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Cationic Antimicrobial Peptides: Thermodynamic Characterization of Peptide-Lipid Interactions and Biological Efficacy of Surface-Tethered Peptides

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Cationic Antimicrobial Peptides: Thermodynamic Characterization of Peptide-Lipid Interactions and Biological Efficacy of Surface-Tethered Peptides Cationic Antimicrobial Peptides: Thermodynamic Characterization of Peptide-Lipid Interactions and Biological Efficacy of Surface-Tethered Peptides Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Mojtaba Bagheri aus Marvdasht (Iran) Juni 2010 Hereby, I declare that I have prepared this work independently under supervision of Dr. Margitta Dathe in the time period from October 2006 until April 2010 at Leibniz-Institut für Molekulare Pharmakologie (FMP) in Berlin. To best of my knowledge, this thesis contains no previously published materials by another person. 1. Gutachterin: Prof. Beate Koksch, Freie Universität Berlin 2. Gutachter: Prof. Michael Bienert, Leibniz-Institut für Molekulare Pharmakologie Disputation am 09 September 2010 To my parents Acknowledgments I would like to show my appreciation to Prof. Dr. Michael Bienert (FMP), the head of Department of Chemical Biology of FMP, and my PhD supervisors Dr. Margitta Dathe (FMP) and Prof. Dr. Beate Koksch (Freie Universität Berlin) for giving me the opportunity to work in the interesting field of antimicrobial peptides and their support during my doctoral thesis research. I would especially like to thank Dr. Michael Beyermann (FMP), and Prof. Dr. Sandro Keller (University of Kaiserslautern) for their informative discussions and suggestions. Furthermore, Mrs. Annerose Klose, Mrs. Angelika Ehrlich, Mrs. Dagmar Krause, and Mr. Bernhard Schmikale are thanked for their assistance in the field of peptide synthesis, HPLC and mass measurements. I would also like to acknowledge Mr. Rudolf Dölling (Biosyntan GmbH) for the synthesis of c-(1MeW)F(1MeW), c-(5MeoW)F(5MeoW), and c- (5fW)F(5fW). I would also like to thank Mrs. Heike Nikolenko and Christof Junkes for their support in maintaining cell cultures. I am very grateful for the generous technical support of Nadin Jahnke, Gerdi Kemmer, and Katharina Grimm (all FMP) with ITC and CD expriments. Special thanks to Ewan St. John Smith (Max Delbrück Center for Molecular Medicine) and Gesa Schäfer (FMP) for the time they spent on correcting my thesis as well as for their fruitful suggestions. Finally, I would like to thank my parents for their understanding and patience and their spiritual support in my life. Content Content ABBREVIATIONS AND SYMBOLS .....................................................................................I Abbreviations .........................................................................................................................................................I Symbols ..................................................................................................................................................................V 1 INTRODUCTION................................................................................................................. 1 1.1 CAPs................................................................................................................................................................. 1 1.1.1 Structure diversity and basis of activity..................................................................................................... 1 1.2 Cellular basis of activity and bacterial selectivity ........................................................................................ 4 1.2.1 Membrane composition ............................................................................................................................. 4 1.2.2 Bacterial LPS............................................................................................................................................. 6 1.3 Mechanims of action ....................................................................................................................................... 9 1.3.1 Membrane permeabilization ...................................................................................................................... 9 1.3.2 Alternative mechanisms of action............................................................................................................ 11 1.4 Small CAPs .................................................................................................................................................... 11 1.4.1 Significance of short CAPs...................................................................................................................... 11 1.4.2 Particular properties of RW-rich hexapeptides........................................................................................ 13 1.5 Surface-tethered peptides............................................................................................................................. 14 1.5.1 Inhibition of biofilm formation................................................................................................................ 14 1.5.2 Peptide-based biofilm .............................................................................................................................. 18 2 AIMS OF THE STUDY...................................................................................................... 20 2.1 Structural basis of anti-E. coli activity of cyclic RW-rich hexapeptides .................................................. 20 2.2 Preparation and perspectives of surface-tethered peptides....................................................................... 21 3 RESULTS AND DISCUSSION.......................................................................................... 23 3.1 W-substituted c-WFW analogs .................................................................................................................... 23 3.1.1 Description and physicochemical properties of W-analogs..................................................................... 23 3.1.2 Cyclic peptide synthesis and their HPLC characterization ...................................................................... 25 3.1.3 Characterization of cyclic peptides by CD .............................................................................................. 26 3.1.4 Antibacterial and hemolytic activities ..................................................................................................... 29 3.1.5 Cyclic peptide binding to lipid bilayers determined by ITC.................................................................... 31 3.1.5.1 Lipid bilayers as model of biological membranes............................................................................ 31 3.1.5.2 Peptide accumulation as reflected by apparent binding.................................................................... 32 Influence of lipid composition upon binding ........................................................................................... 32 Role of sequence composition upon binding ........................................................................................... 34 3.1.5.3 Peptide partitioning in lipid bilayers ................................................................................................ 37 Influence of lipid composition upon binding ........................................................................................... 37 Role of sequence composition upon binding ........................................................................................... 38 3.1.5.4 Effect of ionic strength upon c-WFW binding to r-LPS and s-LPS lipid systems ........................... 42 Content 3.1.5.5 Heat capacity change on membrane partitioning of c-WFW............................................................ 44 3.1.6 Summary.................................................................................................................................................. 46 3.2 Site-specific immobilization of CAPs........................................................................................................... 49 3.2.1 Physical and chemical properties of PEGylated resins as model solid surfaces ...................................... 50 3.2.2. Activity of surface tethered membrane-active CAPs - role of tethered peptide site ............................... 51 3.2.2.1 Characterization of KLAL and MK5E peptides by HPLC and CD.................................................. 51 3.2.2.2 Preparation and characterization of tethered KLAL and MK5E peptides........................................ 53 3.2.2.3 Biological activities of free and tethered KLAL and MK5E peptides.............................................. 55 Antimicrobial activity of the free peptides............................................................................................... 55 Antimicrobial activity of the tethered peptides........................................................................................ 57 Hemolytic activity of the free and tethered peptides................................................................................ 59 3.2.2.4 Bilayer permeabilizing activities of free and tethered KLAL and MK5E peptides.......................... 59 The free peptides...................................................................................................................................... 60 The tethered peptides ............................................................................................................................... 61 3.2.3 Influence of physical characteristics of solid surfaces upon biocidal activity
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