Discovery and Development of Peptide Antibiotics
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Resistance is Not Futile: Discovery and Development of Peptide Antibiotics Composed of D Amino Acids A dissertation submitted by Emel Adaligil In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry May 2016 Advisor: Prof. Krishna Kumar ABSTRACT Since the discovery of penicillin in 1928, many antibiotics targeting different kinds of bacterial species have been developed. Extensive and improper use of antibiotics has triggered a “bacterial resistance” problem. The biggest challenge in developing new antibiotics is that the bacterial strains may become resistant to newly developed drugs even during laboratory development before clinical trials in humans. Vancomycin is one of the “last resort” antibiotics used in the treatment of life threatening hospital infections. Since the isolation and identification of a vancomycin-resistant strain in 1988, it is of the highest and gravest of concern that vancomycin-resistance can be spread to multi-drug resistant pathogenic bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA) that is responsible for lethal infections in immune-compromised subjects, such as those afflicted with AIDS, and organ transplant patients. In such a scenario, there will be no more available drugs for the treatment of such patients and the biggest advantage the modern medicine has thrived upon would be taken away. The aim of this dissertation is to develop stable, protease resistant peptides composed of D-amino acids as antibiotics that bind D-Alanyl-D-Alanine termini of vancomycin- sensitive bacteria and D-Alanyl-D-Lactate termini of vancomycin-resistant bacteria with a mechanism of action similar to that of vancomycin based on mirror-image phage display concept. We have identified several L-peptides that bind the enantiomer of vancomycin-sensitive and resistance bacterial cell wall termini by screening phage display libraries displaying 7-12 residue linear, cyclic and bicyclic random peptides against enantiomers of derivatives of the β-lactam antibiotic cephalosporin, that mimic the D-Ala-D-Ala termini of peptidoglycan structures in bacterial cell walls, crucial for ii crosslinking and providing structural integrity to the cell. In addition, enantiomers of the cell wall crosslinking pentapeptide precursors, including those that contain D-Ala-D-Lac were also used. After evaluating the binding specificity of each identified peptide to its target molecule via ELISA, a total of 94 peptides (linear, cyclic, and bicyclic) identified from such screen were synthesized as their mirror image versions (D-peptides). Antibacterial activity assays established that some of these D-peptides (linear, cyclic and bicyclic) have antibacterial activity against vancomycin-sensitive and vancomycin- resistant bacteria with a MIC values ranging from 8 µg/ml to 64 µg/ml. Taken together, we have a used a hitherto unused unified chemical-biological approach that can be used to generate a wide range of D-peptides that can act as antibiotics and will pave the way for a new family of antimicrobial compounds. iii Dedicated to my parents, my brother and my sister (Adaligils) iv ACKNOWLEDGMENTS To begin with, everyone has involved in my journey here has a different place in my heart. Getting a Ph.D. degree is very long and time-to-time tiring journey with ups and downs, and I realized at the end that how important is to have people who support you unconditionally during that time. I would like to express my deepest and sincere appreciation and gratitude to my advisor Prof. Krishna Kumar for his supports and his great mind. His intelligence is enough to inspire someone for working hard and reaching the goal. He is a great mentor who thinks always “out-of-box”, a good friend who cares and supports all the time, and now he is indispensible member of my extended family. I am also grateful that he treats Kumar group as a family not only a research group. Thanks to all former and current members of Kumar family who I met during my journey at Tufts for always being good friends and supportive colleagues and making our group distinguishable than other groups with our such a great working environment addition to our great science. Special thanks to Dr. Vittorio Montanari and Dr. Venkat Raman for their scientific discussions and helps, friendships and of course for sharing dark chocolates the last three years, and also thank to Marissa Rodenstein for her help during the last intense peptide synthesis of thesis work. I should also thank to other current members of Kumar group, John Paul Issa, Kathleen Sicinski, and Jasper Du, for making everyday full of science and full of fun at the same time, and to former members of our family for their help and putting one brick to make my wall, Gizem, Diren, Deniz, Kalyani, Subbu, Zho, Tao, Sofia. I have to thank to my two other committee members, Prof. Samuel Thomas and Prof. Joshua Kritzer, for being in my thesis committee for more than five years and for their v helpful advises and feedbacks during that time. And my special thanks to Dr. Tomi Sawyer, not only being in my thesis committee as outside member, also for his great support all the time since we met at the American Peptide Society Conference. I feel that it is privilege to meet such an innovative scientist in the area I am working. Also I feel grateful to meet and get know Prof. Christian Heinis. His humble nature as a person, his intelligence as a scientist, and his work inspired me to finish the last part of thesis work. “If one wants an accounting of one’s worth, count his/her friends.” I am indeed fortunate to meet primarily Dr. Eugenia Marin, Dr. Alberto Lopez, and Brad Nissenbaum here in Boston who have been there whenever I needed a friend/a family in difficult times, to care and to share joyful moments, to travel as great buddies everywhere. The meaning of their friendships is indescribable. Not only to my friends in Boston, also I thank my two other sisters lives in CA, Beliz Iristay and Senem Aktuccar, from the bottom of my heart for being second-sisters for me and their emotional supports for the last ten years. You have taken care of me and have considered me as a part of your own families. I love you both so much. Lastly, I should thank my all friends from the department, from Boston, from California, from Turkey for making my life better and better every day. In all reverence, I dedicate my work to my family, my parents Nejla and Acar Adaligil, my sister Nazmiye Adaligil and my brother Fatih Adaligil, for their endless love. Thank you for instilling in me a love of life, for always encouraging me to do my best in whatever I tried, and for making me feel that I am the luckiest person on the earth to have you guys every day. There is no strong word to express my love for you both. vi Table of Content Abstract……………………………………………………………………………………ii Acknowledgements………………………………………………………………………..v Table of content….………………………………………………………………………vii List of Figures…………………………………………………………………………….xi List of Tables……………………………………………………………………………xiii List of Schemes………………………………………………………………………….xvi CHAPTER 1 INTRODUCTION……...…………………………………………………..1 1.1. Brief History of Antibiotics………………………………………………………2 1.2. How Bacteria Develop Resistance to Antibiotics………………………………...4 1.3. Strategies to Overcome Antibiotic Resistance……………………………………9 1.4. Peptide Therapeutics…………………………………………………………….10 1.5. Antimicrobial Peptides as Alternatives to Conventional Antibiotics………...…12 1.6. Phage Display Selection of Peptide Ligands as Novel Therapeutics…………...14 1.6.1. Phage Display: Overview………………………………………………….14 1.6.2. Linear and Cyclic Phage Display Peptide Libraries……………………….19 1.6.3. Phage-coded Bicyclic Peptide Libraries…………………………………...20 1.7. Application of Phage Display in Infectious Disease…………………………….25 1.8. The Scope of Thesis……………………………………………………………..30 CHAPTER 2 DISCOVERY AND DEVELOPMENT OF D-PEPTIDE ANTIBIOTICS TARGETING METHICILLIN-RESISTANT S. AUREUS……………………………..31 2.1. Emergence of Staphylococcus aureus as a Major Infectious Agent…………… 32 2.2. Phage Display in Staphylococcus aureus Infectious………………………….....34 2.3. Peptide Therapeutics Containing D-amino Acids……………………………….36 2.4. Elegant Approach to Design D-peptide Ligands: Mirror Image Phage Display…………………………………………………………………………..38 2.5. Designing The Target Molecules for Mirror Image Phage Display…………….43 2.6. Results and Discussions…………………………………………………….…..50 vii 2.6.1. Control Phage Display Experiments with Streptavidin as Model Target Molecule…………………………………………………………………...50 2.6.2. Linear and Cyclic Peptide Ligands Binding Bacterial Cell Wall of S. aureus……………………………………………………………………54 2.6.3. Binding Confirmation of Selected Peptides via Phage-ELISA...………….93 2.6.4. Bicyclic Peptide Ligands of Bacterial Cell Wall Precursor of S. aureus through Phage-Coded Peptide Libraries………………………………….101 2.6.5. Antibacterial Activity of Selected D-peptides as Antibiotics…………….113 CHAPTER 3 OVERCOMING VANCOMYCIN RESISTANCE BY PHAGE-DERIVED D-PEPTIDE ANTIBIOTICS…………………………………………………………...124 3.1. “The Antibiotic of Last Resort”, Vancomycin…………………………….....125 3.2. Bacterial Resistance to Vancomycin…………………………………………128 3.3. Resistance Mechanism of Vancomycin-resistant Bacteria…………………...128 3.4. What Has Been Done to Overcome Vancomycin Resistance?........................ 131 3.5. Results and Discussions………………………………………………………136 3.5.1. Peptide Ligands of Vancomycin-resistant Strains by Screening Linear and Cyclic Phage Display Peptide Libraries……………….…………………136 3.5.2. Bicyclic Peptide Ligands