff erentiation, cytokine pro- component of innate immune responses to duction and gene expression (Dorschner et al., microbial infection (Deretic, 2009, 2010). 2001; Heilborn et al., 2003; Carretero et al., Because of darker pigmentation and 2008; Segaert and Simonart, 2008). Cell- reduced cutaneous vitamin D synthesis, signalling events that underlie wound healing African-Americans are known to possess also to lead to a substantial induction of signifi cantly reduced serum 25D concen- CYP27B1 and subsequently increase LL-37 trations (Stead et al., 1990; Nesby-O’Dell et expression. Moreover, LL-37 induces keratino- al., 2002). Consistent with previous studies, cyte migration, vital to re-epithelialization of Liu et al. (2007) found that serum from the wound, by transactivating epidermal African-Americans generally contained growth factor receptors responsible for lower levels of 25D than that of Caucasians, cellular architecture (Dorschner et al., 2001; and induced correspondingly lower levels of Heilborn et al., 2003). LL-37 in TLR2-stimulated macrophages. Induction of dermal cathelicidin However, normal physiological function expression by 1,25D is not always benefi cial, could be restored through proper sup- as elevated levels of LL-37 have been plementation of 25D. These studies provide implicated in several infl ammatory skin compelling evidence that TLR2-stimulated conditions. Rosacea is a condition char- macrophages produce a substantial AMP acterized by chronic erythema (redness of response only under conditions of vitamin D the skin) in affl icted patients. Notably, suffi ciency. rosacea is also characterized by abnormal In summary, the above fi ndings are processing of hCAP-18 peptides, leading to signifi cant for a number of reasons. First, an abundance of truncated cathelicidin they clearly establish a central role for peptides in keratinocytes, which contributes vitamin D signalling in primary innate to infl ammation (Yamasaki et al., 2007). immune responses. Secondly, they empha- Additionally, UV light aggravates rosacea, size the importance of maintaining vitamin possibly in part via 1,25D-induced LL-37 D suffi ciency for optimal immune system expression. Indeed, it has been speculated function, and suggest why some ethnicities that azole antimycotics (ketoconazole, may be more predisposed to certain itraconazole and metronidazole), used infectious diseases. And fi nally, they signify clinically to treat rosacea, may work partly Vitamin D and AMP Gene Expression 189

by blocking CYP450-driven vitamin D meta- al., 2009, Peric et al., 2009). Furthermore, the bolism (Yamasaki et al., 2007). synergistic enhancement of LL-37 expression Vitamin D analogues are widely used to is induced by the combination of vitamin D treat psoriasis, an autoimmune infl ammatory and bile salts, suggesting a novel therapeutic skin condition. It is thus paradoxical that approach to treating infl ammatory biliary LL-37 is overexpressed in psoriasis and may disease (D’Aldebert et al., 2009). contribute to its pathogenesis. Cathelicidin Similarly, VDR regulation of the innate expression is limited to keratinocytes immune response is essential for maintaining exposed to injury, stimulating immune lung homeostasis. The epithelial lining of the responses and promoting wound healing lungs is constantly exposed to a variety of (Dorschner et al., 2001; Heilborn et al., 2003). environmental pathogens, and induction of During psoriasis, this tightly regulated antimicrobial gene expression is vital in process is altered, leading to overexpression providing a front line of defence against of the LL-37 peptide and triggering immune inhaled microbes. 1,25D has been shown to responses by activating plasmacytoid DCs strongly induce LL-37 expression in Calu-3 (Lande et al., 2007). Plasmacytoid DCs are cells, a human epithelial airway cell line specialized skin cells that sense viral and (Wang et al., 2004). Moreover, treatment with bacterial DNA through TLR signalling. In 1,25D yielded a signifi cant reduction in the particular cases of autoimmune disease, a microbial activity of either Escherichia coli or breakdown in the sensory apparatus occurs Pseudomonas aeruginosa in infected Calu-3 allowing for self-DNA to trigger an immune cells. P. aeruginosa is an opportunistic patho- response. In psoriatic skin, Lande et al. (2007) gen associated with the vast majority of established that LL-37 is the mediating factor respiratory infections in patients affl icted triggering plasmacytoid DC activation. with cystic fi brosis (Speert et al., 2002). Lending to the cationic, amphipathic nature Notably, patients with cystic fi brosis have of protein, LL-37 directly binds to DNA in also been observed to have low circulating plasmacytoid DCs, forming aggregated and serum 25D levels (Hecker and Aris, 2004). condensed structures that are delivered to Expanding upon these results, Yim et al. TLR-9 receptors, thereby activating immune (2007) documented the presence of the VDR, response mechanisms leading to chronic skin and robust induction of cathelicidin, in infl ammation. normal human bronchial epithelial cells. VDR-regulated signalling is not limited Additionally, this particular study demon- to dermal epithelial cell types. One recent strated the direct contribution of cathelicidin study showed that both LL-37 and the VDR to antimicrobial activity through preincub- are expressed in hepatic biliary epithelial ation of infected normal human bronchial cells (D’Aldebert et al., 2009). Under normal epithelial cells with an anti-LL-37 antibody. physiological conditions, the biliary tract Active vitamin D metabolism has also maintains a number of defence mechanisms, been established in the endometrium and preserving a microbe-free environment. The placenta during gestation. Beginning in the authors found that antibacterial activity in 1980s, studies conducted with rats connected the biliary tract is maintained by signalling vitamin D defi ciency with signifi cantly actions of bile salts through the VDR and a diminished rates of reproduction (Halloran related farnesoid X receptor (FXR). FXR is a and DeLuca, 1980; Hickie et al., 1983; member of the nuclear receptor family Kwiecinksi et al., 1989). Recently, one activated by bile salts. Remarkably, the VDR intriguing study linked high levels of has also been established as a functional bile placental 1α-hydroxylase expression during acid receptor (Makishima et al., 2002). early gestation with the pleiotropic actions of Activation of either the FXR or VDR by bile vitamin D signalling beyond fetal musculo- acids has been found to mediate innate skeletal development (Zehnder et al., 2002). immune function through cathelicidin Expanding upon these results, another study induction in both hepatic epithelial cells and correlated vitamin D signalling with normal human keratinocytes (D’Aldebert et att enuated immune function during the early 190 J.H. White and A.J. Bitton

stages of pregnancy in maternal decidual autoimmune conditions such as type I tissue (Evans et al., 2006). In upholding an diabetes and arthritis (Larsson et al., 1998; immunosuppressive role, localized synthesis van Ett en et al., 2003). of 1,25D was found to modulate maternal Given the central role of 1,25D in immune function, supporting implantation stimulating innate immune responses to at the fetal–maternal interface. Furthermore, infection, we speculate that vitamin D N. Liu et al. (2009) documented the analogues may also fi nd a role in drug 1α-hydroxylase conversion of 25D to 1,25D, cocktails designed to combat infectious as well as robust cathelicidin induction, in diseases. Recent fi ndings raise the possibility placental human trophoblast cells. However, that vitamin D analogues that strongly unlike human macrophages, LL-37 induction induce expression of the NOD2/CARD15– in trophoblasts is independent of TLR DEFB2/hBD-2 innate immune pathway may activation. Taken together, these results be effi cacious in combating Crohn’s disease clearly demonstrate the broad spectrum of (Wang et al., 2010). It is noteworthy in this in vivo activity for vitamin-D-mediated regard that the NOD2/CARD15 patt ern- signalling, moving beyond calcium homeo- recognition receptor is particularly sensitive stasis and bone metabolism. to a modifi ed form of MDP produced by mycobacteria (Coulombe et al., 2009). Therefore, vitamin D analogues may also fi nd a role as modern-day successors to Hip- 11.11 Therapeutic Role for Vitamin D pocrates’ heliotherapy in the treatment of TB. Analogues

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Matthew L. Mayer, Donna M. Easton and Robert E.W. Hancock*

Abstract

Host defence peptides (HDPs) are powerful modulators of human innate immunity, and can modify the outcome of the endogenous host response to infection. The progressive development of pathogen resistance to conventional antimicrobial agents has lead to a new appreciation of HDPs for their ability to fi ght infection, enhance vaccine responses, limit infl ammation and promote wound healing, within the context of human disease. HDPs are a family of cationic, short, amphipathic peptides that include the classical mammalian antimicrobial peptides, cathelicidins and defensins, as well as non-antimicrobial peptides with similar immunomodulatory properties. This chapter reviews our current basic understanding of the anti-infective and immunomodulatory properties of both endogenous HDPs and synthetic derivatives (termed innate defence regulators) with regard to their ability to selectively fi ne tune the responses of host cells and physiology. The clinical application of these molecules is also discussed, with a focus on past and ongoing clinical trials of HDPs and innate defence regulators as novel therapeutics for infectious and infl ammatory diseases.

Infectious diseases have been the primary neoplastic disease) as the most signifi cant cause of morbidity and mortality for most of killers facing Western society (WHO, 2008). human history. At the end of the 19th Following the introduction of penicillin century, the average human life expectancy and the relatively rapid development of was 25 years (Casanova and Abel, 2005). It other antibiotic classes, we have come to rely was the development of Pasteur’s insights on the availability of eff ective antibiotics to into the nature and role of microbes in combat infections. However, having now infections that paved the way for our fi rst well and truly entered the era of antibiotic medical breakthroughs in hygiene, vac- resistance, it is necessary to develop novel cination and antibiotics. These discoveries antimicrobial therapies and strategies to led to a rapid acceleration in life expectancy overcome the growing problem of multidrug- around the world, and infectious causes of resistant bacterial strains. The discovery of mortality have been falling behind the naturally occurring antimicrobial peptides diseases of old age (cardiovascular and (AMPs) provided hope that these could

* Corresponding author.

become the basis for a new class of antibiotic from autotoxicity related to the strong compounds, although clinical trial results to cationic charge and amphipathicity of the date have not yet confi rmed this potential. mature peptide (the latt er also being Research in this area has increasingly begun managed in part by sequestration into to focus on the immunomodulatory prop- granules). Neutrophil α-defensins are stored erties of these peptides and their applications within azurophilic granules for release by for human disease, specifi cally their use as fusion with cell phagosomes containing novel anti-infective therapeutics that circum- ingested microbes; this results in very high vent antibiotic resistance by selectively local concentrations of defensins and, despite enhancing the benefi cial aspects of the their relatively weak antimicrobial activity, endogenous host response to pathogens. concentrations are suffi cient to mediate direct microbicidal activity (Selsted and Ouellett e, 2005). Intestinal α-defensins are constitutively 12.1 Mammalian Host Defence expressed and secreted from Paneth cells, (Antimicrobial) Peptides with secretion being enhanced by contact with bacterial components such as CpG Mammalian peptides with direct anti- oligodeoxynucleotides (ODNs). Contact with microbial activity are generally classifi ed into bacterial components can also stimulate the two main families of cathelicidins and α-defensin secretion by natural killer (NK) defensins, although other peptides exist that cells (Agerberth et al., 2000), indicating that, do not fall into these categories. The in addition to their intracellular antimicrobial defensins are cationic peptides of 18–45 function within neutrophils, such defensins amino acids and are further divided into also have important extracellular functions. three subfamilies: the α-, β- and θ-defensins. β-Defensins are produced by a range of The α- and β-defensins are genetically tissues, mainly in response to pro- distinct, with similar structures dominated infl ammatory cytokines induced by Toll-like by β-strands that diff er in the placement of receptor (TLR) agonists. For example, their six conserved cysteine residues that β-defensins produced by keratinocytes play form three intramolecular disulfi de bridges important roles in the host defence functions (Selsted and Ouellett e, 2005). The θ-defensins of the skin and in wound healing (Niyonsaba are cyclic in structure, are only found in non- et al., 2007). human primates and have antiviral activity While the defensins can demonstrate against human immunodefi ciency virus-1 antibacterial activity in vitro at low micromolar (Gallo et al., 2006). Defensins are produced concentrations, most α- and β-defensins are by leukocytes in many mammals, including only weakly eff ective in the presence of salt humans, rats and rabbits, but not mice. concentrations approaching those found in However, mice, like humans, also produce vivo; this can be over-ridden by extremely α-defensins in their intestinal crypts and high concentrations of these molecules such these include a subclass termed cryptdins as the mg ml–1 concentrations found in (Amid et al., 2009). Conversely, mice do not phagocytic granules or the crypts of the produce leukocyte α-defensins like humans, intestine. At least one β-defensin, human while bovines are devoid of all α-defensins. β-defensin (hBD)-3, is more cationic than the others, less aff ected by cation antagonism and probably has meaningful activity at the 12.1.1 Defensins surface of the skin. Similarly, the θ-defensins are active at these higher salt concentrations, The α- and β-defensins are produced as although only when in their cyclic form (Tang prepropeptides that are cleaved to form et al., 1999). This suggests that, while direct propeptides, which in turn require additional antimicrobial activity is important in some processing to form the active peptide. The physiological niches, where the concentration propiece, which is cleaved to release the of peptide is high enough to overcome active peptide, acts to protect defensins from antagonism by salts, in many other anatomical proteolysis inside cells and protect the cell locations the alternative, immunomodulatory Clinical Applications of Host Defence Peptides 197

functions of these peptides probably pre- with hBD-1, also infl uences susceptibility to dominate. Crohn’s disease, a microbial-induced infl am- Defensins modulate the functions of matory condition of the intestines (Kocsis et many cell types, infl uencing immune cell al., 2008). Similarly, a predisposition to recruitment, activation and maturation, Crohn’s disease has been observed in indi- wound healing and angiogenesis. For viduals with a lower copy number of the example, human α-defensins 1–3 have been hBD-2 gene, and a correspondingly lower shown to infl uence the maturation and expression of hBD-2 at the mRNA level diff erentiation of monocyte-derived dendritic (Fellermann et al., 2006). cells (DCs), with diff erent outcomes induced by either high or low peptide concentrations (Rodriguez-Garcia et al., 2009). Human 12.1.2 Cathelicidins neutrophil α-defensins 1–3 act as direct chemoatt ractants for T cells and DCs (Yang et Cathelicidins are structurally distinct from al., 1999; Hubert et al., 2007), whereas hBD1–4 defensins, containing a characteristic cathelin induce chemotaxis of macrophages and prodomain and a signal sequence. Despite calcium fl ux in mast cells, while having no this related N-terminal region, cathelicidins eff ect on T cells or DCs (Soruri et al., 2007). vary markedly in structure and include α-Defensins also have mitogenic functions, α-helical, β-hairpin, β-turn and extended promoting wound healing through the peptides. They are produced by a wide range stimulation of fi broblast and epithelial cell of species, including mammals, birds and fi sh, proliferation (Murphy et al., 1993; Nishimura with some species producing up to seven et al., 2004). In addition to aff ecting mast cell diff erent cathelicidins (Durr et al., 2006). chemotaxis and calcium fl ux, hBD-3 and Humans have only one cathelicidin, which is hBD-4 have also been shown to induce mast synthesized as the proprotein hCAP-18 and cell degranulation and prostaglandin D2 cleaved extracellularly to form the active production, with mast cell activation being protein known as LL-37. Mice also have a dependent on signalling through the single cathelicidin, known as CRAMP, which mitogen-activated protein kinase (MAPK) has 67% identity to LL-37. Cathelicidins are p38 and extracellular signal-regulated kinase produced by a range of cell types, including (ERK)1/2 pathways (Nishimura et al., 2004). epithelial cells and leukocytes such as As these immunomodulatory functions are neutrophils, monocytes, T cells, B cells and generally seen at peptide concentrations NK cells. They appear in many body locations lower than those needed for direct (e.g. at mucosal and skin surfaces) and fl uids antimicrobial activity, and are not inhibited (e.g. gastric juices, saliva, semen, sweat, by physiologically relevant salt concen- plasma, airway surface liquid and breast trations (being evident under tissue culture milk). Like the defensins, cathelicidins exhibit conditions and oft en in animal models), these a broad range of biological activities that immunomodulatory functions are likely to include both direct antimicrobial activity and be more broadly physiologically relevant immuno modulatory functions. LL-37 is than direct antimicrobial activity. present in many diff erent tissues and The in vivo signifi cance of defensins in secretions, including sweat and breast milk, the context of innate immune functions is and is stored at high concentrations in its supported by observations in humans with proform hCAP-18 in the azurophilic granules genetic alterations in defensin production. of neutrophils. The physiological concen- For example, while defensins are usually trations of LL-37 are estimated to be up to induced in response to interferon (IFN)-γ, in 1 μM (5 μg ml–1) in airway surface fl uid and at those with a particular single nucleotide mucosal surfaces, and under pathological polymorphism expression of hBD-1 and conditions can increase dramatically, with hBD-3 is constitutive and this confers concentrations up to 300 μM reported in enhanced protection from candidiasis (Kalus psoriasis (Ong et al., 2002; Schaller-Bals et al., et al., 2009). This same single nucleotide poly- 2002; Bals and Wilson, 2003). The biological morphism, together with another associated functions of LL-37 have been proposed to 198 M.L. Mayer et al.

vary depending upon the local conditions; activated receptors epidermal growth factor for example, direct antimicrobial activity receptor and nucleotide scavenging receptor requires quite high concentrations of the P2X7 in various properties of LL-37 have been peptide and/or low salt concentrations. described (Tjabringa et al., 2003; Tomasinsig Conversely, wound-healing, angiogenic and et al., 2008). Recent studies (Zhang et al., anti-endotoxic activity and synergistic 2009) indicate that LL-37 can act as an induction of certain chemokines together alternative ligand for the IL-8 receptor with endogenous signalling molecules are (CXCR2) on neutrophils, while Mookherjee all seen at very low (<1 μM) peptide et al. (2009a) reported a physical and concentrations. functional inter action between LL-37 and The ability of LL-37 to augment many intracellular glyceraldehyde 3-phosphate aspects of the innate immune response has dehydrogenase (GAPDH). Many other been well established in the literature. For studies have described the signal trans- example, LL-37 modulates the production of duction pathways, transcription factors and interleukin (IL)-8 by keratinocytes and eff ector proteins and pathways involved in epithelial cells (Filewod et al., 2009) and peptide activity (Mookherjee et al., 2009b). inhibits the action of IFN-γ on monocytes, Studies on cathelicidin activity in vivo macrophages, DCs and B lymphocytes (Nĳ nik and ex vivo have been undertaken using et al., 2009). Studies on human peripheral transgenic mice lacking CRAMP, with notable blood mononuclear cells have shown that observations including that the peptide is LL-37 acts in synergy with immune mediators involved in NK-cell-mediated antitumour such as granulocyte–macrophage colony- responses (Buchau et al., 2010), partial stimulating factor and IL-1β to enhance the protection from bacterial infections (Nizet et production of cytokines (e.g. IL-6 and -10) and al., 2001; Braff et al., 2005; Iimura et al., 2005; chemokines (e.g. monocyte chemotactic Huang et al., 2007) and viral infections protein-1 and -3). This synergy is mediated (Howell et al., 2006) and the antimicrobial through modulation of intracellular signalling activity of mast cells (Di Nardo et al., 2003). pathways, such as via phosphoinositide However, these studies have not always 3-kinase and MAPK, and subsequent changes discriminated between direct and indirect in the activation of associated transcription (modulation of other immune mechanisms) factors (Yu et al., 2007). The immune modes of action. Treatment with exogenous modulation mediated by the cathelicidin leads CRAMP has also been shown to reduce the to a more eff ective and balanced innate severity of colitis induced by dextran sulfate response, with synergistic upregulation of the sodium ingestion, with enhanced intestinal chemokine response accompanied by anti- mucus production and reduced apoptosis endotoxic activity, mediated both by (Tai et al., 2007). In addition to the lipopolysaccharide (LPS) binding and immunomodulatory eff ects it exerts on modulation of TLR signalling cascades to mammalian cells, LL-37 is able to prevent reduce the production of proinfl ammatory biofi lm formation by the bacterial pathogen cytokines such as tumour necrosis factor Pseudomonas aeruginosa at a concentration far (TNF)-α. The anti-endotoxic activity of LL-37 below (at least 1/16th) its minimal inhibitory is supported by experiments showing that it concentration by specifi cally modulating can protect mice from death aft er challenge bacterial gene expression (Overhage et al., with LPS alone (i.e. any bactericidal activity is 2008). irrelevant) and that it can reduce leukocyte responses to LPS ex vivo (Mookherjee et al., 2007). The mechanism of action is as yet 12.2 The Role of Endogenous Host incompletely understood, but several cellular Defence Peptides in the Response to components have been proposed as specifi c Infection interactors. The roles of surface formyl peptide receptor-like 1 as a direct binding The natural human host response to infection receptor in chemotaxis, of other undetermined cannot be modelled on an agar plate, tissue G-protein-coupled receptors and of the trans- culture dish or nutrient-broth-fi lled test tube. Clinical Applications of Host Defence Peptides 199

Rather, it is a dynamic and complex process defence against infection. Although the bulk that involves all of the physiological systems of HDP research has focused on the direct and organs necessary for human life. Cationic antimicrobial nature of these molecules, peptides are responsible for fi ne tuning partly due to their location in professional immunological and physiological homeostatic immune cells and the relative ease of parameters in the context of the host response measuring such activities, other non- to infectious diseases. In light of the evidence antimicrobial activities have recently gained that such peptides have a broad range of att ention for their role in orchestrating global biological functions that go well beyond physiological and biochemical changes that direct antimicrobial activity in vitro modulate the host response, hence the use of (Oppenheim and Yang, 2005; Hancock and the broader designation HDP when consider- Sahl, 2006; Mookherjee et al., 2006), they are ing activities other than direct actions on perhaps more accurately termed host defence microbes. peptides (HDPs) rather than AMPs (Box 12.1). Immunomodulation by HDPs is oft en The creation of synthetic innate defence understood to occur at local tissue sites of regulator (IDR) peptides that lack appreciable infection. Diff erential production of defensins antimicrobial activity yet show similar and cathelicidins has been described to occur physical properties to AMPs and powerful in epithelial, connective tissue and leukocyte (indirect) anti-infective effi cacy mediated cells against the background of an through immunomodulatory mechanisms infl ammatory milieu, while local concen- adds further support to this idea. trations can also increase rapidly by The human innate immune response to degranulation of phagocytic cells at the site infection is a complex physiological process of infection, suggesting that the immuno- that orchestrates non-specifi c responses to modulatory function occurs in a paracrine or infectious threats. Within this dynamic series autocrine manner. Numerous studies on of events, HDPs play a critical role in endogenous HDPs and equivalent synthetic coordinating and fi ne tuning cellular, tissue- IDR peptides have shown that administering specifi c and global physiological changes these molecules in infection models, either that accompany the innate immune response. locally or systemically, results in alterations Considering the critical functions of en- in global immune function (Table 12.1). Other dogenous HDPs beyond direct antimicrobial host-produced peptides, classically grouped killing is essential to an expanded under- together as hormones, play a similar role in standing of the important roles played by the face of infection, allowing the integration these molecules within the context of host of local and systemic host defences with

Box 12.1. Deﬁ nitions. Antimicrobial peptides (AMPs): Host-produced or synthetic peptides, and their derivates or mimetics, with typical cationic and amphipathic structural motifs, that mediate direct killing against bacterial cells in vitro with a readably measurable minimal inhibitory concentration (MIC). This term is appropriate when antimicrobial activity is being examined. Host defence peptides (HDPs): Host-produced peptides (and their derivates), with typical cationic and/or amphipathic structural motifs, that exert anti-infective properties in vivo through their ability to enhance or modulate the normal host immune response to infectious agents. Innate immunity is the major target but effects on adaptive immunity have been observed. This in vivo anti-infective effect can be mediated by one or more of the following: selective immune modulation, modulation of global host physiology or direct antimicrobial activity. Innate defence regulators (IDRs): Synthetic peptides that exert anti-infective properties in vivo by their ability to enhance, complement or modulate the normal host response to infectious agents. This in vivo anti-infectious effect is mediated predominantly by their immunomodulatory abilities and includes a subset of activities observed for the HDPs. Structurally, these peptides share features such as cationicity and amphipathicity with HDPs. 200 M.L. Mayer et al.

Table 12.1. Small cationic peptides that have no antimicrobial activity or activity only in very dilute medium or buffer, but that appear to play a role in the human host response to infection. HDP family Physical Site(s) of Role in host response and Cellular (immunomodulatory) and prototype properties production therapeutic applications function Histatins: 24 amino Parotid and Play a broad role in maintaining Attenuate IL-6- and IL-8- histatin 5 acids, +5 sublingual oral cavity homeostasis in the mediated gingival net charge salivary face of oral and periodontal infl ammation (Imatani et al., glands pathogens such as 2000) induced by P. Porphyromonas gingivalis. gingivalis and prevent LPS- Potent mediators of mucosal induced apoptosis in gingival wound healing and fi broblasts (Imatani et al., re-epithelialization. The 2004). Wound healing occurs histatin-derived peptide P-113D via an ERK1/2-dependent has shown effi cacy in animal mechanism (Oudhoff et al., models of Pseudomonas 2008). aeruginosa sepsis (Cirioni et al., 2004), and has been in Phase II clinical trials (Table 12.2). Iron-binding 25 amino Liver Iron homeostasis is a Circulating proinfl ammatory peptides: acids, +2 cornerstone of host defence cytokines induce hepatic hepcidin net charge (Schaible and Kaufmann, synthesis (Nemeth et al., 2004). Iron-binding peptides 2004). Phagocytic cells imit the utility of bacterial produce the peptide following haemolysins in iron acquisition, TLR2 or TLR4 activation via while simultaneously activating STAT1 and NF-κB (Sow et host antibacterial defences al., 2009), leading to such as phagocytic cells and improved innate immune complement (Ganz, 2009). recognition of bacterial pathogens (Wang et al., 2009a). Leads to impaired incorporation of iron into haemoglobin (Rivera et al., 2005) and infl ammation- associated anaemia (Nemeth, 2010). Melanocortins: α-MSH is Anterior Interface endocrine system with Bind to MC receptors found MSH 13 amino pituitary the resolution of infl ammation. on most human cells, with acids, +1 gland Enhance anti-infl ammatory MC-1R mediating net charge; programme in leukocytes and immunomodulation. MC-1R γ-MSH is 11 lymphocytes (Sarkar et al., ligation leads to large amino acids, 2003; Yoon et al., 2003; Cooper increases in cellular cAMP +2 net et al., 2005), without impairing via PKA, activation of the charge the ability to clear infection. MAPK pathway and altered Exhibit signifi cant anti-endotoxin NF-κB responses. In PBMCs, activity (Scholzen, 2003; Yoon MSH causes downregulation et al., 2003) and promote of TNF-α, IL-1β, IFN-γ, IL-6 wound healing (Zou et al., and IL-8, with simultaneous 2004; Yoon et al., 2008). induction of IL-10 and NO. The MSH-derived peptide The immunomodulatory AP214 has shown effi cacy in functions of MSH are diverse, preventing organ failure in cell-type-dependent and well animal models of sepsis (Doi reviewed elsewhere (Böhm et al., 2008) and is in clinical et al., 2008). trials (Table 12.2). Clinical Applications of Host Defence Peptides 201

Table 12.1. Continued. HDP family Physical Site(s) of Role in host response and Cellular (immunomodulatory) and prototype properties production therapeutic applications function GI peptides: Ghrelin is GI tissue Regulate energy and nutrition Ghrelin acts via the ghrelin, VIP 28 amino and PNS homeostasis, linking GHSR-1a receptor, in a acids, +5 ganglions metabolism to the endogenous MAPK phosphatase-1- net charge; that anti-infective response, dependent manner, to inhibit VIP is 28 regulate especially to enteric pathogens LPS and noradrenaline- amino GI function (Conlin et al., 2009; Coron mediated proinfl ammatory acids, +3 et al., 2009). VIP enhances cytokine release (Jacob et net charge innate immunity to P. al., 2010). Acts directly on aeruginosa (Szliter et al., effector cells to inhibit TNF- 2007). Ghrelin plays an α- and LPS-induced release important role in host defence of HMGB1; also enhances against pulmonary pathogens their bactericidal function by regulating both nutritional (Chorny and Delgado, 2008; state and neutrophil recruitment Chorny et al., 2008). (Kodama et al., 2008; Kim VIP-mediated et al., 2010). Both are in clinical immunomodulation is trials for sepsis, where they incredibly complex and inhibit toxic infl ammation and dynamic and is well reviewed organ dysfunction (Chorny and elsewhere (Delgado et al., Delgado, 2008; Huang et al., 2004). Briefl y, VIP binds to 2009; Wang et al., 2009b; Wu the VPAC receptor expressed et al., 2009), with ghrelin also on nearly every cell (Ceraudo being investigated in cystic et al., 2008; Lv et al., 2009). fi brosis (Table 12.2). Immune-specifi c effects occur by modulating the function of lymphocytes (Ganea, 1996) and innate immune cells (Szliter et al., 2007) to balance pro- and anti-infl ammatory tone. Abbreviations: cAMP, cyclic AMP; ERK, extracellular signal-regulated kinase; GHSR, growth hormone secretagogue receptor; GI, gastrointestinal; HDP, host defence peptide; HMGB1, high mobility group protein B1; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MC, melanocortin; MC-1R, melanocortin 1 receptor; MSH, melanocyte-stimulating hormone; NF-κB, nuclear factor κB; NO, nitric oxide; PBMC, peripheral blood mononuclear cell; PKA, protein kinase A; PNS, peripheral nervous system; STAT1, signal transducer and activator of transcription 1; TLR, toll-like receptor; TNF, tumour necrosis factor; VPAC, vasoactive intestinal peptide receptor; VIP, vasoactive intestinal peptide. cardiovascular function, pyrogenic and discharge of sodium through the urine neuroendocrine responses, iron homeostasis (natriuresis), as well as having potent and gastrointestinal motility. These peptides vasodilating activities. NPs are implicated in are also summarized in Table 12.1, and are body fl uid homeostasis and blood pressure discussed below to further illustrate this control, playing a role in the regulation of host concept. cardiovascular, haemodynamic and renal function, and are used today as clinical markers in the diagnosis of congestive heart 12.2.1 Natriuretic peptides failure (Eleuteri and Di Stefano, 2009). Three members of the NP family are found in Natriuretic peptides (NPs) are a family of humans, namely atrial (or A-type), B-type and cationic, ring-shaped autocrine/paracrine C-type, abbreviated as ANP, BNP and CNP, peptide hormones secreted by neuronal, respectively (Pott er et al., 2009). Showing high cardiac and endothelial cells. They cause sequence homology across mammalian 202 M.L. Mayer et al.

species, NPs are secreted as 126–151 amino has been found to do just the opposite, acid precursors and cleaved into 22–32 amino increasing mobilization of polymorpho- acid active peptide forms with net positive nuclear neutrophils and macrophages and charges. In addition to their traditional roles, enhancing endothelial–leukocyte interactions. these peptides have now been identifi ed as ANP is also able to broadly activate immune playing a critical role in fi ne tuning cells, increasing leukocyte production of cardiovascular and innate immune responses bactericidal reactive oxygen species and in the face of severe infection and sepsis inducing the maturation and proliferation of (Mohapatra, 2007; Eleuteri and Di Stefano, T cells and DCs (Pott er et al., 2009). 2009; Vesely and de Bold, 2009). Conversely, ANP has been shown to have One of the most devastating (and oft en more of an anti-infl ammatory/immuno- lethal) complications of sepsis is septic shock, suppressant eff ect in lung tissue, in both consisting of haemodynamic and cardio- animal models (Wang et al., 2009c; Sekino et vascular collapse leading to renal and al., 2010) and patient trials (Oda et al., 2009). multiorgan failure. Consequently, elevated Mechanistically, ANP can enhance the barrier BNP (which has the longest half-life of the eff ect of endothelial tight junctions (and thus NP family members) has been identifi ed as limit leukocyte recruitment) in response to an early clinical marker of sepsis (Rudiger et both infl ammatory (LPS) (Irwin et al., 2005) al., 2006; Kandil et al., 2008). Surprisingly, and allergic (ovalbumin (OVA)) stimuli elevated BNP in sepsis has been found to (Mohapatra, 2007), probably via the att enu- precede the onset of septic shock (Vila et al., ation of LPS- and TNF-α-induced MAPK, 2008). This was correlated with increased ERK1/2, NF-κB and Rho pathway signalling systemic infl ammatory cytokines rather than (Xing and Birukova, 2009). haemodynamic changes, and thus constituted The NPs and other small cationic HDPs part of the early systemic innate immune that exhibit no or minimal direct anti- response to the infectious agent. This fi nding microbial activity (i.e. active only in dilute was reiterated in vitro, with IL-1β and TNF-α medium or buff er), but play an important inducing BNP production in cardiomyocytes role in the endogenous host response to (Vesely and de Bold, 2009), and in animal infection, are listed in Table 12.1. These models, where an ANP/BNP hybrid antagon- peptides and their IDR derivatives (Box 12.1) ist was shown to enhance LPS- and IL-1β- have formed the basis of many induced fever (Miyoshi et al., 2006). LPS immunomodulatory peptide agents currently injection in healthy humans was found to in preclinical or clinical trials (Table 12.2). cause rapid and substantial increases in circulating pro-BNP (Vila et al., 2008), suggesting that this peptide is part of the 12.3 Potential Therapeutic Uses early host response to sepsis, perhaps beyond Anti-infective Activity alerting the host physiology to prepare for impending septic shock. The range of potential therapeutic Whereas BNP-directed immunomodula- applications of HDPs and their synthetic tion has largely been studied in the context derivatives has been greatly broadened by of cardiac infl ammation, the related peptides the discovery of their immunomodulatory ANP and CNP have demonstrated immuno- properties. Beyond their use as novel anti- modulatory eff ects on vascular and lung infectives against bacteria (Scott et al., 2007), tissue. CNP, which has a net charge of +2, viruses (Gallo et al., 2006; Falco et al., 2009) or was shown to inhibit leukocyte recruitment fungi (Benincasa et al., 2006; Kollef et al., and rolling across endothelial surfaces that 2006), they also have signifi cant potential as was driven by IL-1β or histamine in mice, adjuvants (Garlapati et al., 2009), anti- and to inhibit platelet–leukocyte interactions, infl ammatories and anti-endotoxic agents primarily through the modulation of (Andra et al., 2006) and are showing P-selectin (Scotland et al., 2005). Interestingly, substantial promise in wound-healing ANP, which has a higher net charge of +6, applications (Steinstraesser et al., 2008). Clinical Applications of Host Defence Peptides 203

Table 12.2. Immunomodulatory host defence peptides, innate defence regulators and their derivatives in clinical trials. Most advanced References and Peptide Description Intervention progress trial registriesa AP214 (Action Synthetic derivative of the Sepsis and Phase II Doi et al. (2008); Pharma) HDP α-MSH (Table 12.1). postsurgical organ NCT00903604 Parent peptide is fused to a failure hexalysine sequence at its C-terminus CD-NP Chimeric synthetic NP (37- Organ failure Phase II Rose (2010); mer) able to bind to the NCT00482937 receptors for all three endogenous NPs. Modifi ed to lack haemodynamic activity DiaPep277 HSP60 derivative (24-mer Autoimmune- Phase III NCT00644501; (DeveloGen) peptide) that induces mediated diabetes ISRCTN55429664 T-regulatory cells EA-230 Oligopeptide fragment from Sepsis Phase II van den Berg et (Exponential β-hCG (4-mer, LQGV) al. (2009); NAb Biotherapies) Ghrelin Endogenous HDP Airway Phase II (all) Table 12.1; JPRN- infl ammation, UMIN000002599; chronic respiratory JPRN- infection, cystic UMIN000001598; fi brosis NCT00763477 Glutoxim/NOV-002 Hexapeptide with a stabilized Tuberculosis, Market Sokolova et al. (Pharma BAM/ disulfi de bond (bis-(γ-L- myelodysplastic (Russia); (2002); Novelos) glutamyl)-L-cysteinyl-bis- syndromes Phase III (N. NCT00960726 glycine disodium salt) America) Heptapeptide-7 Peptide fragment (7-mer), Wound healing, Phase II Falla and Zhang (Helix BioMedix) derived from the synthetic skin regeneration (2010); NR prototype HB-107 (which is itself derived from cecropin B) hLF1–11 Derivative of the cationic Bacteraemia and Phase I/II van der Does et (AM-Pharma) AMP human lactoferricin fungal infections in Earlier anti- al. (2010); (amino acids 1–11) immuno- infective trials NCT00509938; compromised terminated NCT00509847 haematopoietic (withdrawn); stem-cell transplant NCT00509834 recipients (terminated) IMX942 Immunomodulatory peptide Nosocomial Phase IA NA (Inimex) lacking antimicrobial activity, infections, febrile derived from the prototype neutropenia IDR-1 (which is itself derived from indolicidin) Iseganan (IB-367) Synthetic protegrin-1 Oral mucositis in Phase III NCT00022373; (Ardea derivative (17 amino acids) radiation therapy Earlier anti- NCT00118781 Biosciences) patients infective trials (terminated) terminated Continued 204 M.L. Mayer et al.

Table 12.2. Continued. Most advanced References and Peptide Description Intervention progress trial registriesa Omiganan Synthetic derivative of Topical antiseptic, Phase III NCT00000435, (MX-226) indolicidin (12-mer). Retains acne vulgaris, NCT00027248 (Migenix/BioWest both antimicrobial and papulopustular Therapeutics) immunomodulatory activity rosacea of parent peptide OP-145 (OctoPlus) Synthetic derivative of LL-37 Chronic bacterial Phase II ISRCTN84220089 optimized for maximal LPS otitis media and LTA binding activity (24- mer) Opebacan 21 amino acid peptide Endotoxemia in Phase II, NCT00454155; (Xoma) derivative of bactericidal/ haematopoietic Phase I NCT00462904 permeability-increasing stem-cell protein) transplant recipients, burn wounds PAC-113 Synthetic cationic HDP (12- Antifungal Phase IIB NCT00659971 (Pacgen Bio- mer), histatin derivative pharmaceuticals) RDP58 Semisynthetic D-amino acid Inﬂ ammatory Post Phase II Travis et al. (Genzyme) decapeptide derived from bowel disease (2005); NRc HLA class I B2702 Vasoactive Endogenous HDP Respiratory tract Phase I Table 12.1; intestinal peptide infections, sepsis NCT00004494 a International clinical trial registration number as indexed on the World Health Organization’s global trial database (http://apps.who.int/trialsearch/Default.aspx). b Trial is registered, but in a country that lacks publicly searchable trials. c Clinical trial is registered. Abbreviations: AMP, antimicrobial peptide; hCG, human chorionic gonadotrophin; HLA, human leukocyte antigen; hLF, human lactoferrin; HDP, host defence peptide; HSP, heat-shock protein; IDR-1, immune defence regulator 1; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MSH, melanocyte-stimulating hormone; NA, not available; NP, natriuretic peptide; NR, not registered.

12.3.1 Adjuvant potential system to respond to non-protein antigens and it would be a great step forward to create Adjuvants are components of vaccines that an adjuvant that would enable single-dose are generally not immunogenic themselves, vaccines that are eff ective in infants. but enhance the immunogenicity of the Additionally, adjuvants that can enhance the antigenic components. The inclusion of mucosal immune response to vaccines, adjuvants thus enhances the effi cacy of allowing them to be taken orally rather than vaccines, such that lower doses of antigen are injected, are also actively sought in the fi ght required to achieve eff ective immune against a vast range of diseases. responses and lasting immunological memory. Human neutrophil defensins have Appropriate adjuvants may also decrease the shown adjuvant activity in murine models, number of doses required, thus not only enhancing both humoral and cell-mediated reducing the cost of vaccines but also antigen-specifi c immune responses. For enhancing patient compliance (Nicholls et al., example, the inclusion of defensins with 2010). The development of vaccines to prevent ovalbumin for intranasal immunization of many childhood diseases is frustrated by the mice enabled the enhancement of immuno- limited ability of the immature immune globulin (Ig)G antibody responses; despite Clinical Applications of Host Defence Peptides 205

the mucosal route of administration, deoxyinosine/deoxycytosine, referred to as however, IgA responses were not induced IC31, has shown the ability to enhance cellular (Lillard et al., 1999). Additionally, CD4+ T and humoral immune responses in mice, cells isolated from the immunized mice including the activation of DCs and demonstrated enhanced antigen-induced proliferation and diff erentiation of naive CD4+ proliferation and increases in the archetypal T cells (Schellack et al., 2006). Hurtado and T-helper (Th)1 cytokine IFN-γ, as well as Peh (2010) recently demonstrated that LL-37 secretion of the classical Th2 cytokines IL-5, is able to enhance the responses of B cells and IL-6 and IL-10, demonstrating that defensins DCs to CpG ODNs, and it may well be are capable of stimulating a balanced mimicry of this natural immunomodulatory immune response. Tani et al. (2000) observed property that provides the adjuvant activity of similar adjuvant activity when defensins IDRs. were administered in combination with the model antigen keyhole limpet haemocyanin, showing that IgG1, IgG2a and IgG2b 12.3.2 Wound-healing activity antibody responses were all enhanced by inclusion of the defensins, while ex vivo LL-37 exhibits angiogenic and wound-healing splenocytes from immunized mice produced properties, promoting neovascularization and more IFN-γ and IL-4 and demonstrated re-epithelialization in animal models greater proliferative responses to antigen. (Koczulla et al., 2003; Steinstraesser et al., This same study also demonstrated that 2006, 2008; Carretero et al., 2008). The defensins could be used to boost the immune physiological role of peptides in wound response to tumour-specifi c antigens and healing is supported by the observations that aff ect the outcome of a tumour challenge HDPs such as psoriasin and hBD-2 are more model. highly expressed in tissues surrounding Synthetic IDRs are being investigated as chronic wounds (Dressel et al., 2010; Harder components of vaccine formulations, with et al., 2010) and that the expression of promising results. CpG ODNs have been cathelicidins dramatically increases aft er investigated for their adjuvant potential, but cutaneous injury (Dorschner et al., 2001). cause a signifi cant release of pro-infl ammatory Additionally, treatment with exogenous cytokines. However, the addition of poly-l- hBD-3 was able to enhance re-epithelialization lysine in formulation with CpG ODNs of wounds in a porcine model (Hirsch et al., prevents this undesirable infl ammatory 2009). Many authors have suggested that the response while retaining antigen-specifi c ability of HDPs to enhance wound healing is immune responses (Lingnau et al., 2002). mediated through direct antimicrobial Similarly, a combination of IDR-HH2 and activity, with the presence of HDPs prevent- CpG ODN enhanced antigen-specifi c ing or limiting infection of the wounds. While antibody responses in mice, including the antimicrobial activity may well play a role in production of high titres of IgA, IgG1 and the wound-healing activity of HDPs, the IgG2a. This combination also appeared to ability of these molecules to modulate the increase surface markers on DCs and induce expression of growth factors and cytokines is cytokine and chemokine responses ex vivo likely to be more important. (Kindrachuk et al., 2009). Formulations HDPs are able to stimulate the including HDPs, CpG ODNs and poly- production of cytokines and growth factors phosphazene-enhanced antigen-specifi c cellu- in epithelial cells and keratinocytes. For lar and humoral responses in both mice and example, LL-37 stimulates the release of IL-8 catt le (Kovacs-Nolan et al., 2009a,b,c) and a from airway epithelial cells by a mechanism similar combination showed promise in a that appears to involve transactivation of neonatal pig model, suggesting applicability epidermal growth factor receptor (Tjabringa of this technology to infant vaccines (Garlapati et al., 2003). LL-37 and hBD2–4 are able to et al., 2009). Additionally, immunization with induce the secretion of IL-18 from keratino- a formulation of the peptide KLKL5KLK with cytes; inhibitor studies have demonstrated 206 M.L. Mayer et al.

that this involves modulation of p38 and wide ranging, giving a vast potential for ERK1/2 MAPK pathways, but is independent designer synthetic peptide drugs targeting of JNK (c-Jun N-terminal kinase) (Niyonsaba diff erent clinical conditions. et al., 2005). IL-18 is a pleiotropic cytokine that is classically thought of as an inducer of IFN-γ, but is also able to promote 12.4 Recent Clinical Advances in angiogenesis (Park et al., 2001). LL-37 and Therapeutic Application of Host hBD-2–4 also promote the proliferation and Defence Peptides migration of keratinocytes and the production of IL-6, IL-10, 10-kDa IFN-γ- The introduction of insulin in the 1920s induced protein, monocyte chemotactic marked the beginning of a new age in protein-1, macrophage infl ammatory protein pharmaceutical drugs, where endogenous (MIP)-3α and RANTES (regulated on agents (or their mimetics) could be activation, normal T-cell expressed and administered with the therapeutic aim of secreted) by these cells (Niyonsaba et al., modulating human physiology. These 2007). In a subsequent study, the HDP biopharmaceutical agents, typically proteins dermcidin (DCD-1L) stimulated keratino- or peptides and more commonly referred to cytes to produce a similar range of cytokines, as ‘biologics‘, have become a mainstay in the including TNF-α, IL-8, IFN-inducible protein treatment of endocrine, autoimmune and 10 (IP-10) and MIP-3α (Niyonsaba et al., other diseases with pathophysiology or 2009), demonstrating that a wide range of dysfunction involving human systems with HDPs are able to activate keratinocytes. In an complex feedback and feedforward regu- in vivo system, hBD-2 has also been found to lation. At the forefront of cutt ing-edge stimulate chemotaxis of endothelial cells and research and development into novel promote capillary-like tube formation as biologics are compounds that act at multiple eff ectively as vascular endothelial growth points within the dynamic regulatory factor (Baroni et al., 2009). This same HDP networks underlying disease states, moving also promotes the migration, but not proliferation, of intestinal epithelial cells beyond the classic one drug–one receptor (Ott e et al., 2008), as does LL-37 (Ott e et al., model of traditional pharmaceuticals. 2009). A synthetic acetylated and amidated Peptide-based immunotherapies are still 24-mer derivate of LL-37, termed P60.4-Ac in their infancy, but the use of immune- (Nell et al., 2006), has been shown to inhibit targeting therapeutics is well established disruption of the respiratory epithelium within the practice of Western medicine ultrastructure by bacterial pathogen- (Waldmann, 2003). To date, the majority of associated molecular patt erns in vitro (Vonk these biologicals with immunomodulatory et al., 2008). Although some of these activities action have aimed at the suppression or occur at quite high concentrations of disruption of normal immune function, rather peptides, it has been demonstrated that very than its enhancement. As a result, these drugs low concentrations of peptides synergize have become the mainstay of treatments for with endogenous host molecules such as chronic infl ammatory and autoimmune IL-1β and granulocyte–macrophage colony- diseases, but show litt le appreciable value as stimulating factor, as well as with TLR adjuvants or therapeutics in treating infectious agonists such as fl agellin and poly-IC diseases. Such applications are also limited by (Bowdish et al., 2005a,b; Filewod et al., 2009). an inability to selectively target the eff ector Thus, the range of biological activities of subsets that mediate the necessary com- HDPs extends well beyond direct bacterial ponents of innate and adaptive immunity killing, with an ability to modulate (Feldmann and Steinman, 2005), resulting in intracellular signalling and gene expression side eff ects that can include enhanced in a wide range of cell types. The susceptibility to infections (Botsios, 2005), downstream eff ects of such activities are also signifi cantly high rates of anaphylaxis (Corren Clinical Applications of Host Defence Peptides 207

et al., 2009) and, in extreme cases, toxic While no clinical data are yet publicly cytokine storms (Suntharalingam et al., 2006). available, IMX942 (which is a derivative of Immunostimulatory drugs are beginning IDR-1 and lacks direct antimicrobial activity) to make inroads as viable options for the has completed Phase I safety trials in treatment of infectious diseases, oft en as chemotherapy patients with febrile neutro- adjunctive therapy to mainstay antibiotics. A penia (Table 12.2). In addition, OP-145, a recent Cochrane clinical trial meta-analysis structural derivate of LL-37, has been (Del-Rio-Navarro et al., 2006), examining the reported to show effi cacy in clinical trials for use of 21 diff erent biological immuno- the treatment of chronic otitis media in adult modulatory agents in the treatment of patients (Table 12.2). Whether immuno- pediatric acute respiratory infections, modulatory (immunostimulatory) peptides provided fascinating data on the safety and will become mainstays in the treatment of effi cacy of these drugs. The Cochrane review bacterial antibiotics remains to be seen. found that the use of immunostimulatory However, the successes in clinical trials to drugs reduced the incidence of respiratory date demonstrate signifi cant promise for tract infections by an average of 40% across these peptides in playing, at minimum, an the studies, with adverse eff ects not being adjunctive role to conventional antibiotic signifi cantly diff erent to those associated therapy. with placebo groups. Many of the peptides with documented Due to the complexities inherent in immunomodulatory and antimicrobial studying innate immunity and the cor- activity that have entered clinical trials have responding diffi culty in selecting appropriate done so as topical antimicrobials for clinical readouts or endpoints in preclinical infectious, infl ammatory or wound-healing and Phase I trials, clinical trials of applications (Table 12.2). The choice of immunomodulatory drugs oft en evaluate moving forward with these agents as topical compounds in manners that avoid excessive interventions likely refl ects diffi culties in responses or systemic absorption. For selecting appropriate systemic biomarkers as example, RDP58, which is under investi- safety readouts. Thus, a series of peptides gation for the treatment of ulcerative colitis have been tested topically, including (Travis et al., 2005), was structurally modifi ed omiganan (MX-226), iseganan (IB-367), to prevent both proteolytic degradation and human lactoferrin (hLF)1–11 and HB-107 systemic absorption (Holtmann, 2003). Two (Table 12.2), despite the fact that most of Phase II trials of RDP58 in a combined cohort these peptides have demonstrated immuno- of 127 patients found the drug to be well modulatory activity in vivo (Giacomett i et al., tolerated (adverse eff ects equivalent to 2003; Lee et al., 2004; Falla and Zhang, 2010; placebo) and effi cacious in improving sig- van der Does et al., 2010). Interestingly, the moidoscopy scores in colitis patients majority of the peptide (and peptide-derived) receiving medium or high doses of the drug drugs under investigation as immuno- (Travis et al., 2005). The pathogenesis of modulatory anti-infectives are the classical ulcerative colitis is thought to be at least neuroendocrine peptides described in Table partially due to a dysregulated host response 12.1. This is probably because these peptides to intestinal microbiota (Xavier and Podolsky, have been under investigation for decades 2007) and, although RDP58 is being (albeit in other contexts) and have well- investigated for its anti-infl ammatory prop- defi ned receptors and cellular mechanisms. erties, it may also alter host responses to This is in contrast to HDPs, which are microbiota in a manner similar to HDPs and relatively new discoveries. Based on these IDRs. observations, we feel there is a strong An exciting area in which the dual anti- possibility that, in the future, immuno- infective and anti-infl ammatory actions of modulatory activity will play a substantial peptide immunotherapy have potential is the role in any clinical benefi t demonstrated by treatment of bacteraemia and septicaemia. HDP- and IDR-based drugs. 208 M.L. Mayer et al.

12.5 Innate Defence Regulators as matory sequelae of severe acne and a non- Anti-infective Therapeutics infectious infl ammatory skin disease, rosacea, demonstrating promise for the treatment of The increased awareness of the natural infl ammatory conditions independent of immunomodulatory functions of HDPs and antimicrobial activity. Systemic safety has the role they play in the endogenous anti- been shown in Phase I clinical trials for hLF1– infective response has created an exciting 11, a truncated form of lactoferrin, which was new avenue for their application: the use of examined in immunocompromised haemato- synthetic peptides that selectively modulate poietic stem-cell transplant recipients. Recent the innate immune response as an anti- work with hLF1–11 in animal models has also infective therapeutic strategy. One of the fi rst demonstrated its effi cacy as a systemic anti- synthetic immunomodulatory peptides infective through immunomodulatory based on a HDP, immune defence regulator 1 mechanisms (van der Does et al., 2010). (IDR-1), is able to mediate in vivo protection OP-215, a synthetic 24 amino acid against bacterial challenge in the absence of derivate of LL-37, has also been shown to be direct antimicrobial activity (Scott et al., effi cacious and safe (compared to placebo) in 2007). The sequence of IDR-1 is based on the a randomized, double-blind, placebo- 12 amino acid bovine cathelicidin bactenecin, con trolled, multicentre Phase II study when which has immunomodulatory activities that applied topically in ear drops to chronic refl ect a subset of those apparent in the 37 suppurative otitis media patients (F.A.W. amino acid human peptide LL-37 (Bowdish Peek et al., 2009, unpublished results). et al., 2005a). The functional mechanism of Although litt le information on this peptide is action of IDR-1 in animal infection models available, it is claimed that it binds LPS and appears to involve the concomitant lipoteichoic acid, degrades biofi lms and has enhancement of chemokine production and antimicrobial actions, while its direct moderation of proinfl ammatory cytokines chemotactic activity is said to be low. such as TNF-α, leading to enhanced cellular Another peptide, RDP58, which is a cationic clearance of bacteria without excessive peptide derived from the heavy chain of infl ammation. These eff ects appear to be human leukocyte antigen (HLA) class I mediated through modulation of specifi c molecules, has also demonstrated safety in intracellular signalling pathways via intra- clinical trials (Travis et al., 2005). RDP58 cellular binding partners such as sequesto- reduced the production of the pro- some-1/p62 (Yu et al., 2009) or GAPDH, infl ammatory cytokines TNF-α, IFN-γ and which leads to altered activation states of key IL-12, but did not aff ect the production of transcription factors (Scott et al., 2007; several other cytokines including IL-4, -6, -8 Mookherjee et al., 2009a). Such activities and -10. The proposed mechanism of action provide a powerful rationale for designing involves interference with intracellular synthetic peptides with optimized immuno- signalling pathways; however, the specifi c modulatory activities based upon HDPs (e.g. details and interactors are currently Nĳ nik et al., 2010) and demonstrate that unknown (Travis et al., 2005). IMX942, which direct antimicrobial activity is not a necessary is based on IDR-1, has completed Phase I characteristic for the effi cacy of such safety trials in chemotherapy patients with peptides. febrile neutropenia (see www.inimexpharma. Five immunomodulatory cationic pep- com). tides have already demonstrated safety in While IDR-1/IMX942 and RDP58 are human clinical trials. Two of these, MX-226 both cationic peptides that modulate innate and hLF1–11, were originally developed as immunity, they appear to do so via distinct AMPs but also demonstrate immuno- mechanisms. Both peptides suppress the modulatory activity (van der Does et al., induction of particular proinfl ammatory 2010). Topical delivery of MX-226 has cytokines, such as TNF-α, but their eff ects on demonstrated safety and effi cacy in Phase II other cytokines vary. For example, RDP58 clinical trials that targeted both the infl am- does not aff ect IL-6 or -10 production, while Clinical Applications of Host Defence Peptides 209

the treatment of cells with IDR-1 tends to in vivo, as isolated cell systems cannot enhance the production of these two capture the complexity of the innate immune cytokines. The distinct responses to these response. Thus, compared to testing of peptides indicate the value of diff erent antimicrobial activity, for which there is oft en design strategies, since RDP58 is based upon a strong correlation between in vitro and in HLA class I molecules while IDR-1 is a vivo activity, it is much more labour-intensive derivative of a bovine cathelicidin. Import- and costly to generate the large datasets antly, this also indicates that if we can necessary for applying computational understand the intricacies of immuno- methods to immunomodulatory peptide modulatory peptide interactions with host design. cell components and pathways, it may be Despite this constraint, we now have a possible to design peptides with customized few precedents where rational peptide immunomodulatory eff ects to target diff erent design based on structure–function para m- diseases. eters has proven successful. For example, synthetic endotoxin-binding peptides can be optimized for protection in vivo (Dankesreiter et al., 2000) and a fragment of LL-37 can 12.6 Rational Design of be optimized for anti-endotoxin activity Immunomodulatory Peptides through appropriate amino acid substitutions (Nagaoka et al., 2002). It is also possible to The development and validation of screening create truncated peptides based on HDPs techniques is an important aspect of the IDR such as LL-37 that retain either the parent design process. High-throughput in vitro peptide’s antimicrobial or immuno- screening of antibacterial activity has been modulatory properties (Braff et al., 2005; used for the validation and iterative Chapter 9). Taken together, these fi ndings optimization of rationally designed AMPs demonstrate that particular domains are (Cherkasov et al., 2009; Fjell et al., 2009). The necessary for these functions and suggest technical simplicity of in vitro testing for that it will be possible to identify particular direct antibacterial activity has made it characteristics associated with protection. possible to collect large datasets using high- Other studies have optimized the activities throughput screening techniques and use of smaller bactenecin- and indolicidin-like this information to identify particular synthetic peptides as components of adjuvant physical characteristics that correlate with formulations by screening for chemokine- antimicrobial activity. By contrast, the inducing activity followed by in vivo analysis complexity of the interactions between (Garlapati et al., 2009; Kindrachuk et al., 2009; multiple cell types and eff ector molecules of Kovacs-Nolan et al., 2009a,c). An iterative the immune system mean that it is not design process has also successfully possible to thoroughly assess subtle immuno- produced new IDRs with greater anti- modulatory activities in vitro, and simplifi ed infective effi cacy than IDR-1 (Nĳ nik et al., markers of this activity must be used. For 2010). example, IDR-1 was selected based on markers such as synergistically enhanced release of chemokines from LPS-stimulated 12.7 Limitations and Challenges human peripheral blood mononuclear cells (Scott et al., 2007) and chosen for further All drug candidates must overcome potential analysis aft er demonstrating an ability to toxicities and questions of in vivo stability protect mice against bacterial infections. and appropriate delivery routes. Clearly it is Another possible screen would be the ability possible to design IDRs that protect without to suppress TNF-α induction in response to substantial in vitro or animal-model toxicity treatment with TLR agonists (Mookherjee et (Scott et al., 2007; Nĳ nik et al., 2010). al., 2006). Even with the use of such markers, However, no systemic toxicology and candidate peptides must ultimately be tested pharmacokinetic studies have been described 210 M.L. Mayer et al.

for any peptide and questions still remain proposed for enhancing the in vivo stability regarding potential in vivo toxicities. This is and long-term shelf-life of peptides. particularly important when considering the Additionally, the cost of manufacturing transition from murine models to use in synthetic peptides is very high, so strategies humans, considering the many diff erences that enhance bioavailability or reduce the between the rodent and human immune size or number of doses required for systems and possible requirements for therapeutic benefi t are also important for multiple dosing. While small-animal models decreasing the cost of treatment. provide very valuable information regarding One way of enhancing peptide stability in vivo activity, dosing, appropriate for- is to induce chemical modifi cations that mulation and routes of administration, the provide resistance to proteolytic digestion intricate regulatory networks and feedback without altering the functional characteristics loops that make up the mammalian immune of the peptide. For example, the use of system are such that even small diff erences d-amino acid isomers of peptides is one between hosts can be magnifi ed quite approach used for RDP58 (Table 12.2), substantially. In cases where severe problems exploiting the specifi city of proteases for have occurred with immunomodulators l-amino acid forms of the peptide. Since the (Ponce, 2008), few in vitro or ex vivo data three-dimensional structure of the peptides involving human cells were available and the may be important for their interactions with problems may have been predicted had such specifi c receptors, retro-inverse peptides experiments been undertaken prior to human comprised of d-amino acids in the reversed trials (Dayan and Wraith, 2008) or with bett er sequence have been investigated; this pre- regulatory oversight (Gott lieb, 2008; Shuch- serves the spatial positions of the side chains man, 2008). Several peptide-based drugs, and while maintaining the protease resistance of many immunomodulatory drugs of varying the d-amino acid forms (Fischer, 2003). compositions, have successfully passed Recently, a synthetic peptide, M33, was clinical testing and entered the market, shown to resist proteolytic degradation when demonstrating that it is possible to ensure constructed in a tetra-branched form (Pini et the safety of such drugs. al., 2009). The peptide demonstrated anti- The reduction of the in vivo stability of bacterial activity against a range of Gram- peptides by proteolytic processing is also of negative species and protected against concern. While small peptides are probably endotoxemia in mice. Conversely, creating a degraded within minutes in many com- cyclic structure – as for defensins – may partments of the host (e.g. blood, where enhance in vivo stability. The use of unnatural proteases abound), results with IDR-1 amino acid side chains or modifi ed peptide indicate that treatments up to 48 h prior to or backbones (peptidomimetics) has substantial 6 h aft er bacterial challenge are protective in potential in this regard. animal models (Scott et al., 2007). This raises A potential limitation of any novel anti- the question of how a peptide that is rapidly infective is the development of microbial degraded can have long-lasting eff ects and resistance. Thus, a very important char- whether enhancing peptide stability is acteristic of IDRs is that there is a lower desirable in this context. One possibility is likelihood of microorganisms developing that the peptides prime immune cells; resistance to them than is the case with direct indeed, their ability to readily translocate AMPs (Steinstraesser et al., 2009). For the into immune cells (Lau et al., 2005) may AMPs, it has been argued that their physical protect them from proteolytic degradation. action on membranes or their multiple This is consistent with the observation that targets in a single cell make resistance the peptides are active when delivered by unlikely. However, several studies have many diff erent routes, possibly indicating demonstrated resistance to AMPs mediated that these locally primed cells (or cells by the diff erential expression of a range of containing peptide) migrate to the infection genes, oft en through blocking uptake site. Regardless, several methods have been (McPhee et al., 2003; Kraus and Peschel, 2008; Clinical Applications of Host Defence Peptides 211

Ernst et al., 2009; Majchrzykiewicz et al., 2010; antitumour activity, since some HDPs reduce Warner and Levy, 2010). Nevertheless, IDR angiogenesis rather than promote it peptides do not act directly on bacteria, but (Economopoulou et al., 2005; Krylov et al., rather through modulation of the host 2007), while others promote apoptosis of immune system. Since the innate immune tumour cells or some have even demon- system has co-evolved with pathogenic strated anti-proliferative activity (Sutt mann microbes, subtly augmenting this response et al., 2008). Current research is investigating should not enhance the selective pressure on the feasibility of designing membrane- bacterial immune evasion mechanisms. disrupting peptides that exploit diff erences Additionally, enhanced innate clearance of in membrane fl uidity to permit them to be microbes mediated by IDRs should avoid the cytotoxic to tumour cells but not to healthy creation of bacterial debris that continues to cells (Mader and Hoskin, 2006; Fadnes et al., promote infl ammation in the absence of live 2009). Indeed, many of the HDP-based bacteria (Andra et al., 2006), providing yet immunotherapies in clinical trials are being another advantage over traditional antibiotic explored as cancer therapies (Table 12.2). therapies; indeed, some of these peptides Therefore, as with any novel immuno- actually suppress such septic infl ammatory modulatory drug, while it is certainly responses. important to remain conscious of the A potentially serious undesirable possibility of tumour promotion as a side outcome of treatment with peptides based eff ect, with the appropriate testing and upon LL-37 is the promotion of tumori- rational design it is possible to ensure that genesis. As discussed previously, LL-37 has this scenario never becomes a reality. demonstrated wound-healing activity, which includes enhanced angiogenesis and proliferation of keratinocytes and epithelial 12.8 Conclusions cells. These functions alone suggest the possibility of tumour promotion as a HDPs and their synthetic derivatives have potential side eff ect of the inappropriate demonstrated signifi cant potential as anti- presence of LL-37 and such concerns are infective therapeutics and current research compounded by the observation that LL-37 promises to reveal crucial mechanistic can act as a growth factor for lung cancer information that should greatly enhance the cells (von Haussen et al., 2008), promote a development of optimized peptides. These metastatic phenotype in breast cancer cells peptides exert powerful actions in the human (Weber et al., 2009) and infl uence the body, including modulation of innate progression of ovarian tumours (Coff elt et al., immunity, suppression of harmful infl am- 2009). However, the very nature of the mation and promotion of adaptive immunity, altered characteristics of cancer cells means and roles in the enhancement of wound that any immunomodulatory molecule could healing and resolution of the infl ammatory inadvertently become a growth factor for process. New insights and awareness about such cells. It is not surprising that LL-37 structure–function activities, cellular binding could be co-opted in such a way, especially if partners and dynamic functional roles of it is a ligand for CXCR2 considering that this these peptides will allow the rational design receptor is involved in melanoma growth of synthetic peptides targeted for diff erent and invasion (Singh et al., 2009). However, purposes, not all of which will necessarily be this limitation is not grounds to assume that anti-infective. The ability to subtly alter the certain HDP-based treatments are inherently normal balance between protective innate problematic. Rather, these concerns illustrate immune responses, such as chemokine that tailored synthetic analogues with some, production and concomitant leukocyte but not all, of the natural functions of recruitment, and the potentially harmful particular HDPs should be considered the eff ects of excessive infl ammation provides a optimal design output. Indeed, it seems unique opportunity to promote eff ective likely that one can design IDRs with innate immune defences against pathogens. 212 M.L. Mayer et al.

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J.J. (1999) β-Defensins: linking innate and against H2O2-induced inhibition of wound adaptive immunity through dendritic and T cell restitution in IEC-6 cells via a Syk kinase- and CCR6. Science 286, 525–528. NF-κB-dependent mechanism. Shock 22, Yoon, S., Goh, S., Chun, J., Cho, E., Lee, M., Kim, 453–459. K., Kim, J., Kim, C. and Poo, H. (2003) Index

Note: page numbers in italic indicate tables, page numbers in bold indicate ﬁ gures. For antimicrobial peptides not listed, please visit the online database at htt p://aps.unmc.edu/AP/main.html.

A3-APO 101, 136 DNA binding 130–131 abaecin 7 HIV inhibition 14 ABC transport system 23 hydrophobic residue content 11 Ac-AMP1 and 2, 46 identifi cation 1 actagardine 26 length 11 acyl-lysyl oligomers (OAKs) net charge 10 antibacterial properties 107–109 nomenclature 5 antiparasitic properties 109–110 structure 14, 150–152 charge and hydrophobicity 105 synergistic eff ect 9 design 104 amphipathic peptides xvi, xvii, 141, 155, 158 most selective 105 AMSDb see Antimicrobial Sequences Database structure–activity relationships 104, 106–107 angiogenesis 132, 143 adjuvants: for vaccines 204–205 animal AMPs 6, 15 AFM see atomic force microscopy (AFM) anionic peptides 9 African clawed frog xv ANN see artifi cial neural network (ANN) agar diff usion assay 17, 91–92, 93 antibacterial activity 8 alanine-scanning mutagenesis 30 anticancer activity 8 alginase 173 antifungal activity 8 ALL-38 75 ANTIMIC database 2–3 alpha (α)-helices 150–152 antimicrobial, defi nition 199 alpha (α)-peptide 102 amidation 12, 82 Antimicrobial Peptide Database (APD) 2–3, 41 amino acids classifi cation fl uorinated 82 by amino acid sequence 15–16, 17 frequently occurring residues 74 by binding targets and mechanisms of properties used in AMP prediction and action 14–15 design 74 by biological activity 8–9 selectivity index 74 by peptide characteristics 9–14 sequence analysis 15–16, 17 by peptide family 6–8 single (three)-lett er code 74 by source organism 5–6 transfer free energy 74 by structure 14 AMPer prediction tool 3, 75–76 NMR structures 14, 143 amphibian AMPs 6 nomenclature 3–5 cancer therapy 131 in prediction of novel peptides 72–76 classifi cation 7 screening for HIV-inhibitory activity 76–77 d-amino acids 13 see also design

221 222 Index

Antimicrobial Sequences Database 2, 41 therapeutics 131 in prediction of novel AMPs 75–76 and vitamin D 182, 190 antiparasitic activity 8 Candida albicans 103 antiviral activity 8 Candida elegans high-throughput assay 92 APD see Antimicrobial Peptide Database (APD) CAP see cationic antibacterial peptides (CAP) apidaecin 133–134 carbohydrate-binding AMPs 15, 45, 159 apoptosis 132, 143 carbohydrate-binding molecule, family III archaea AMPs 6 (CBM3) 144 arginine–PG interaction by solution NMR 156–158 carbonate and activity 143 artifi cial neural network (ANN) 74 cardiolipin (CL) 120, 122 arylamides 103–104 carpet model 128 atomic force microscopy (AFM) 124 carrier proteins for AMP expression 144 aurein 1.2 151, 152, 157 cateslysin 124 avian AMPs see bird AMPs cathelidicins xvi, 4 function and characteristics 8, 185–186, bacterial AMPs 15 197–198 see also bacteriocins induction of dermal expression by vitamin bactericidal propensity value (PV) 74 D 188–189 bacteriocins 158 structure 142–143, 151, 197 activity spectrum 8 see also LL-37 BACTIBASE database 3 cation–π interaction 154 charge, net 10 cationic antibacterial peptides (CAPs) 171–172, circular 12 173, 174, 175–176 classifi cation 6–7, 24 cationic peptides 199 defi nition 22 natriuretic peptides 201–202 hydrophobic residue content 11 with no antimicrobial activity, but regulating identifi cation and classifi cation 22–23 response to infection 200–201 length 11 cationic steroid antibiotics (CSAs) 175 potency 22 cations, polyvalent 170–171 prediction of novel peptides 76 CBM3 see carbohydrate-binding molecule, family see also lantibiotics; microcins III (CBM3) BACTIBASE database 3 CD see circular dichroism (CD) BAGEL for bacteriocin prediction 76 cecropins xvi, 1, 109 barrel-stave pore model 88 source of cecropin P1 6 beta (β)-peptide 102 cell-cycle inhibition 159 beta (β)-sheets 16, 152–154 ceragenins see cationic steroid antibiotics bicelles 147 CF see cystic fi brosis (CF) biliary tract 189 charge (Q) binding: classifi cation by binding target 14 acyl-lysyl oligomers 105 biofi lms 171–172 as basis for classifi cation 9–10, 11 bioinformatics 2–15, 31–32, 52, 72-81 cyclotides 58–59, 61 BioMagResBank 149 density 125 bird AMPs 6 neutralization and inactivation 169–171 BMAP-27 and -28 76, 80 statistical analysis 9–10 bombinins 12 cheese: starter cultures 29 brazzein 5 chemical cleavage 145 brevinin 7 chemical modifi cations 10–13 broth dilution assays 90–91, 92 chemistry, combinatorial see combinatorial physiological conditions 143 chemistry buforin II 9, 129, 130–131 chemokines: nomenclature 5 chemotaxis 5, 143, 153 caenopore-5 150 chitin-binding peptides 14, 45–48, 158–159 Caenorhabditis elegans 150 cholic acid derivatives 175 calcipotriol, treatment of psoriasis 190 cinnamycin 28 CAMP database 3 circular dichroism (CD) 106, 124, 130, 135, 143 cancer circulin A and B 7, 56–57, 58 and LL-37 143, 176, 211 CL see cardiolipin (CL) Index 223

classifi cation cystic fi brosis (CF) xv, xvii, 169–170 by binding target and mechanism of eff ects of DNase, gelsolin, alginase on action 14–15 sputum 173 by biological activity 8–9 eff ects of polyaspartate on sputum 174 of databases 2 F-actin and DNA in sputum 171 of lantibiotics 24–25 sequestration and inactivation of LL-37 172–173 by peptide characteristics and vitamin D 189 charge, length and hydrophobic cytokines 205–206, 208 residues 9–10, 11 cytolysin 28–29 chemical modifi cations 10, 12–13 cytotoxic eff ect 8 three-dimensional structures 13–14 by peptide family 6–8 D8PG see dioctanoyl PG (D8PG) of plant AMPs 7, 41 d-amino acids by source organism 5–6 eff ect on structure 159 cleavage 145 in natural AMPs 12–13 clinical trials 203–204, 207 in peptide design 80–82 CNBr see cyanogen bromide (CNBr) modifi cation enzymes 13 Cochrane reviews 207 dairy industry: use of lacticins 29 cod-liver oil and vitamin D 182 databases colic acid derivatives 175 in AMP prediction and design see design: of colicin 22 novel peptides; discovery: of novel combinatorial chemistry peptides chronological list of major databases 2 accomplishments 96 classifi cation 2 and high-throughput screening 91 nomenclature 3–5 indexed methods 90 of plant AMPs 41 library synthesis 88–89 scope and overview 2–3 non-indexed methods 89–90 use in screening for new lantibiotics 31–32 CP-11 159 DCs see dendritic cells (DCs) CpG oligodeoxynucleotides (ODNs) 196 defensins xvi, 158–159 CRAMP 197 activities and function 8, 186 Crohn’s disease xvii, 187, 197 adjuvant potential 204–205 crustacean AMPs 6 circular 153 see also shrimp AMPs Defensins Knowledgebase 3 cryptdins 196 HIV inhibition 14 CSAs see cationic steroid antibiotics (CSAs) identifi cation 1 Cy-AMP1 46–47 mammalian cyanogen bromide (CNBr) 145 activities and function 196–197 Cybase cyclotide database 3, 41, 51–52 human: structural characteristics 141–142, cyclization 12, 62, 63 152–154 cyclopeptide alkaloids 49, 51 production and secretion 196–197 cyclotides nomenclature 4 activity plants 44–45 anti-HIV 56–57 dendritic cells (DCs) 189, 197 antibacterial and antifungal 57, 60, 61–62 deoxyribonucleic acid (DNA) 130, 131, 133 pesticidal 56 in cystic fi brosis sputum 171 range 49, 56 sequestration and inactivation of LL-37 172 biosynthesis 54–56 depolarization, membranes 129 Cybase database 3, 51–52 dermaseptins 77, 150 database 41 dermcidin 4, 8–9 detection and isolation 54 design: of novel peptides diversity 49, 50, 53, 54 amino acid properties used 74 in drug design 62 database-aided enhancement of desired engineering and mass production 62–63, 64 activity 80 statistical analysis of amino acid sequences 17 database screening for desired activity 76–77 structure 51–53 de novo peptide design 78 surface characteristics and charges 58–59, 61 of host defence peptide mimics see under cycloviolacin 58 host defence peptides 224 Index

design: of novel peptides continued database-aided 76–80 hybrid and grammar-based approach 78 of lantibiotics 30–31 immunomodulators 209–211 structure-based 159–160 improvement of cell selectivity 80–81 enterocin AS-48 12 improvement of stability to proteases 82 Enterococcus faecalis, vancomycin-resistant see VRE resistance to inactivation by (vancomycin-resistant E. faecalis) polyelectrolytes 174–175 enterokinase 145 sequence shuffl ing 77–78 enzyme cleavage 145 detergents 146 epidermin-like lantibiotics 26–27 Dha, dehydrated serine 23 medical applications 30 Dhb, dehydrated threonine 23 epilancin 15X 26 diff erential scanning calorimetry (DSC) 107, 120, ericin 25 124 erythrocytes see red blood cells (RBCs) dioctanoyl PG (D8PG) 146, 158 Escherichia coli dipicolinic acid (DPA) 96 as bacterial expression system for protein discovery: of novel peptides production 143–146 activity datasets 72 as lab strain for antibacterial activity amino acid properties used 74 assays 94–95, 105, 119 combinatorial chemistry 88–90 esculentin 7 prediction based on highly conserved exopolysaccharides and biofi lm formation 171 propeptide sequences 75 see also alginate prediction based on mature peptides expression, bacterial 143–146 AMP sequence motifs 75 eukaryotes 6 Fourier transformation method 73–74 information used 73 F-actin 170 knowledge-based 72 in cystic fi brosis sputum 171 machine-learning methods 73–74 sequestration and inactivation of LL-37 172 prediction based on processing enzymes 76 factor Xa 145 prediction based on propeptides and mature FALL-39, initially predicted human cathelicidin 75 peptides farnesoid X receptor (FXR) 189 AMPer model 3, 75–76 ‘fi ght or fl ight’ defence reponse 40 prediction: genomic-based of fi sh AMPs 6 bacteriocins 76 FK-13 77, 151–152 distinctin 150–151 food chemistry: use of lantibiotics 29 divercin V41 6 food preservative see nisin Dm-AMP1 44–45 formic acid 145 DNA see deoxyribonucleic acid (DNA) formyl peptide receptor-like 1 (FPRL-1) 143 DNA-binding AMPs 15, 130–133 Fourier transform infrared spectroscopy (FT- DnaK heat-shock protein 134–135 IR) 143 DNase 173 frog AMPs see amphibian AMPs dodecylphosphocholine (DPC) 146 fungal AMPs 6 double-quantum-fi ltered correlation spectroscopy FXR see farnesoid X receptor (FXR) (DQF-COSY) 147 DPA see dipicolinic acid (DPA) gamma (γ)-core motif 5, 16, 75, 152 DPC see dodecylphosphocholine (DPC) gassericin A 13 DQF-COSY see double-quantum-fi ltered gastrointestinal peptides 201 correlation spectroscopy (DQF-COSY) gelsolin 173–174 drosocin 133–135 gestation 189–190 DSC see diff erential scanning calorimetry (DSC) GF-17 77, 123 DSS see sodium 2,2-dimethylsilapentane-5- ghrelin 201 sulfonate (DSS) GI-20 158 dynamics by NMR 148, 150, 152 glucosylceramide 159 glutathione S-transferase (GST) 144 endotoxin see lipopolysaccharide (LPS) glycoprotein 41 (gp41) 14 engineering glycoprotein 120 (gp120) 153 of A3-APO 101 glycosaminoglycans 170 of cyclotides 62–63 glycosylation 12 Index 225

gomesin 153 HPLC see high performance liquid gramicidins 10 chromatography (HPLC) granulysin 8 HSQC see spectra, heteronuclear single-quantum Greek-key motif 49 coherence (HSQC) human AMPs 1, 6, 8, 142, 153, 169, 186–188, H see hydrophobicity (H) 196–199 haemolytic activity 8, 103 see also cathelicidins; defensins; dermcidin; haloduracin 26, 28, 76 hepcidin; histatins; granulysin halogenation 12 human beta defensins (hBDs) 142, 186, 196 hBDs see human beta defensins (hBDs) human immunodefi ciency virus (HIV): anti-HIV hCAP-18 4, 143, 186, 197 activity 56–57, 133 HDPs see host defence peptides (HDPs) database-aided enhancement 80 healing, wound 205–206 database screening for inhibitory heat-shock protein DnaK 134–135 peptides 76–77 helices, alpha (α) 150–152 sequence shuffl ing in peptide design 76–77 heliomicin 7, 14 human neutrophil defensins (HNPs) 142 helix bundle 150 hydrophobicity (H) helix-hinge-helix 150 acyl-lysyl oligomers 104, 105, 106 hepcidin 200 basis for classifi cation 9–10, 11 hevein 45–46, 159 enhancement 175 high performance liquid chromatography measurement by HPLC 105, 159 (HPLC) 105, 144, 159 reduction 80–81 high-throughput screening (HTS) hydroxylamine 145 accomplishments 96 hydroxylation 12 biological assays 90–92 and combinatorial library synthesis 91 IDRs see innate defence regulators (IDRs) example data 95 IDR-1 208, 209 non-biological assays, e.g. vesicle-based 94, immunity proteins 24 96–97 immunomodulators scope 90 antimicrobial and host defence peptides 185– selection of broad-spectrum peptide 186, 197, 198, 199, 201–202 antibiotics 92, 94 clinical use and clinical trials 206–209 histatins 8, 200 rational design 209 HIV see human immunodefi ciency virus (HIV) limitations and challenges 209–211 HMMER in AMP prediction 75 vitamin D 184–185 HNPs see human neutrophil defensins (HNPs) IMX-942 207, 208 host defence peptides (HDPs) indexing: of combinatorial libraries 90 clinical trials 203–204 indolocidin 132–133, 154, 155 de novo-designed peptidomimetics infl ammation 132 arylamides 103–104 innate defence regulators (IDRs) 199 hairpin-shaped 102 adjuvant potential of synthetic IDRs 205 OAKs (oligomers of acylated lysines) see as anti-infective therapeutics 208–209 acyl-lysyl oligomers clinical trials 203–204, 207 peptoids 103 rational design 209 representative building blocks 102 limitations and challenges 209–211 discovery 100–101 insecticidal activity 8, 63 limitations on use 101 insects role in response to infection 198–199 classifi cation of AMPs 7 source of new antibiotics 128 pests 56 strategies to alleviate shortcomings 101–102 proline-rich insect peptides 133–135 therapeutic potential 101, 202, 208–209 in silico peptide screening 78 adjuvant potential 204–205 interfacial activity model 87–88 recent clinical advances 206–207 interleukin 8 (IL-8) 205–206 wound healing 205–206 invertebrates, marine see also cathelidicins; cationic peptides; classifi cation of AMPs 7 defensins see also cyclotides 226 Index

iron-binding peptides 200 lipid-transfer proteins (LTPs) 41–43 isothermal titration calorimetry (ITC) 107 lipids isotope labelling 145–146 anionic lipid clustering 120–121 basis for bacterial species specifi city of japonicin 7 toxicity 121–124 properties favouring clustering 125 kalata B1 5, 49 putative mechanism of action 124–125 antimicrobial activity 61 sources of energy 125 batch cultivation process 64 and antimicrobial sensitivity 118–119 biosynthesis 55–56 lipopolysaccharide (LPS) 156, 172 insecticidal activity 56 anti-endotoxic activity of LL-37 198 structure 51 chain length 119 kalata B2: precursor 55 inhibition of LPS transport 102 keratinocytes 206 interaction with LL-37 by NMR 156 knott ins 50 LL-37 4 KR-12 77, 143, 157 activities and functions 142–143, 186, 198 aggregation, pH-dependent 156–157 lactic acid bacteria (LAB) 22 bacterial expression systems 144 lacticins 26 electrostatic properties 169–170 engineering 30–31 engineering selective peptides 159 lacticin-like lantibiotics 27–28 expression of LL-37 187–188 use in dairy industry 29 implication of regulation by vitamin lactocin 6, 28–29 D 188–190 mechanism of action 15 inhibition 171 lactoferrin 154, 155, 208 interactions of polyvalent cations with linear lantibiotics polyelectrolytes 170–171 applications interactions with bacterial membranes 155–158 foods 29 NMR spectra 157 medical 30 recombinant 144 biosynthesis 23 release from polyelectrolyte bundles 172–173 classifi cation 24–25 sequestration and inactivation 172 defi nition 23 structure 151 engineering 30–31 therapeutic potential 175–176, 189, 205–206, post-translational modifi cations 23–24 207, 208 regulators 24 and tumorigenesis 143, 176, 211 screening for new peptides 31–32 LPS see lipopolysaccharide (LPS) sequence analysis 17 LTPs see lipid-transfer proteins (LTPs) structures 26 lupus vulgaris (cutaneous TB) 182 subgroups lysines, acylated: oligomers see acyl-lysyl epidermin-like 26–27 oligomers lacticin-like 27–28 mersacidin-like 28 machine-learning methods 74 nisin-like 25–26 magainins xvi, xvii, 1, 9, 14, 109, 130, 150 other types 28–29 magic angle spinning NMR Pep5 27 (MAS/NMR) 120–121 planosporicin- and streptin-like 27 maltose-binding protein (MBP) 144 lantipeptide, defi nition 23 marine AMPs 4, 7, 13 latarcin 2a 150 MAS/NMR see magic angle spinning NMR lebocin 7 (MAS/NMR) leucocin A 6 maximum tolerated dose (MTD) 109 libraries, combinatorial see combinatorial mechanism of action 14–15 chemistry medicine lichenicidin 32, 76 advantages of cyclotides in drug design 62 light therapy 182 applications of lantibiotics 30 linguistic model 78 immunomodulators see immunomodulators lipid bilayers 94, 118–121, 147 regulatory role of vitamin D see vitamin D lipid II, cell wall precursor 158 selectivity for cancer cells 131 Index 227

therapeutic potential of cationic antibacterial mutacins 30 peptides 175–176 MX-226 208 therapeutic potential of PR-39 132 see also human immunodefi ciency virus (HIV) natriuretic peptides 201–202 melanocortins 200 nematodes: pests 56 membrane curvature 151 nigrocin 7 membrane perturbation potential, protein surface nisin 158 properties 155 engineering 31 membranes, cancer cell 131 medical applications of nisin F 30 membranes, microbial use of nisin A as food preservative 29 AMP action nisin-like lantibiotics 25–26 anionic lipid clustering see under lipids nitric oxide 187, 200 basis for AMP classifi cation 14, 24 NMR see nuclear magnetic resonance (NMR) importance of amphipathic structure 61 NOESY see nuclear Overhauser eff ect spectroscopy inhibition of LPS transport 102 (NOESY) interaction with specifi c components nomenclature 158–159 peptide-based 4 interfacial activity model see interfacial and searching 3 activity model source-based 4 lipid transfer 42–43 source + peptide-based 4–5 LL-37 155–158 non-αβ structures 154–155 outer membrane of Gram-negative non-classical amphipathic structure 159 bacteria 119–120 nuclear magnetic resonance (NMR) 120–121, 124 transmembrane ionic pores 10, 43 of buforin II 130 usually non-specifi c interaction 129 of lipid-transfer proteins 42–43 voltage-dependent pores 27 methods depolarization 129 heteronuclear 3D studies 148–149 diff erences between Gram-positive and Gram- natural abundance NMR spectroscopy 148 negative bacteria 116, 117, 118 NMR structure determination, validation lipid composition and antimicrobial and deposition 149 sensitivity 118–119 two-dimensional (2D) 147 membrane-mimetic models 146–147 in structure determination target of most plant AMPs 40–41 fl ow chart 144 mersacidin-like lantibiotics 15, 26, 28 major technique 143 MiAMP1 49, 50 studies with LL-37 MIC see minimal inhibitory concentration (MIC) interactions with membranes 155–158 micelles 147 of thionins 43 microbeads: for combinatorial peptide use of membrane-mimetic models 146–147 libraries 89–90 nuclear Overhauser eff ect spectroscopy microbisporicin 32 (NOESY) 147, 148, 157–158 microcins (e.g. MccV, MccC7, MccE492) 5, 12 nukacin 31 classifi cation 7 microplusin 150 OAKs (oligomers of acylated lysines) see acyl-lysyl mimics: of host defence peptides see under host oligomers (OAKs) defence peptides (HDPs) oligomerization of AMPs 15, 152, 153–156 minidefensins (θ-defensins) 141, 153 oligomers: of acylated lysines see acyl-lysyl minimal inhibitory concentration (MIC) 122–123 oligomers (OAKs) models, membrane-mimetic 146–147 OP-215 208 modifi cation enzymes (Lan B, LanC, LanL, open reading frames (ORFs) 76 LanM) 23 oxidation and lantibiotics inactivation 12 modifi cations, chemical 10, 12–13 molecular simulation 103, 154 1-palmitoyl-2-oleoyl-PE (POPE) 120 mollusc AMPs 6 paenibacillin 26 MRSA 30 pallicourein 59 MSI peptides 123 palustrin 7 MTD see maximum tolerated dose (MTD) pathogens see, e.g. HIV; MRSA; Pseudomonas mucins 172 aeruginosa 228 Index

pardaxin 151 POPE see 1-palmitoyl-2-oleoyl-PE (POPE) patents porins 116 CAMP database 2–3 PR-39 4, 131–133 Defensin Knowledgebase 2–3 preservatives, food 29 PE see phosphatidylethanolamine (PE) PROCHECK 149 Penbase shrimp AMP database 3 production technology 62–63, 64 nomenclature 4 prokaryotes 6 Pep5 27 proline 129 medical applications 30 key feature for unique properties of buforin Peptaibol database 2 II 130 peptide defi nition 2, 40 proline-rich peptides 101, 129, 133–135 peptidoglycans 118 proteases: improvement of AMP stability 82 peptidomimetics see under host defence peptides protegrin-1 15, 102, 154, 203 (HDPs) protein-binding potential 142 peptoids 102, 103 Protein Data Bank (PDB) 149–150 pests: cyclotide pesticide activity 56 protozoan AMPs 6 PG see phosphatidylglycerol (PG) Psd1 45, 159 PG-1 see protegrin-1 Pseudomonas aeruginosa 78, 109, 170–171, 198, 200 phenylene ethynylene 102 psoriasis xvii, 189, 197 pheromones 24 purifi cation tags 144–145 phosphatidylethanolamine (PE) 103, 118–119, 120 PW2 154, 155 phosphatidylglycerol (PG) 122, 146 pyrrhocoricin 133–135 PhytAMPdatabase 3, 41 planosporicin (lantibiotic 97518) 26, 27, 32 Q see charge (Q) plant AMPs quorum sensing system 24 charge, net 10 chitin-binding peptides 45–48 ranacyclin 7 classifi cation 7, 41 Ramachandran plot 149 cyclopeptide alkaloids 49, 51 RAPD database 3 databases 3, 41 Raphanus sativus antifungal proteins 45, 46 defi nition 40 RBCs see red blood cells (RBCs) hydrophobic residue content 11 RDP58 207, 208–209 Ib-AMPs 47 reactive oxygen species (ROS) 159 length 11 recombinant AMPs lipid-transfer proteins 41–43 expression and purifi cation 143–146 mechanism of action 40–41 LL-37 144 MiAMP1 49, 50 RAPD database 3 PhytaAMP database 3 red blood cells (RBCs) 103, 106 structure 41 regulators 24 thionins 43, 44 reptile AMPs 6 see also cyclotides resistance, microbial 101, 210–211 plantaricin 6, 28 impetus for new antibiotic classes 128 plectasin xvii, 136 retinoid X receptor (RXR) 183–184 pleurocidin 150 reutericin 6 13 polyaspartate 174 rickets 182 polyelectrolytes RMSD see root mean square deviation (RMSD) design of inactivation-resistant antimicrobial RNase 7 14 agents 174–175 root mean square deviation (RMSD) 149 eff ects of DNase, gelsolin, alginase 173–174 royalisin 7 eff ects of small multivalent anions 174 Rs-AFP2 45 interactions with polyvalent cations 170–171 RTD-1 153 and invasive bacteria 174 RXR see retinoid X receptor (RXR) and LL-37 172–173 production by host and microbial sources SAPD database for synthetic antibiotic peptides 2 171–172 sapecin 7 polymerase chain reaction (PCR) 144 saposin-like proteins 150 polyphemusin 153 SAR see structure–activity relationships Index 229

screening activity data quality 8 high-throughput see high-throughput screening of acyl-lysyl oligomers 104, 106–107 in silico 78 peptide fragments 143 for new lantibiotics 31–32 role of proline 130 see also Antimicrobial Peptide Database (APD) three-dimensional structure 151–155 SDS see sodium dodecylsulfate (SDS) structures selectivity 80–81, 121–124 amphipathic 79 d-amino acid incorporation 81 buforin II 130 peptoid incorporation 80 chitin-binding peptides 47 structural basis 159 classifi cation 13–14 tuning hydrophobicity 80 cyclotides 51–53 sepsis 202 defensins 46, 142, 153 sequence analysis 15–16, 17 deposition of coordinates 149 cyclotides 54 determined by NMR see under nuclear of non-membrane-active peptides 129 magnetic resonance (NMR) in prediction of novel AMPs 73–76 indolocidin 14, 132, 155, 159 sequence shuffl ing in peptide design 77–78 lipid-transfer proteins 41–43 sesquin 4 non-membrane-active peptides 129 shark 175 obtained from NMR measurements 149 sheep myeloid AMP-29 (SMAP-29) 4, 15 peptides 41 shrimp AMPs structural basis of selectivity 159 nomenclature 4 structure-based engineering 159–160 PenBase 2 thionins 43, 44 SMAP-29 see sheep myeloid AMP-29 (SMAP-29) three-dimensional sodium 2,2-dimethylsilapentane-5-sulfonate alpha (α)-helical 149–152 (DSS) 147 beta (β)-sheet 152–154 sodium dodecylsulfate (SDS) 146 classifi cation 149 spectra, heteronuclear single-quantum coherence non-αβ structures 154–155 (HSQC) 148, 157 styelin D 13 spectroscopy, double-quantum-fi ltered correlation sublancin 28 see double-quantum-fi ltered correlation SUMO protease 1 145 spectroscopy (DQF-COSY) sunlight and vitamin D synthesis 182 spectroscopy, NMR see nuclear magnetic support vector machine (SVM) 74 resonance (NMR) surface plasma resonance (SPR) 107 spectroscopy, nuclear Overhauser eff ect see Swiss-Prot database 2, 75 nuclear Overhauser eff ect spectroscopy synergy 8–9 (NOESY) synthesis: advantages of bacterial expression 143– spectroscopy, total correlation see total correlation 144 spectroscopy (TOCSY) synthetic AMPs 31, 88, 154 sphingolipids, fungal 159 SAPD database 2 spider AMPs 6 see also design; selectivity; stability to protease spinigerin 150 SPOT synthesis 90 Tachyplesin 153 stability to protease 82, 210 TALOS program 148 acetylation, N-terminal 82 TB infection 187 amidation, C-terminal 82, 158 temporins 7, 9, 15, 152 branched peptides (dendrimers) 210 termicin 7 cyclization 82, 159 tetraoleoyl-cardiolipin (TOCL) 120 d-amino acids 82, 210 TEV protease 145 fl uorinated amino acids 82 thanatin 153 non-standard amino acids 82 therapeutic index (TI) xvii, 57, 76 peptide mimicries 102, 160 thionins 43, 44 staphylococcin C55 28 thioredoxin 144 Staphylococcus aureus: methicillin-resistant see thrombin 145 MRSA TLRs see Toll-like receptors (TLRs) streptin-like lantibiotics 27 TOCL see tetraoleoyl-cardiolipin (TOCL) structure–activity relationships TOCSY see total correlation spectroscopy (TOCSY) 230 Index

Toll-like receptors (TLRs) 186, 187–188, 196 receptor (VDR) 183–184, 185 toroidal pore model 88, 150–151 regulation of gene expression 183–184, 185 total correlation spectroscopy (TOCSY) 147 DEFB2/hBD-2 186–187 toxicity 8, 95, 151, 210 LL-37 187–188 transporter 24 regulation of immune system 184–185 tricyclon A 59 regulation of LL-37 expression: trifl uoroethanol (TFE), membrane-mimetic 134 implications 188–190 tritrpticin 154, 155 sources 182 ‘Trojan horse’ trick 12 structure 183 tryptophan 13, 79, 132, 154–155 therapeutic role of analogues 190 tumorigenesis 143, 176, 211 vitamin D response elements (VDREs) 183–184 two-component signal transduction system 24 in LL-37 187–188 winter 182 ulcerative colitis 207 VRE (vancomycin-resistant E. faecalis) 30 UniProtKB database 41 WLBU2 176 vaccines 204–205 worm AMPs 6 VDR (vitamin D receptor) see under vitamin D wounds: healing 205–206 VDREs (vitamin D response elements) see under vitamin D X-ray crystallography 143, 152–153 vesicle-based screening 94–96 viruses see human immunodefi ciency virus (HIV) yeast 103 vitamin D yield, recombinant LL-37 144 defi ciency xvii, 182 history 181–182 zinc-binding peptides 15 hydroxylation 182–183