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Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications Yazan H

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Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications Yazan H University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2013 Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications Yazan H. Akkam University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Biochemistry Commons, Medicinal and Pharmaceutical Chemistry Commons, and the Molecular Biology Commons Recommended Citation Akkam, Yazan H., "Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications" (2013). Theses and Dissertations. 908. http://scholarworks.uark.edu/etd/908 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Biology by Yazan H. Akkam Jordan University of Science and Technology Bachelor of Science in Pharmacy, 2001 Al-Balqa Applied University Master of Science in Biochemistry and Chemistry of Pharmaceuticals, 2005 August 2013 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. Dr. David S. McNabb Dissertation Director Professor Roger E. Koeppe II Professor Gisela F. Erf Committee Member Committee Member Professor Ralph L. Henry Dr. Suresh K. Thallapuranam Committee Member Committee Member ABSTRACT Candida species are the fourth leading cause of nosocomial infection. The increased incidence of drug-resistant Candida species has emphasized the need for new antifungal drugs. Histatin 5 is a naturally occurring human salivary antifungal peptide and the first line of defense against infections of the oral cavity. This research has focused on understanding the activity of histatin 5, and subsequently designing novel peptides that may serve as models for the further development of therapeutics to treat fungal infection. This objective has been achieved in three steps: studying the structural requirement of histatin 5 involved in antifungal activity, the identification of a short peptide sequence, referred to as KM motif, important for fungicidal activity, and finally, the development of a novel antifungal peptide with potent activity. In the initial phase of this work it was demonstrated that reversing the sequence of histatin 5 C-16 peptide to create a retro peptide did not interfere with the fungicidal activity or secondary structure of the peptide. This suggested that the spatial arrangement of amino acid residues was more relevant for fungicidal activity than the actual peptide sequence. In the second phase of the work, we identified and characterized a five amino acid sequence, termed the KM motif, within histatin 5 that maintained fungicidal properties. Although this short peptide was less active than histatin 5, the data suggested it was killing fungi via a mechanism similar to histatin 5. In the final phase, a novel antimicrobial peptide, termed KM-12, was generated containing two KM motifs dimerized via disulfide bonds. The activity of KM-12 on C. albicans was approximately fifteen times more potent than the monomeric peptide and ten times more active than the native histatin 5. KM-12 was shown to have antifungal activity with several Candida species, including fluconazole- resistant species. In conclusion, KM-12 is promising antifungal peptide that will serve as a lead candidate for the development of antifungals peptide for pharmaceutical applications. ACKNOWLEDGMENTS The achievement and final outcome of this project required a lot of guidance and assistance from many people and in so different ways, and I am extremely fortunate that to have got this all along the completion of my project work. Whatever I have done is only due to such guidance and assistance, and I would not forget to thank them. I owe a debt of gratitude to many people for the following pages. Above all I must thank my advisors, the inimitable Dr. David McNabb and Dr. Suresh Kumar. Without their invaluable guidance none of this tremendously hard work would be achieved. Also, my deep gratitude and extended thanks go to Dr. Douglas Rhoads, who is the reason of why I am here. I am profoundly grateful to him for his unfailing encouragement and many hours of help. Also at the University of Arkansas, my sincerest thanks go to Dr. Ralph Henry, Dr. Gisela Erf, and Dr. Roger Koeppe who serve as my committee members and who have made my time here such an intellectual and personal delight. My thanks go also to Dr. Ines Pinto who kept both me and my work throughout my years here interesting, thoughtful, and productive. In addition, I would like to send my true thanks to the members of Dr.Koeppe lab, especially Dr. Denise Greathouse for their time and guidance in peptide synthesis. For their support and friendship, my unending thanks go to all members of Dr.McNabb and Dr.Pinto Labs, especially Marsha Rhoads. DEDICATION I dedicate this dissertation to my family, especially my wife who has been fathering my three riotous kids along five years. Their constant love, support, and encouragement have sustained me throughout my career and life. TABLE OF CONTENTS ABSTRACT.......................................................................................................................ii ACKNOWLEDGMENTS.................................................................................................iii DEDICATION..................................................................................................................vi TABLE OF CONTENTS ..................................................................................................v CHAPTER 1. Introduction and literature review. ............................................................ 1 1.1 Candida Species ................................................................................................... 2 1.1.1 Candida albicans ................................................................................................. 2 1.1.2 Epidemiology and risk factors .............................................................................. 4 1.1.3 Candida and Human immunity ............................................................................. 5 1.1.4 Treatment of fungal infections .............................................................................. 6 1.1.4.1 Azoles ............................................................................................. 6 1.1.4.2 Polyenes ........................................................................................ 9 1.1.4.3 Echinocandins ............................................................................... 10 1.1.4.4 Antimetabolites ............................................................................. 11 1.2 Antifungal peptides (AFPs) ................................................................................ 12 1.2.1 Classification........................................................................................................ 13 1.2.1.1 Cationic ........................................................................................ 13 1.2.1.2 Anionic ......................................................................................... 20 1.2.1.3 Neutral ......................................................................................... 22 1.2.2 Post-translation modification in natural AFPs ..................................................... 23 1.2.3 Disadvantages of AFPs and proposed solutions ................................................. 26 1.3 Histatins .............................................................................................................. 29 1.3.1 Histatin 5 .............................................................................................................. 30 1.3.1.1 Unique features ............................................................................ 31 1.3.1.2 Antimicrobial activities ................................................................ 31 1.3.1.3 Secondary structure ...................................................................... 32 1.3.1.4 Amphipathicity and hydrophobicity ............................................ 32 1.3.1.5 Charge .......................................................................................... 33 1.3.1.6 Amino acids composition ............................................................. 33 1.3.1.7 Structure function relationship ..................................................... 34 1.3.1.8 Structural modifications ............................................................... 35 1.3.1.9 Mechanism of action .................................................................... 37 Figures .......................................................................................................................... 39 Tables ............................................................................................................................ 43 References ..................................................................................................................... 49 CHAPTER 2. Analysis of Wild-type ,enantio,
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