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 for Bacterial Cell Wall Precursor of Vancomycin resistant Enterococci…………………………………………..………….147 3.5.3. Antibacterial Activity Assays of D-Peptides against Vancomycin-resistant Strains…………………………………………………………………….152

CHAPTER 4 EXPERIMENTAL PROCEDURES………………………………..……156 4.1. Synthesis of Target Molecules……………………………………………….157 4.1.1. Synthesis of Enantiomer of Cephalosporin……………………………...157 4.1.2. Synthesis of Enantiomer of Pentapeptide Cell Wall Precursor of S. aureus…………………………………………………………………..167 4.1.3. Synthesis of Enantiomer of Cell Wall Precursor of Vancomycin-resistant

viii Enterococci…………………………………….…………………………169 4.2. Phage Display Screening with Linear and Cyclic Phage Display Peptide Libraries………………………………………………………………………...171 4.2.1. Materials and Buffers…………………………………………………...171 4.2.2. Maintenance of E. coli ER2738 Host Cells…………………………….173 4.2.3. Checking Phage Titers of Linear and Cyclic Phage Display Libraries...173 4.2.4. Control Biopanning Experiment with Streptavidin as Model Target…..174 4.2.4.1.Solid-phase Biopanning in 96-well plate…………………………...174 4.2.4.2.Solid-phase Biopanning in 60 x 100 mm petri-dish………………..176 4.2.4.3.Solution-phase Biopanning with Streptavidin-coated Magnetic Beads…………………………………………………………...…..178 4.2.5. DNA sequencing and Analysis…………………………………………179 4.2.6. Biopanning Experiments with Target Molecules using Linear and Cyclic Phage Display Peptide Libraries………………………………………..180 4.2.6.1.The More Stringent Biopanning Selection………………..……..…180 4.2.6.2.Phage Amplification and Purification………………………………181 4.2.6.3.Additional Rounds………………………………………………….182 4.2.6.4.The High Yield Biopanning Selection……………………………...183 4.2.6.5.Phage Amplification and Purification………………………………184 4.2.6.6.Additional Rounds………………………………………………….185 4.2.6.7.Negative Selections for both Biopanning…………………………..185 4.2.6.8.Blue/White Plaque Assays for Phage Titering……………………...186 4.2.6.9.Plaque Purification for DNA sequencing and Phage ELISA……….187 4.2.6.10. DNA Sequencing and Analysis………………………………...... 187 4.3.Phage ELISA for Binding Confirmations to Target Molecules………………..188 4.3.1. Buffers………………………………………………………………….188 4.3.2. Phage ELISA via Sandwich ELISA Protocol…………………………..189 4.4.Biopanning Experiments with Bicyclic Phage Display Peptide Libraries……..190 4.4.1. Buffers and Materials……………………………………………………190 4.4.2. Maintenance of Host E. coli TG1 cells………………………………….192 4.4.3. Bicyclic Phage Library Production and Amplification………………….192

ix 4.4.4. PEG purification of Amplified Phage Library…………………………..193 4.4.5. Reduction of Cysteine Residues in Phage Library………………………193 4.4.6. Bicyclization of Phage Library…………………………………………..194 4.4.7. Phage Titering…………………………………………………………...195 4.4.8. Biopanning………………………………………………………………195 4.4.9. Additional Rounds……………………………………………………….197 4.4.10. Negative Selections as Control Biopanning……………………………..197 4.4.11. DNA Sequencing and Analysis………………………………………….198 4.5.Synthesis of Enantiomer of Identified L-peptides: D-peptides…………………198 4.5.1. Synthesis of Linear D-peptides………………………………………….198 4.5.2. Synthesis of Cyclic D-peptides………………………………………….199 4.5.3. Synthesis of Bicyclic D-peptides………………………………………..201 4.6.Antibacterial Activity Assays of Synthesized D-Peptides……………………...202 4.6.1. Bacterial Strains and Medium…………………………………………...202 4.6.2. Determination of Minimum Inhibitory Concentration (MIC) with Broth Micro-dilution Method……………………………………………..……203 4.6.3. Determination of Minimum Bactericidal Concentration (MBC)…..……204

CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS……………………....205

CHAPTER 6 REFERENCES…………………………………………………………..212

CHAPTER 7 APPENDIX……………………………………………………………...256

x List of Figures

Figure 1. Five major targets for common antibiotics……………………………………..5 Figure 2. Resistance mechanisms to antibiotics…...………………………………….…..7 Figure 3. Schematic representation of cyclization reaction of Bicyclic Phage Display Peptide Libraries…………………..……………………….…………...………………..22 Figure 4. The structure of three thiol-reactive reagents…….………………………...…24 Figure 5. Schematic representation of mirror image phage display concept……….…...40 Figure 6. The last step of bacterial cell wall biosynthesis………………………...…….45 Figure 7. Structural similarities between peptidoglycan cell wall termini D-Ala-D-Ala and penicillin……………………………………………………………………………..46 Figure 8. Structure of target molecules used in phage display experiments…………….49 Figure 9. Schematic representation of affinity selection in solution-phase biopanning………………………………………………………………………………..53 Figure 10. The structure of target molecules used in the biopanning experiments……..57 Figure 11. The schematic representation of sandwich phage-ELISA…………...………94 Figure 12. Phage-ELISA results of target EA-1-CEP after the more stringent selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer ………………..…95 Figure 13. Phage-ELISA results of target EA-2-ALA after the more stringent selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer…………………...96 Figure 14. Phage-ELISA results of target EA-3-ALA after the more stringent selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer…………………...97 Figure 15. Phage-ELISA results of target EA-1-CEP after the high yield selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer……………………...…99 Figure 16. Phage-ELISA results of target EA-2-ALA after the high yield selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer……………………….100 Figure 17. Schematic representation of biopanning experiments with bicyclic phage display peptide libraries………………………………………………………………...107 Figure 18. The structure of vancomycin…………………………………………...…..127 Figure 19. Vancomycin resistance……………………………………………………..129 Figure 20. The structure target molecule EA-4-LAC……………………………….....136

xi Figure 21. Phage-ELISA results of target EA-4-LAC after the high yield selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer……………………….146

xii List of Tables

Table 1. Selected phage display research in infectious disease…………………………27 Table 2. Association constants for the binding of vancomycin with various peptides….48 Table 3. Peptide sequence results of control biopanning with Streptavidin…………….52 Table 4. Initial input of each naïve phage display library…………………………...…..56 Table 5. Phage display peptide libraries differ in size with the same complexity…...….56 Table 6. Phage titers of target EA-1-CEP with linear 12-mer library…………...………63 Table 7. Phage titers of target EA-1-CEP with linear 7-mer library……...……………..63 Table 8. Phage titers of target EA-1-CEP with cyclic 7-mer library………...………….63 Table 9. Phage titers of target EA-2-ALA with linear 12-mer library…………………..64 Table 10. Phage titers of target EA-2-ALA with linear 7-mer library……………...…...64 Table 11. Phage titers of target EA-2-ALA with cyclic 7-mer library………...…...…...64 Table 12. Phage titers of target EA-3-ALA with linear 12-mer library………...…….…65 Table 13. Phage titers of target EA-3-ALA with linear 7-mer library……………...…...65 Table 14. Phage titers of target EA-3-ALA with cyclic 7-mer library……………...... 65 Table 15. Peptide sequences isolated from EA-1-CEP biopanning……………...……...66 Table 16. Peptide sequences isolated from EA-1-CEP biopanning……………...……...67 Table 17. Peptide sequences isolated from EA-1-CEP biopanning……...……………...68 Table 18. Peptide sequences isolated from EA-2-ALA biopanning………...…………..69 Table 19. Peptide sequences isolated from EA-2-ALA biopanning…………...………..70 Table 20. Peptide sequences isolated from EA-2-ALA biopanning………………….....71 Table 21. Peptide sequences isolated from EA-3-ALA biopanning……………...……..72 Table 22. Peptide sequences isolated from EA-3-ALA biopanning…………...………..73 Table 23. Peptide sequences isolated from EA-3-ALA biopanning………...…………..74 Table 24. Phage titers of target EA-1-CEP with linear 12-mer library…………...……..76 Table 25. Phage titers of target EA-1-CEP with linear 7-mer library……...…………....76 Table 26. Phage titers of target EA-1-CEP with cyclic 7-mer library…………………..76 Table 27. Phage titers of target EA-2-ALA with linear 12-mer library………………....77 Table 28. Phage titers of target EA-2-ALA with linear 7-mer library…………...……...77 Table 29. Phage titers of target EA-2-ALA with cyclic 7-mer library………………….77

xiii Table 30. Peptide sequences isolated from EA-1-CEP biopanning……...……………...78 Table 31. Peptide sequences isolated from EA-1-CEP biopanning……...……………...79 Table 32. Peptide sequences isolated from EA-1-CEP biopanning………...…………...80 Table 33. Peptide sequences isolated from EA-1-CEP biopanning………....…………..81 Table 34. Peptide sequences isolated from EA-1-CEP biopanning………....…………..82 Table 35. Peptide sequences isolated from EA-1-CEP biopanning………………...…...83 Table 36. Peptide sequences isolated from EA-2-ALA biopanning…...………………..84 Table 37. Peptide sequences isolated from EA-2-ALA biopanning…...………………..85 Table 38. Peptide sequences isolated from EA-2-ALA biopanning……...……………..86 Table 39. Peptide sequences isolated from EA-2-ALA biopanning………...…………..87 Table 40. Peptide sequences isolated from EA-2-ALA biopanning…………...………..88 Table 41. Peptide sequences isolated from EA-2-ALA biopanning…………...………..89 Table 42. Known consensus sequences of target-unrelated peptides……………………92 Table 43. Phage-coded bicyclic peptide libraries used in the biopanning……………..102 Table 44. Library A and Library B with different loop lengths………...……………...102 Table 45. Selected bicyclic peptides for EA-1-CEP and EA-2-ALA……………...…..105 Table 46. Phage titers of EA-1-CEP and negative controls after each round of biopanning with Library A and enrichment factors….……………………………………………...107 Table 47. Phage titers of EA-2-ALA and negative controls after each round of biopanning with Library A and enrichment factors.…………………………………....107 Table 48. Phage titers of EA-1-CEP and negative controls after each round of biopanning with Library B and enrichment factors …………………………………………….…..108 Table 49. Phage titers of EA-2-ALA and negative controls after each round of biopanning with Library B and enrichment factors…………………………………….108 Table 50. Peptide sequences from EA-2-ALA biopanning…………………………….109 Table 51. Peptide sequences from EA-2-ALA biopanning…………………………….110 Table 52. Peptide sequences from EA-1-CEP biopanning…………………………….111 Table 53. Peptide sequences from EA-1-CEP biopanning……………...……………..112 Table 54. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains…………………………………………………………………115 Table 55. Minimum bactericidal concentration (MBC) of potent D-peptides on

xiv S. aureus…………………………………………………………………………………………..123 Table 56. Phage titers of target EA-4-LAC with linear 12-mer library………………..139 Table 57. Phage titers of target EA-4-LAC with linear 7-mer library…...…………….139 Table 58. Phage titers of target EA-4-LAC with cyclic 7-mer library…...…………….139 Table 59. Peptide sequences isolated from EA-4-LAC biopanning……...……………140 Table 60. Peptide sequences isolated from EA-4-LAC biopanning….…………...…...141 Table 61. Peptide sequences isolated from EA-4-LAC biopanning…………...………142 Table 63. Peptide sequences isolated from EA-4-LAC biopanning…………...………143 Table 64. Peptide sequences isolated from EA-4-LAC biopanning…………...………144 Table 65. Peptide sequences isolated from EA-4-LAC biopanning…………...………145 Table 66. Phage titers of EA-4-LAC after each round of biopanning with Library A with negative controls and enrichment factor………………………………………………..148 Table 67. Phage titers of EA-4-LAC after each round of biopanning with Library B with negative controls and enrichment factor………………………………………………..148 Table 68. Peptide sequences isolated from EA-4-LAC biopanning………………...…149 Table 69. Peptide sequences isolated from EA-4-LAC biopanning…………...………150 Table 70. Bicyclic peptides synthesized for antimicrobial activity assays………….....151 Table 71. Minimum inhibitory concentrations (MIC) in mg/ml of D-peptide antibiotics on several bacterial strains…………...…………………………………………………153 Table 72. Common peptide sequences isolated from all the biopanning of three commercially available phage display peptide libraries against target molecules EA-1- CEP, EA-2-ALA, EA-3-ALA and EA-4-LAC…………………………………………208

xv List of Schemes

Scheme 1. Synthesis scheme of enantiomer of cephalosporin…………………………157 Scheme 2. Synthesis scheme of enantiomer of biotinylated pentapeptide cell wall precursor D-Ala-L-γ−Glu-D-Lys-L-Ala-L-Ala………...... ……………………………167 Scheme 3. Synthesis scheme of the enantiomer of bacterial cell wall analogue of vancomycin-resistant enterococci (VRE) starting………………………………...……169

xvi

Chapter 1

Introduction

1 1.1. Brief History of Antibiotic Discovery

The road from discovery of an antibiotic to its deployment in the clinic is a long and expensive journey.1 Since the 1980s, only two new novel antibiotics, linezolid and daptomycin, have been released to market. Society now faces not only the emergence of rapidly growing multidrug-resistant pathogens, but also significant decline in the development of new antimicrobials. Factors including low hit rates in high-throughput screens, nonspecific antibacterial modes of action even in the development stage, and failure to display activity in vivo have all contributed to the pharmaceutical industry to downsize or eliminate research programs for discovery of new antibacterial agents.2 The global marketplace for antibacterial drugs is estimated to be worth more than $25 billion per year, however the cost associated with bringing compounds to market and the high failure rate have reduced appetite for development.3 Therefore there is a clear and present need to develop new antibacterial agents and it has never been greater. It is only a matter of time that is when and not if, for the specter of widespread untreatable pathogenic infections rears its ugly head and threatens the basic tenet on which modern medicine is based.

Although penicillin was the first discovered antibiotic in 1928, when Alexander Fleming observed the antibacterial activity of a Penicillium mould against S. aureus, the first antibiotic released to market was a synthetic suphonamide named “Protonsil” used in the treatment of Streptococcal infections.4-7

In 1940s the search for new antibiotics relied mostly on the isolation and identification of natural antimicrobials from soil organisms. In 1943, streptomycin, an aminoglycoside,

2 was isolated from Streptomyces griseus by Waksman and Schatz for the treatment of tuberculosis.8 Another β-lactam, cephalosporin, was discovered by Florey and his co- workers during an era when wide spectrum compounds with a very low toxicity were viable.9

After isolation of 6-aminopenicillanic acid produced during the fermentation of P. chrysogenum, large-scale production of β-lactam antibiotics, ampicillin, methicillin, flucloxacillin, amoxycillin, ticarcillin and carbenicillin, followed later by mezlocillin, azlocillin, piperacillin and mecillinam, saw quick entry to the market as therapeutics during the 1960s.10

Chlorotetracycline, representative of the tetracycline class of antibiotics, was first prescribed in 1948 as the second most commonly used antibiotic after β-lactams due to its broad-spectrum action, and inexpensive production method.11

In 1962, Lesher and his co-workers isolated nalidixic acid as a by-product of chloroquine production, which became the first quinolone antibiotic to be developed. The first- generation quinolones were active against Gram-negative Bacillary infections, especially in the human urinary tract. In the 1980s the second-generation fluoroquinolones were introduced with 1000-fold increased potency and therefore a concomitant decrease in the dose.12

More than 20 novel classes of antibiotics were discovered between 1930 and 1962. Since then, only four new classes of antibiotics have seen clinical use. Interestingly, none of recently clinically used compounds is really “new”: daptomycin, approved in 2000, was discovered in the early 1980s; linezolid, approved in 2000, derives from a synthetic lead

3 discovered in the 1970s; pleuromutilins, approved in 2007, have been widely used for about 30 years in veterinary medicine; fidaxomicin, approved in 2011, was first reported in the 1970s.

1.2. How Bacteria Develop Resistance to Antibiotics

Despite the wide range of chemical diversity, antibiotics mainly work by targeting five major pathways (Figure 1): interfering with DNA and RNA synthesis, inhibiting protein synthesis by exploiting the differential specialization and structures of bacterial ribosomes, interfering with the bacterial cell wall biosynthesis, disrupting and compromising the integrity of cell membranes and arresting growth by depletion of folate by inhibition of biosynthetic pathways.13

Bacteria have been present on earth for more than 3 billion years, and therefore it is not surprising that they become resistant to antibiotics, as many of compounds are frequently products of their own secondary metabolism synthesized to gain advantage against other bacteria. Improper use of antibiotics triggers and exacerbates the “bacterial resistance” problem.14-18 Every antibiotic in principle is vulnerable to this problem and most have engendered resistance after being employed clinically. Penicillin-resistant Staphylococci was reported only three years after its introduction as a new antibiotic into clinical use, and more than 80% of Staphylococcal isolates are resistant to penicillin.19

Antibiotic resistance can be either intrinsic or acquired. Intrinsic resistance is due to

4

Figure 1. Five major targets for common antibiotics in inhibiting bacterial infections: the bacterial cell wall, the cell membrane, protein translation mediated by the ribosomes, DNA and RNA synthesis, and folic acid metabolism.13

5 genes that bacteria inherently have.20-21 Evolutionarily this is expected as many of the compounds are those that bacteria employ in inter-species warfare, and they must develop mechanisms to remain viable while eliminating competitors. Gram-negative bacteria are inherently less permeable to many antibiotics as their outer membrane forms an impermeable barrier. Hydrophilic antibiotics usually cross the outer membrane by diffusing through porins. For instance, Mycobacteria are naturally resistant to many antibiotics due to their dense lipid layer surrounding the cell wall, and Gram-negative bacteria have intrinsic resistance to the glycopeptide antibiotic vancomycin because of the presence of the additional outer membrane.22 Therefore, reducing the permeability of the outer membrane and limiting antibiotic entry into the bacterial cell is achieved by the down-regulation of porins or by the replacement of porins with more-selective channels.

On the other hand, acquired resistance in susceptible bacteria is gained by either mutations in bacterial genes, or acquisitions of new genetic material, causing them to develop new mechanisms of resistance. Bacteria can develop acquired resistance in several different ways: enzymatic inactivation, chemical modification, prevention of antibiotic access to the target site or alteration of the antibiotic target site (Figure 2).23-27

The best example of this kind of resistance is exhibited by vancomycin-resistant

Enterococci that bacteria gain by changing the D-Alanyl-D-Alanine terminal end of the cell wall to D-Alanyl-D-Lactate.28

Enzymatic inactivation of antibiotics is accomplished when acetyl, adenyl or phosphoric groups are enzymatically transferred to specific sites on the antibiotics.29-31 β-lactamase produced by bacteria cleave the labile amide bond embedded in the ring of most penicillins and cephalosporins, and thereby inactive the compounds.

6

Figure 2. Resistance Mechanisms to Antibioics. Major resistance mechanisms in bacteria for developing acquired resistance: enzymatic inactivation, chemical modification, prevention of antibiotic access to the target site or alteration of the antibiotic target site.

7 Down-regulation or inactivation of transport proteins, such as porins, may prevent permeation of antibiotics across the outer membranes of Gram-negative bacteria. The uptake of fosfomycin is mediated by the glycerolphosphate transporter; mutations of this non-essential transporter cause development of resistance and tend to occur rapidly.32

Bacterial efflux pumps actively flush out antibiotics from the bacterial cytoplasm before onset of action and mediate resistance to the tetracyclines, chloramphenicol and the fluoroquinolones.33,34 Many of these efflux systems, such as AcrAB/TolC transporters, are not selective for a specific class of antibiotic, and are able to pump out many toxic compounds, thereby providing innate resistance to antibiotics. Other efflux systems are highly specific and can be triggered by exposure to antibiotics. Unlike the innate resistance efflux proteins, the TetA efflux pump is selective for tetracycline antibiotics.35

Bacteria also develop resistance through the acquisition of new genetic material from resistant organisms. This kind of selection is termed horizontal evolution, and may occur in an intra- or interspecific way or even among different genera and may be facilitated by transferrable elements such as transposons, which contain resistance genes.36-39 During conjugation, a Gram- negative bacterium transfers a plasmid carrying resistance genes to a recipient bacterium through a mating bridge, which joins the two bacteria. In Gram- positive bacteria, exchange of nucleic acid material by conjugation is usually triggered by sex pheromones, which facilitate clumping of donor and recipient cells. During transduction, resistance genes are transferred via bacteriophage. Finally, the so-called competent bacteria may acquire and incorporate resistance genes from other bacteria that have released their DNA into the environment after cell lysis, by transformation.

Through genetic exchange mechanisms, many bacteria become resistant to multiple

8 classes of antibacterial agents, and these multidrug-resistant pathogens are a serious threat to public health.40,41

1.3. Strategies to Overcome Antibiotic Resistance

The understanding of the molecular evolution of antibiotic-resistance is the starting point in developing new antibacterial agents. These resistance mechanisms can be used to modify promising molecules to avoid enzymatic modification or to develop enzyme- specific inhibitors of resistance. For instance, development of aminoglycosides lacking hydroxyl groups, such as in tobramycin and gentamicin, prevents inactivation by kinases.42 Developing resistance enzyme inhibitors has also been successful approach; it is used in the clinic exemplified by β-lactamase inactivators of clavulanic acid, such as sulbactam and tazobactam, to overcome resistance to the β-lactamase penicillinases.43

Identification and cataloguing of the genes giving rise to resistance and understanding their origins, and combining this information with combinatorial chemistry, high- throughput screening and rational drug design may also provide new avenues to overcome the resistance problem. Although several bacterial proteins were used as targets for high-throughput screening, and the leads subjected to rational design to develop new antibacterial agents, none of these efforts have yielded purchase as new antibacterial agents due to their inability to sufficiently penetrate the bacterial cell wall to reach targets, especially in Gram-negative bacterial species. One of the main reasons of this failure might be that even though high-throughput screening has been very successful

9 in identifying hits against targets, these libraries eliminate potent lead compounds not obeying Lipinski’s rules of five resulting in undesirable pharmacokinetic properties and poor oral bioavailability. Several other approaches have been utilized for antibiotic discovery, such as, developing semi-synthetic and synthetic antimicrobial peptides, phage therapy, targeting virulence factors, and using monoclonal antibodies.

1.4. Peptide Therapeutics

Peptides as therapeutics fill the gap between small molecules and antibodies. Since the first chemical synthesis of the therapeutic peptide oxytocin in 1953, more than 60 peptide drugs have gained entry to market, and more than 270 compounds are in clinical trials and approximately 400 more are under advanced preclinical investigation.44

Peptide therapeutics have several advantages compared to small molecules and antibodies.45-48 Most of biological processes are governed by protein-protein/peptide- peptide interactions, and developing small molecules modulating these interactions is difficult due to lack of good surface complementarity for the “hot-spots” spread over a large and shallow interaction interface. Compared to small molecules, peptides have greater selectivity and specificity for their target molecules, and they are also less toxic as the byproducts are composed of amino acids. Peptides can be considered better drug candidates than antibodies owing to their higher activity per mass, greater stability at room temperature, and better tissue and/or tumor penetration.

10 In spite of the extensive diversity and their exquisite specificity, peptides are poor drug candidates due to their low oral bioavailability, immunogenicity, poor pharmacokinetics, low serum stability and poor membrane permeability.49-51 Pharmacokinetic (PK) and pharmacodynamic (PD) properties of peptide drugs are more complicated than those of small molecules.

The major disadvantage of peptide as therapeutics is enzymatic degradation by exo- and endopeptidases of the gastrointestinal tract (GI).52 Modification of the N- and C-termini as the N- terminal pyroglutamate, C-terminal amide, and by acetylation, PEGylation, or glycosylation improves the stability of peptides.53-57 In addition, substitution with unnatural amino acids and cyclization may also confer improved stability and permeability.340 Peptide ligands made up of D-amino acids of the CXC- chemokine receptor type 4 did not show any degradation after 72 h, whereas natural CXC-4 was degraded within 24 h.58 Cyclization provides conformational restraint, and hence diminishes proteolytic lability. Disulfide bridged oxytocin and insulin are also potent peptides with good stability profiles.59

Their size and hydrophilic character limit peptides from entering the cell. Utilization of a pro-drug strategy, lipidation of peptides and using liposomes as a drug delivery method may improve the uptake of peptides.60-62

The route of administration of peptides is another challenge in developing them as therapeutics. The most common delivery method is parenteral. There are liposome- based technologies for transdermal and pulmonary delivery although there are usually limited to sites close to where the compounds are delivered.63,64 Transdermal delivery through skin has the advantage in stringent control of the drug concentration over generic

11 parenteral administration. Lately, micro-projection patches have facilitated the delivery of peptides into the epidermis with 1/30 of dose needed for intramuscular injection.65

1.5. Antimicrobial Peptides as Alternatives to Conventional Antibiotics

The ever-increasing threat and the emergence of many pathogenic bacterial strains resistant to commonly used antibiotics is a growing concern for public health. Patients with weakened immunity because of chemotherapy, AIDS or organ transplantation or patients undergoing acute care in hospitals are significantly and increasingly at risk for acquiring opportunistic bacterial infections. The discovery and development of novel antibiotic compounds has been slow and our arsenal of effective antibiotics is dwindling.

There are several approaches currently being undertaken in attempts to overcome bacterial resistance. Modification of existing antibiotics, finding new leads from nature, and the development of new synthetic classes through rational and target based design are proving the most interesting and most successful in the race to overcome multidrug- resistance.66-70

On the other hand, Nature has found its own way to fight bacterial infections by producing small cationic antimicrobial peptides (AMP) as a part of its innate immune system.71-73 Although these peptides have bactericidal activity against viral, bacterial and fungal infections, the level of antibiotic resistance occurrence that is observed is very low. Therefore, synthetic derivatives of these natural AMP can be good alternatives to solve the antibiotic resistance problem.74-81

12 Many natural peptide antibiotics produced by plant, insect, humans are both non- ribosomally, e.g. gramicidins, glycopeptides, and ribosomally synthesized, e.g. bacteriocins, defensins. Gramicidin is a well-known antimicrobial peptide on the market isolated from Basillus brevis in 1939 and is active against wide range of Gram-positive bacteria.82,83 Although their extensive diversity in structure, antimicrobial peptides are typically short cationic peptides having fewer than 50 amino acid residues exerting their antibacterial activity by disrupting cell membranes.84-94 Most antimicrobial peptides have cyclic structures, as well as unnatural amino acid residues.95-101 Some of these peptides have been shown to posses anti-viral activity, and others exhibit anti-cancer activity and the ability to modulate the innate immune response.102

Antimicrobial peptides differ in amino acid composition, spectrum of antimicrobial activities, hemolysis, cytotoxicity and mode of action.103-106 Although there are more than 2000 known natural AMPs, they are classified in four major groups based on their structure: α-helical, loop, β-sheet and extended peptides and having net charge between 0 to +7 besides being linear or cyclic. Regardless of their structure, AMPs usually have half of their sequence composed of hydrophobic residues with the highest frequency seen for L, and G and the cationic K conferring them with the ability to fold into amphiphilic structures.107

The most striking feature of AMPs is the low-level of observed bacterial resistance observed to commonly used conventional antibiotics.108,109 The action mechanism of

AMPs involves multiple bacterial cellular targets, and hence it is less likely the bacteria can evolve quickly to develop the resistance. For instance, it takes 30 passages of P. aeruginosa in broths containing sub-MIC peptide to develop the resistance by 2-to-4-

13 fold, while the resistance to the aminoglycoside gentamicin can increase by 190-fold under the same conditions.110 However, what AMPs gain in avoidance of resistance, they give up in potency with reported MIC values usually in the 1 µM range for the best of the compounds. (ref more in meng/kumar jacs paper)

During the past several decades, development of natural, semi-synthetic and synthetic

AMPs, has not resulted in a therapeutic in the clinic. Nevertheless, the membrane active compound daptomycin could be classified as an AMP, since it is a cyclic amphipathic peptide produced by Streptomyces roseosporus. Although the potency of these antimicrobial peptides against the more susceptible pathogens is normally not as strong as certain conventional antibiotics, one of their major strengths is their ability to kill multidrug-resistant bacteria at similar concentrations, hence they can be administered in synergy with commonly used antibiotics to improve their potency.111,112

1.6. Phage Display Selection of Peptide Ligands as Novel Therapeutics

1.6.1. Phage Display: Overview

George P. Smith introduced the concept of “phage display” by presentation of short peptide sequences on the surface coat protein of filamentous phage, and established the concept by isolating peptide binders for an antibody in 1985.113,114 The technique provides rapid and efficient high-throughput screening of peptide-peptide, peptide-small molecule, and peptide-protein interactions. Phage are DNA-containing viruses that are capable of displaying polypeptides on one of the coat proteins. Each phage clone carries a

14 different foreign DNA, and therefore displays a different peptide on the surface. The system allows discovery of ligands for a wide range of targets, including proteins, cells, tissues, inorganic materials, and others. The bacteriophage contains the linkage between the phenotype (displayed peptide) and genotype (encoding DNA), and therefore allows rapid identification of the peptide displayed by isolation of the phage by sequencing of phage DNA.115

There are three common phage systems used in phage display screening, namely the non- lytic filamentous phage M13, and the lytic viruses T4 and T7.116-1188 The M13 phage particle is a flexible rod-shaped cylinder consisting of circular single-stranded DNA, and five coat proteins. Two coat proteins, pVIII and pIII, are frequently used in displaying peptides. The major coat protein pVIII has 2700 copies and is suitable for displaying short peptide sequences due to virion packaging reasons. On the other hand, the minor coat protein pIII is displayed as a mere five copies, and is a well-suited option for displaying longer peptides. The pIII display system is much more common because it allows different foreign DNA insertion sites (the N-terminal region of pIII, middle of pIII or replacement of N-terminal domain or C terminus via a leucine zipper) while pVI only allows fusion of foreign peptide at C-terminus, and pVIII at the N-terminus. And linear random peptides varying in length from 6 to 43 amino acids have been successfully expressed at the pIII protein. Additionally, displaying peptides on the major coat protein pVIII might dramatically alter the physical and biological properties of the phage.119

There are three kinds of phage systems: wild-type filamentous phage with ssDNA, phagemid systems with plasmid vectors, and hybrid systems containing both wild-type phage and fusion genome.119-121 The type of phage systems can be chosen according to

15 the nature of target and end products, called “3”, “3+3”, and “33” or “8”, “8+8”, and “88” according to coat protein displayed, respectively. In general, wild-type phage systems allow polyvalent display on the coat protein whereas phagemid and hybrid phage systems only have monovalent display. Since the valency of phage display systems effects the selection of low and high affinity of binders for target molecules, monovalent systems are suitable for selection of the strongest binders while polyvalent display prevents the highest-affinity clones in a selection from being identified because it confers a high apparent affinity on weak-binding clones.

The concept is simple in principle, a library of phage particles expressing a wide diversity of peptides is used to screen and select peptides that bind to the target molecule.122-124

Filamentous bacteriophage have several advantages for use as cloning vectors and for the display of the encoded entities. Their genome can tolerate insertions in the non essential regions without disrupting phage packaging; their size is not constrained by the DNA contained in them; their genome can be isolated as single stranded and double stranded

DNA for cloning and library construction; the phage coat proteins can be modified to display foreign entities as fusion proteins without loss of phage infectivity; phage are stable under broad range of potential selection conditions including a range of pH and temperatures and can accumulate in high concentration in the infected bacterial cells due to non-lytic propagation. Any protein/peptide can be displayed on the surface of phage as long as it is translocated efficiently across the inner membrane into the periplasm for assembly into phage particles. Once in the periplasm, it has to be able to fold correctly in the oxidative environment.

16 There are several factors influencing the success of phage selection, the nature of the target, size of, and conformational space available to, and the quality of library. The size and quality of libraries are the most crucial parameters, and the most of them are produced as random peptide libraries according to the protocol developed by Zoller and

Smith.125,126 Theoretically, it is feasible to display a wide range of sized and structured peptides on phage surface, but some of the displayed peptides can be toxic to host cells or be sensitive to bacterial proteases. Also, cysteine is a rare residue in peptide libraries, and also might have to be removed from the libraries to prevent the formation of unexpected disulfide bonds. There are hundreds of different phage display libraries constructed by several research groups, whereas the commercially available ones are provided by New England Biolabs as linear 12-mer and 7 mer and cyclic 7-mer with a diversity of 109 independent peptides.

A critical aspect of peptide phage-display technology is the complexity of the library used during screening, as successful isolation of peptides for a target requires the presence of the peptide in the library chosen for biopanning. That is, the library should be of sufficient diversity to contain potential binding ligands for the target. Most phage- display peptide library screenings have been conducted using filamentous phage- displayed peptides.

Affinity selection procedure of phage display called “biopanning” generally consist of three main steps; (1) construction of a library with peptide or displayed variants, (2) selections based on affinity to targets of interest, and (3) confirmation of selected binders using biological assays and biophysical analysis.122,127,128 Affinity selections include four critical steps, incubation of target molecule with phage display library, washing off of

17 unbound phage, elution of the bound phage, and amplification of the eluted phage in host cells. After an initial round of biopanning, usually peptides exhibiting strong and moderate binding affinities to target molecules are captured to have higher yield, and the strong binders are selected in successive rounds with increasing stringency. Typically three to four rounds of selections are enough to obtain a consensus sequence depending on the nature of the target molecule, and the selected phage should be low in yield but high in stringency, with the specific and tight binding phage-displayed peptides.

Individual phage clones are isolated, amplified and followed by the DNA sequencing for deducing the sequence of the displayed peptide. Amplification is the most essential step in phage screening; it decreases the diversity and limits the number of binding clones that a screen can identify.

In addition to displaying peptides on phage, other in vitro selection systems have been developed to isolate peptide ligands and antibodies, such as, mRNA display, ribosome display, and yeast display.129-139 Each system has its own advantages and disadvantages.

For instance, mRNA and ribosome display systems provide large libraries, commonly 109 and 1014, without any transformation steps. Also, their biggest advantage is to enable incorporation of unnatural residues albeit with greatly reduced incorporation efficiencies than their natural counterparts.140

Despite its limitations, phage display remains the most commonly used screening system, due to its robustness, its versatility and, in the case of peptides, the availability of commercially available libraries. Phage display peptide libraries have significant advantages over other screening methods. The main advantage stems from its enormous diversity of displayed peptides up to 109 unique sequences. Secondly, it allows screening

18 of large numbers of clones at once in typically three to four weeks. Additionally, phage clones can be amplified by infecting the host cell E. coli without requiring additional purification steps compared to other screening methods.

So far, phage display has been successfully used to isolate peptide ligands binding the active sites of several enzymes, for instance, β-glucosidase, β-lactamase, dihydrofolate reductase, HIV transcriptase, and tRNA synthetases, protein targets, such as, calmodulin, oxizided thioredoxin, proteases, and for small molecules, with examples including dinitrotoluene, prostaglandin E2, 15-ketocholestane, and taxol.141-144

1.6.2. Linear and Cyclic Phage Display Peptide Libraries

The first developed phage library was a linear polypeptide system. Later, linear peptide libraries from 6-mer to 30-mers were generated to screen against different target molecules. Linear peptide ligands identified through phage display exerted weaker affinities for their targets, and were thus usually used for epitope mapping.145

Cyclic peptides have several advantages compared to their linear forms: they have a conformationally constrained structure enabling a smaller loss of entropy upon binding, and thus providing higher binding affinities.146-150 These constrained structures of cyclic peptides limit the number of possible confirmations during the phage display selection and more likely result in highly specific ligands for their target molecules. Furthermore, cyclic peptides are significantly more resistant to enzyme catalyzed hydrolytic damage.

19 In certain cases, cyclization can additionally enhance membrane permeability.151 Cyclic peptide libraries are produced by flanking a cysteine residue at both the N- and C- terminus of each displayed peptide sequence, and cyclized through disulfide bond formation.152

First a random cyclic-peptide phage library was used to isolate cyclic hexapeptides binding to platelet glycoprotein IIb/IIIa that mediates aggregation through binding of

153 fibrinogen with a Kd in the nanomolar range. Peginesatide, an erythropoietin mimetic, was the only phage-derived cyclic peptide that has seen clinical use in 2012, and was withdrawn shortly after its release due to undesired side effects.

1.6.3. Phage-coded Bicyclic Peptide Libraries

In nature, bicyclic peptides are produced both ribosomally and non-ribosomally, for instance, the bicyclic sunflower trypsin inhibitor 1 SFT-1, multicyclic plant cyclotides, multicyclic defensins, and other.154-156 In multicyclic peptides the above described advantages for cyclic peptides are even more pronounced. Currently, there are two bicyclic peptide drugs on the market, one is the antibiotic actinomycin-D, and other is and anti-cancer agent romidepsin that inhibits histone deacetylases.

Phage display libraries have been modified for the selection against specific target molecules. Since cysteine is the least abundant residue in phage libraries, stable cysteine- free mutant libraries can be produced to create through cysteine residues on the

20 phage.157,158 Thus far, linear phage display peptide libraries have been modified with fluorophores, glycan functional groups, and photo-reactive scaffolds to generate light- responsive cyclic peptide ligands.159-164 Derda and co-workers have generated glycopeptide libraries via oxime ligation by oxidation of N-terminal Ser/The residues to aldehydes/ketones to act as reaction partners.165

Phage-coded bicyclic peptide libraries were developed by Christian Heinis and Greg

Winter by chemical modification of the linear phage display libraries in 2009.163, They hypothesized that bicyclic peptides could mimic the complementarity determining regions of antibodies, and thus they could provide high affinity and specificity for their targets. While antibodies have excellent properties as therapeutics candidates such as their highly specificity and stability in human serum, they lack oral bioavailability and have high production cost. On the other hand bicycles are short peptide sequences constrained by a chemical scaffold core to form a structure with two loops of amino acids enabling them to target protein-protein interactions with low nanomolar affinity, and selectivity similar to antibodies.

Phage-coded bicyclic peptide libraries are constructed on mutated M13 phage peptide library having disulfide-free pIII. Linear peptide libraries with the construction of Cys-

(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3, 4, 5 or 6) having three cysteine residues are cyclized to generate bicycles in situ on the surface of phage with the thiol-reactive reagent 1,3,5-tris(bromomethyl)benzene (TBMB), and more than

108 unique bicyclic peptides can be generated through this protocol.166-168

21

Br

HS Br Br S S SH HS S TBMB

Figure 3. The schematic representation of cyclization reaction between a linear phage library having three free cysteines and the thiol-reactive reagent 1,3,5- tris(bromomethyl)benzene (TBMB) to form bicyclic libraries.166

22 Two parameters were used to tune the diversity of these bicyclic peptide libraries: the loop lengths and the structure of cyclization scaffold. The loop length has the fundamental role in the diversity of peptide libraries that modulates binding interactions and specificity.169,170 For instance, a PK15 inhibitor isolated through screening of 6 × 6 bicyclic peptide libraries was effective on human and monkey PK in thet low nanomolar range, rat PK in the micromolar range, and showed no inhibition of paralogous proteases, whereas a PK15 inhibitor from a 5 × 5 bicyclic library inhibited human, monkey and rat

PK in the nanomolar range, and was not effective against paralogous proteases. On the other hand, a bicyclic PK15 inhibitor from a 3 × 3 library inhibited human, monkey, rat

PK, and human factor XIa in the nanomolar range.171

In addition to TBMB, Heinis and co-workers also have developed three new thiol- reactive reagents to perform “bicyclization” of phage peptide libraries to establish further interactions with the backbone and/or side-chains of peptide loops: 1,3,5-triacryloyl-

1,3,5-triazinane (TATA), N,N’,N’’-(benzene-1,3,5,-triyl)tris(2-bromoacetamide)

(TBAB), and N,N′,N′′-benzene-1,3,5-triyltrisprop-2- enamide (TAAB).146,172

Bicyclic peptide ligands were successfully isolated by screening phage libraries for binding to serine proteases present in plasma namely kallikrein, cathepsin G, urokinase- type plasminogen activator (uPA), and Notch1 and Her2 receptors with Kds in the nanomolar range with high target selectivity.163,164,171,173-175

A bicyclic peptide inhibitor UK-18 bearing the sequence of Ac-SRYEVDCRGRGSACG-

NH2 for uPA, a trypsin-like serine protease involved in tumor growth and migration, was isolated through a 6 × 6 bicyclic peptide library modified with TBMB. It showed a selective inhibitory activity for human uPA with a Ki of 53 nM. Also, UK18 showed

23

Br O O

N N O NH O NH

N O O O O Br Br O N N N N H H H H

TATA TBAB TAAB

Figure 4. The structure of three thiol-reactive reagents besides TBMB to develop bicyclic phage libraries 1,3,5-triacryloyl-1,3,5-triazinane (TATA), N,N’,N’’-(benzene- 1,3,5,-triyl)tris(2-bromoacetamide) (TBAB), and N,N′,N′′-benzene-1,3,5-triyltrisprop-2- enamide (TAAB).

24 improved resistance to proteolytic degradation in plasma than its monocyclic and linear counterparts.171 The increased constraints and resulting rigidity enable the peptide to exhibited improved stability to enzyme catalyzed hydrolytic damage. Moreover, substitution of a glycine residue with D-Ser conferred a further 4-fold increased in plasma stability.176

Another bicyclic peptide ligand identified through phage display is an inhibitor of

FXII402 called FXII with a peptide sequence of Ac-GCGGRPCPPAYCG- NH2. It was isolated through 4 × 4 peptide library with a Ki of 1.2 µM showing 100-fold more selectivity than for other similar and related proteases.173

Recently, bicyclic ligands of the Notch receptor that inhibits the Notch signaling pathway associated with a wide variety of cancers was explored by screening 6 × 6 bicyclic peptide libraries, and peptide ligands with low Kd values, such as 150 nM, were identified.175

Although poor oral bioavailability and low membrane permeability still limit the application of bicyclic peptides as peptide therapeutics, they represent promising new drug candidates compared to their linear and monocyclic counterparts.

1.7. Application of Phage Display in Infectious Disease

Since short-synthetic peptides have been gaining importance as probes for microbial detection, phage display is a powerful tool for selecting novel peptides having

25 antibacterial activity by screening molecular targets, such as replication/cell division enzymes and host-pathogen virulence factors, or whole bacterial cells. There are several published reports detailing the use of phage display for the isolation of peptides with antibacterial activity against different Gram-positive and Gram-negative bacteria. Phage display enables of screening of target molecules to obtain high-potency therapeutic candidates in the nM to µM range much faster than what is possible for small molecules.177

Shiua et al. identified five peptides by screening the recombinant histidine-containing phosphocarrier HPr protein by using the linear 12-mer NEB random peptide library.

Only one of the five peptides, called AP1 with the sequence of YQVTQSKVMSHR, was found to inhibit the growth of E. coli cells efficiently with a value of IC50, value of

50 µM.178

One of the important virulence factors Urease of the Gram-negative bacterium H. pylori was used as a target for screening of 24-mer and 6-mer phage display peptide libraries.179

Two peptides, having peptide sequences of TFLPQPRCSALLRYLSEDGVIVPS and

YDFYWW, were shown to inhibit H. pylori. Later, the same group utilized the phage- displayed scFv libraries to select the antibodies inhibiting the same target H. pylori urease.180

Targeting bacterial cell wall biosynthesis is another approach to develop novel antibiotics.181 Peptide inhibitors of P. aeruginosa MurA enzyme, were identified using

182 NEB Ph.D.-12 and Ph.D.-C7C libraries with IC50 values in 200 µM range. The same experiments were carried out to discover inhibitors for the MurC enzyme and two

26

Table 1. Selected phage display research studies in infectious disease.

Target Phage Display Library Application

TEM-1 b-lactamase BLIP library Antimicrobial

RAP Linear 12-mer Anti S. aureus

SEB Linear 12-mer Anti S. aureus

S. aureus SdrC Linear 12-mer Anti S. aureus

S. aureus whole cell Linear 12-mer Anti S. aureus

P. aeruginosa whole cell Linear 9-mer and 12-mer Anti P. aeruginosa

P. aeruginosa MurA Cyclic 7-mer, Linear 12-mer Anti P. aeruginosa

P. aeruginosa MurC Cyclic 7-mer, Linear 12-mer Anti P. aeruginosa

P. aeruginosa MurE and MurF Linear 12-mer Anti P. aeruginosa

P. aeruginosa FtsA and FtsZ Cyclic 7-mer, Linear 12-mer Anti P. aeruginosa

Bacterial Membrane Model T7 and Linear 12-mer Antibiotic design

LPS/Lipid A scFv and peptide libraries Antibacterial

L. monocytogenes scFv library Diagnostics

27 peptides showed the similar IC50 values, a linear 12-mer peptide DHRNPNYSWLKS with 1.5 mM and cyclic 7-mer CQDTPYRNC with 0.9 mM MIC, also showing low permeability and susceptibility to enzymatic degradation in plasma.183 Later, the same group identified two linear 12-mer peptides as inhibitors of P. aeruginosa MurE and

MurF enzymes, NHNMHRTTQWPL and TMGFTAPRFPHY with IC50s of 500 µM and

250 µM, respectively.184,185 Furthermore, inhibitors for P. aeruginosa FtsA and FtsZ, bacterial cell division-related proteins were fished out from the Ph.D.-12 and Ph.D.-C7C libraries.186,187

Whole bacterial cells can be used for phage selection in addition to purified antigens for infectious diseases.188-192 The main advantage of using whole cells over purified antigens is that it can also provide information on unknown antigens, which provide unique binding sites for peptide interactions, as well as antigen presentation in a native environment.

Phage-derived peptide biosensors binding to P. aeruginosa whole cells were evaluated and proved to be efficient in diagnostic assays.193 Pini et al. panned a 10-mer peptide library against E. coli whole cells, and reported an antimicrobial dendrimeric peptide,

QEKIRVRLSA, with good MIC values of 4 to 8 µg/ml against clinical isolates of P. aeruginosa and Enterobacteriaceae.194

Another study done by Bishop-Hurley and his colleagues showed that a 15-mer phage- derived peptide had bactericidal activity against Haemophilus influenza in the biopanning of whole bacterial cells.195 Later, the same group isolated antibacterial peptides specifically active against Campylbacter jejuni but not other Gram-positive and Gram-

28 negative bacteria.189 They identified eleven peptides inhibiting the bacterial growth by up to 99%, and one of them also had bactericidal activity.

Rao et al. used a phage-displayed peptide library to pan against E. coli, and the isolated peptide was able to inhibit growth of both E. coli and P. aeruginosa.196 The isolated peptide after six rounds of biopanning having the sequence of RLLFRKIRRLKR exhibited certain features common to AMPs and was rich in arginine and lysine residues.

However, using whole bacterial cells in phage display screening has some limitations due to the need for washing the cell membrane with detergent, and eluting the bound phage with acidic buffer at pH 2.2. The use of detergents and acidic solutions may damage bacterial cells, resulting in a lower recovery of bound phage. Furthermore, antimicrobial peptides bound to bacterial cells may not be efficiently eluted by use of acidic buffer.

Liposome-conjugated magnetic beads and lipopolysaccharide (LPS)-conjugated epoxy beads were introduced to solve the above issues during phage selection.197,198

Tanaka et al. employed bacterial magnetic particles, obtained from Magnetospirillum magneticum strain AMB-1, and used phospholipase D to recover bound phage in biopanning experiments to isolate peptide inhibitors of B. subtilis.199

These studies showed that phage display technology is highly versatile and a robust tool for isolation of antibacterial peptides. Like other affinity selection processses, phage display has its own problems in the discovery of novel antimicrobials. The selected peptides may or may not show high affinity for target molecules, or may lack antimicrobial activity, and thus require further modification to improve their biological profile and effectiveness.

29 1.8.The Scope of Thesis

Although antibiotics have been used by bacteria in inter-species warfare for a very long time, antibiotic resistant bacteria, so-called “superbugs”, present one of the most challenging problems facing modern medicine.41,200 Superbugs fall under two classes: the first category contains those that have acquired antibiotic resistance genes, such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococci (VRE), and the second feature opportunistic pathogens, such as P. aeruginosa. The World Health

Organization’s 2014 report on antibiotic resistance highlights the risk to the ability to treat common infections in the community and hospitals stems from multi-drug resistant

E. coli and K. pneumoniae infections, and methicillin-resistant S. aureus (MRSA). Now, more than ever, we need to think of ‘out of the box’ solutions in order to win the battle against superbugs.

The emergence of vancomycin resistance calls for an urgent need to develop more potent antibacterial agents possessing new modes of action that make the development of the bacterial resistance difficult as seen in natural antimicrobial peptides. Small peptides mimicking the action mechanism of vancomycin with higher or equal affinities to bacterial cell wall precursors of sensitive and resistance bacterial strains, D-Ala-D-Ala and D-Ala-D-Lac, would be ideal for the development of novel therapeutics with metabolic stability. Therefore, developing D-peptide antibiotics through mirror-image phage display based on the selection of linear, cyclic, and bicyclic phage display would provide a new class of antibiotics. These studies are described in the following chapters.

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

Staphylococci, from the Greek: grape-berry, were first classified in 1882 by Sir

Alexander Ogston.201 Staphylococcus aureus is a Gram-positive bacterium. It is one of the main causes of hospital- and community-acquired infections that can result in serious consequences. Although S. aureus infections are normally endogenous, the hospital- acquired versions are classified as exogenous. S. aureus can infect skin, soft tissue, and result in endovascular infections, pneumonia, septic arthritis, endocarditis, osteomyelitis, foreign-body infections, and sepsis. In addition, it can invade surgical wounds and patients with prosthetic implants due to their compromised immune system face increased risk.

Apart from its ability to cause life-threatening infections, its remarkable potential to develop resistance to compounds is another factor making this pathogen number one in the rankings of the most dangerous to human health. S. aureus gain antibiotic resistance through several different ways: via the enzymatic inactivation of antibiotics by agency of promiscuous penicillinases, and aminoglycoside-modification enzymes, modification of the antibiotic target, such as, PBP2a in methicillin-resistant S. aureus (MRSA) strains, D-

Ala-D-Lac of peptidoglycan precursors of vancomycin-resistant strains, and efflux pumps for fluoroquinolones and tetracycline.202

The misuse of the antibiotics promotes the resistant strains of S. aureus due to its ability to acquire resistance with facility. β-lactam resistant S. aureus strains were reported shortly after deployment of penicillin as an antibiotic in hospitals in the 1940s. Today, more than 80% of S. aureus strains produce β-lactamases, and therefore are resistant to

32 penicillin.19 After the emergence of β-lactam-resistant strains, a β-lactamase-stable penicillin derivative ‘methicillin’ entered clinical use to overcome this shortcoming in

1960. Soon after its release to market, the first MRSA strain was identified in 1961.203

MRSA strains are dependent on the acquisition of the staphylococcal cassette chromosome mec (SCCmec) encoding a low-affinity penicillin-binding protein PBP2a by horizontal DNA transfer from an unidentified donor.204 Hospital-acquired MRSA strains cause serious health problems for the patients having organ-transplant surgery,

HIV/AIDS, and other afflictions. The mortality rate of severe MRSA infections is about

20% which is the leading cause of death by a single infectious agent in the US, exceeding deaths caused by HIV/AIDS making it both a public health and economic societal burden.205,206

After the widespread emergence of MRSA, vancomycin has become the “go to” therapy for MRSA infections since 1956. MRSA infections are currently treated with vancomycin, the so-called “drug of last resort”, which owes its inhibition of bacterial proliferation to prevention of bacterial growth biosynthesis. Over the past decade, the appearance of strains not susceptible to vancomycin, showing either intermediate resistance (vancomycin-intermediate S. aureus (VISA)) or, full resistance (vancomycin- resistant S. aureus (VRSA)) has seen as a worrying increase. VISA have reduced susceptibility with a minimum inhibitory concentration (MIC) of 4-16 µg/ml, whereas

VRSA have MIC values of about 32 µg/ml.207 The presence of a thickened cell wall characterizes VISA strains, because of the modification of the D-Ala-D-Ala terminus of the pentapeptide precursor to D-Ala-D-Lac, the latter resulting from vancomycin having a 1000-fold reduced affinity for the new dipeptide. No characteristic genetic trait has

33 been associated with VISA, although the loss of the accessory gene regulator (agr) locus was observed in these strains, a quorum-sensing gene cluster that regulates virulence, conferring a selective survival advantage in the presence of vancomycin. Although

VRSA strains are fortunately still rare, there is an urgent need to develop new antibacterial agents active against resistant strains.

2.2. Phage Display in Staphylococcus aureus Infectious

There are several research groups working on the identification of small peptides having inhibitory activity against S. aureus, by binding to bacterial membrane proteins and/or to toxins essential for bacterial survival.208-212 Yacoby et al. panned commercially available

12-mer random peptide libraries against S. aureus whole cells to identify a peptide carrier to deliver antibiotics into bacterial cells.213 The drug molecule is released from the phage surface by ester bond hydrolysis in the presence of serum. Inhibition of bacterial growth was significantly improved by incubation with drug-carrying phage versus free drug alone.

S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via phosphorylation of its target protein TRAP, a quorum-sensing molecule. Therefore, inhibition of quorum sensing by antibodies or peptides is being considered as a novel approach for therapeutics alternative to antibiotics that act on proliferative activities. Yang et al. selected RAP-binding peptides from a random linear

34 12-mer phage display peptide library.211 Nine peptides were identified from biopanning experiments, and a peptide with sequence WPFAHWPWQYPR showed the strongest inhibitory effect.

Three peptides with sequences WWRPLTPESPPA, MNLHDYHRLFWY, and

QHPQINQTLYRM, were identified from a linear 12-mer phage display peptide library that had binding affinity to staphylococcal enterotoxin B (SEB), a protein that is responsible for food intoxication and can be used as a biological war agent.210

Α β-neurexin peptides, FFSARG, that binds to the cell-wall anchored proteins was selected through a linear 12-mer library, and demonstrated high affinity and specificity in screens against S. aureus SdrC.208 These peptides play a key role in modulating the interactions between pathogenic microorganism and the host cell.

Chen and his colleagues investigated a peptide mimicking the S. aureus peptidoglycan to enhance the immunogenicity of peptidoglycan as a vaccine candidate.214 Several peptide sequences were obtained from a phage display peptide library using a mAb against a S. aureus peptidoglycan, and a 12-mer linear single peptide (Sp-31) and a four-branch multiple antigen peptide (MAP) (MAP-P31) with a consensus sequence of

ATWxHxLxSAGL showed high reactivity with anti-PGN mAb.

However, little work has been done on, or has been successful with Methicillin-resistant

S. aureus (MRSA) or other resistant strains to identify strain-specific peptides or antibodies. Kaur et al., investigated the effects of protein synthesis inhibiting molecules on the plaque size and morphology of S. aureus phage to develop MRSA-virulent phage as therapeutics.215

35 2.3. Peptide Therapeutics Containing D-amino Acids

Peptide therapeutics with promising pharmacological activities usually suffer from short- half-lives primarily due to their poor metabolic stability in the presence of proteolytic enzymes in the blood, liver, and kidney. Protease stability of peptide therapeutics can be enhanced by several strategies including substitution of natural amino acids by unnatural

D-amino acids, N-methylation or by use of β-amino acids, capping of the N or C-termini of the peptide, esterification, or PEGylation among others. Among these strategies to improve the biological profile of peptides, the introduction of unnatural amino acids, in particular D stereoisomers, in the peptide sequence is widely used. Although D-amino acids disrupt α-helices, and thereby destabilizing secondary structures, other structural properties, such hydrophobicity and charge distribution are maintained. Furthermore, peptides less prone to proteases are obtained since only few enzymes are known to digest amide bonds with amino acids of D-configuration at the cleavage site.216 This strategy has been used to improve the biological activities of synthetic antimicrobial peptides while maintaining the antimicrobial activity, and is especially useful for antibiotics whose mode of action features membrane disruption.217,218

D-amino acids are found in peptides and proteins from all kingdoms of life, in bacterial cell walls, antimicrobial peptides, crustacean peptide hormones, venoms from snails, funnel web spiders and the enigmatic platypus. Two peptides isolated from the skin of the South American leaf frog Phyllomedusa sauvagei containing D-Met, D-Leu or D-Ala in position 2 have been shown to be more potent opioids than their corresponding all-L- peptides on the δ-opioid receptor.219,220 Another study on D-peptides, substitution of a

36 glycine residue with D-Ser conferred a further 4-fold increased in plasma stability.176

LL-37 is a member of cathelicidin family of peptides expressed in humans, and has broad antimicrobial spectrum against bacteria, fungi and viral pathogens.221 The mirror image peptide of LL-37, termed D-LL-37, showed higher potency in simulating of IL-8 release in keratinocytes compared to its natural isomer as well as being less prone to enzymatic degradation.

The most striking feature of D-peptides is their stability in serum. This property stems from the high-energy barrier to reach the transition state from the initial enzyme-substrate complex is set by steric incompatibility.222-225 Proline-rich cell penetrating peptides,

(VRLPPP)3, have been proposed to be efficient intracellular delivery vectors due to lack of cytotoxicity combined with their capacity to be internalized by cells with one limitation, the metabolic instability. The D-enantiomer of this peptide was completely stable for more than 48 hours in highly concentrated trypsin solution and in human serum.223

Contrary to their L-counterparts, peptides solely consisting of D-amino acids can be delivered orally likely because of extended half-lives afforded in the digestive protease rich environment in the stomach.226,227 An octapeptide composed of D-amino acids with a sequence of EASASYSA was absorbed efficiently from the intestine of mice, and excreted in urine without hydrolysis catalyzed by membrane-bound peptidases of brush borders.

Furthermore, D-peptides are either less or non-immunogenic compared to their L- enantiomers.228-231 Immunogenicity of the all D-amino acid protein rubredoxin was

37 compared with its corresponding L-enantiomer.229 It was observed that the multiple administration of L-rubredoxin induced a strong specific IgG antibody response, whereas the D-enantiomer did not. Additionally, D-rubredoxin had a four-fold longer lifetime than the natural L-protein.

Compared with L-peptides, the properties described above provide D-peptides dramatically increased serum half-lives, as well as possible oral bioavailability. These favorable features make them a particularly attractive class of therapeutics, and it is therefore time to consider D-amino acids as alternatives and as modular design elements in the development of peptide therapeutics.

2.4. Elegant Approach to Design D-peptide Ligands: Mirror Image Phage

Display

Peter Kim and co-workers introduced an elegant and clever approach to obtain D-peptide ligands of specific targets based on the traditional phage display technique.232 In this innovative scheme, the selection is carried out against a target has been synthesized in the

D-configuration (the mirror image of the original target) using a phage library of peptides that only carry regular canonical amino acids. Due to symmetry considerations, the mirror images of the selected peptides interact with the target protein in the natural form.

As a proof of concept, the first mirror image phage display was carried out by using enantiomer of c-Src homology 3 (SH3) domain of chicken Src kinase with a cyclic

38 peptide library.232 A cyclic D-peptide ligand with sequence rclsglrlgvpca (note that the small one letter code is used for amino acids in the D-form) was identified for the c-Src

SH3 domain with a dissociation constant a Kd of 63 µM.

One of the most prominent applications of mirror image phage display was the identification of potent D-peptide inhibitors of the human immunodeficiency virus type 1

(HIV-1) entry leading to the development of orally applicable anti-HIV drugs. In 1999,

Eckert et al. designed the peptide called IQN17 resembling the HIV-1 gp41 protein, and thus inhibiting HIV-entry.233 Cyclic D-peptide inhibitors of HIV-1 infection with

GACXXXXXEWXWLCAA motif were isolated. Later, based on this peptide motif,

Welch et al. constructed cyclic phage display peptide library with a CXXXXXEWXWLC motif to find better binding profile. Several D-peptide inhibitors for HIV-entry were isolated and the antiviral potency was increased by up to 40,000-fold compared to the previous study.234 Furthermore, Welch and co-workers reported the first designed D- peptide for the inhibition of HIV-entry with a 100,000-fold increased potency as a 3rd- generation of the HIV gp41 N-trimer pocket-specific D-peptide called PIE12-trimer.235

In 2001, Kozlov et al. used the mirror image forms of the naturally occurring D- configured carbohydrates to identify high affinity D-peptide ligands for the cell-surface carbohydrates.236 As a proof of principle, single-chain Fab sequences and dodecapeptides binding to the D-enantiomeric versions of sialic acid and 3-deoxy-α-L- manno-2-octulosonic acid (L-KDO) with nanomolar to high micromolar affinities were identified. This sugar-protein interaction study provides a new method to develop antibiotics and drug delivery systems.

39

Figure 5. Schematic representation of mirror image phage display concept: the phage display peptide library is screened for identification of L-peptides that bind the mirror image of the target molecule target is D-target. Due to symmetry relations, the mirror images of L-peptides identified through phage display that are D-peptides bind the original target (L-peptide).

40 This method has also been applied to develop a new class of D-peptide therapeutics for the diagnosis and therapy of Alzheimer’s disease caused by abnormalities of the amyloid peptide (Aβ peptide).237-246 In the previous study, a D-peptide called D1, qshyrhispaqv, with binding affinity to Aβ in the micromolar range was identified from a commercially available linear 12-mer phage peptide library. β-sheet structure of this Aβ peptide found in aggregated extended form that build up amyloid plaques shows a high affinity to chromophores and therefore they are stainable. Further in vitro and in vivo biophysical and biochemical studies with FITC-labeled peptide D1 showed that it is capable of both staining of Aβ (1-42) plaques in the brains of transgenic AD mice. In a similar approach, another 12-mer D-peptide with sequence of rprtrlhthrnr, called D3, was selected by screening monomeric and small oligomeric D-Aβ species as target. D-peptide D3 had a strong influence on Aβ aggregation, disaggregation, and cyctotoxicity in vitro while it reduced amyloid plaque load and cerebral inflammation of transgenic mouse models of

Alzheimer’s disease in-vivo by direct application in brain. Later, same group worked on the effect of orally applied D3 peptide on plaque load and on the cognitive behavior of

Alzheimer’s disease transgenic mice. It was observed that brain tissue section of DE treated mice had significantly lower Aβ load as compared to that of untreated one via their drinking water.

Studies on D-peptide inhibitors of p53:MDM2 interactions also have been done by the application of the mirror-image phage display concept.247-250 The two oncoproteins,

MDM2 and MDMX, down-regulate the tumor suppressor protein p53 activity by inhibiting transcriptional activity and promoting p53 degradation, thereby are bolstering tumor development and survival. As a starting point, Pazgier et al. identified a potent L-

41 peptide inhibitor, called PMI (TSFAEYWNLLSP), of the p53-MDM2/MDMX complex by screening a linear 12-mer phage display peptide library binding to MDM2 and

247 MDMX with a Kd of 3.2 nM and 8.5 nM, respectively. Although PMI has two orders of magnitude stronger affinity than the same length wild-type p53 peptide,

ETFSDLWKLLPE, it has limited therapeutic value due to its rapid enzymatic degradation and poor bioavailability. Later, Liu et al. identified D-peptide inhibitors of the p53-MDM2 interaction, called DPMI-α (tnwyanlekll), DPMI-β (tawyanfekllr), and

D 248-250 PMI-γ (dwwplafeallr), with Kd values of 219, 35 and 53 nM, respectively. Despite being resistant to proteolysis, these D-peptides failed to induce p53-dependent tumor cell death because of inability of traversing the cell membrane.

Although all these promising results have come from the studies with the mirror image phage display concept, it has a one major limitation. Since the enantiomer of protein/small molecule/peptide is used as a bait in phage display screening, the mirror image of the target molecule needs to be chemically synthesized and it therefore places a limit of targets accessible by mirror image phage display to relatively small protein targets that are tractable by chemical synthesis.

Three commercial outfits have publicly announced programs currently utilizing mirror- image phage display concept to develop new class of therapeutics. Noxxon Pharma AG, based in Berlin, develops mirror-image nucleic acid molecules known as “spiegelmers” as drugs for the treatment of inflammatory diseases and hematological indications. It has products in Phase-I clinical trials. Cosmix Molecular Biologicals GmbH, a

Braunschweing-based company, is focused on using RNA display technology to develop

42 D-peptide drugs and has compounds in pre-clinical development stage. Reflexion

Pharmaceuticals of San Francisco, works on developing of D-protein drugs that are also in pre-clinical development.

Together, these studies and commercial efforts devoted to mirror image phage display underscore the immense potential of D-peptide based drugs. In the following chapters, outlined are our own research efforts in the search and development of D-peptide antibiotics.

2.5. Designing The Target Molecules for Mirror Image Phage Display

The aim of this research is to identify peptide ligands inhibiting the cross-linking reaction at the last step of the bacterial cell wall biosynthesis with the same action mechanism of vancomycin. Therefore, the target molecule is the pentapeptide (L-Ala-D-γ-Glu-L-Lys-

D-Ala-D-Ala) unit of the bacterial cell wall of S. aureus.

The nature of the target molecule has a pivotal role in the phage display selection. For a successful selection process, the target must retain its native conformation during the affinity selection. A ligand that has exactly same conformation and shape to complement a binding site should have higher affinity for its desired target. Since our target molecule is the pentapeptide precursor of the growing bacterial cell wall of S. aureus, this short free-rotating peptide would have thousands of different conformation in the solution

43 during the biopanning selection process in phage display. Therefore, the selection of peptide ligands might not yield high-affinity binders for the target molecule.

On the other hand, Tipper and Strominger proposed that penicillin, a β-lactam antibiotic, mimics a high energy conformation of D-Ala-D-Ala that bind to the transpeptidase, thus inhibits the cross-linking of bacteria cell wall by irreversibly binding the active site of the peptidoglycan transpeptidase enzyme (Figure 6A and 6B).252-257

According to this proposed model, the highly reactive amide bond of β-lactam ring of penicillin and the amide bond between the D-Ala-D-Ala dipeptide of bacterial cell wall have exactly same conformation (Figure 7).258 Also, two carbonyl groups of both penicillin and D-Ala-D-Ala termini of bacteria cell wall forms hydrogen bonding in same direction as a proof of their common structure. Additionally, the acidity of these two molecules is same, pKa 2.6 to 2.7. These structural similarities are also studied by computational studies on β-lactam ring model of cephalosporin and penicillin.258-260

Cephalosporin is another β-lactam antibiotic that targets the bacterial cell wall with same mechanism as that of penicillin. It has similar structure with penicillin with a difference in bicyclic ring system, a six-member dihydrothiazine ring instead of a five-member thiazine ring. Thus, the enantiomers of cephalosporin and penicillin can be also good target molecules in the selection of D-peptide ligands in biopanning selection experiments in addition to using pentapeptide as the enantiomer of D-Ala-D-Ala termini of bacteria cell wall. Therefore using a β−lactam, cephalosporin, mimicking the bacterial cell wall might be more advantages to get high-affinity peptide binders than using just

44 A GlcNAc MurNAc GlcNAc OH NHAc GlcNAc MurNAc GlcNAc HO NHAc GlcNAc MurNAc GlcNAc O O O O HO O O H O H O O O H H3C NHAc H3C OH OH O H3C O O L-Ala L-Ala L-Ala D-γ-Glu D-γ-Glu D-γ-Glu L-Lys-NH L-Lys-NH2 2 L-Lys-NH peptidoglycan transpeptidase cross-linking reaction 2 NH NH B NH (D) CHCH (D) CHCH3 3 H (D) CHCH3 D-Ala C O O D-Ala C O C O D-Ala Ser O NH B NH L-Lys

H (D) CHCH3 D-γ-Glu D-Ala COOH L-Ala D-Ala O NH2 L-Lys CH3 O H D-γ-Glu GlcNAc MurNAc GlcNAc L-Ala O CH3 O H GlcNAc MurNAc GlcNAc

B GlcNAc MurNAc GlcNAc OH NHAc HO NHAc O O O O HO O O O H O NHAc OH OH H3C O L-Ala H D-γ-Glu peptidoglycan transpeptidase R N S

L-Lys-NH2 O HN No cross-linking reaction D-Ala ✖" O OH Enzyme D-Ala H O R N S

O N O O OH Penicillin

Vancomycin

GlcNAc MurNAc GlcNAc OH NHAc HO NHAc O O O O HO O O O H O NHAc OH OH H3C O L-Ala D-γ−Glu

L-Lys-NH2 D-Ala No cross-linking reaction D-Ala ✖"

Figure 6. A) The last step of bacterial cell wall biosynthesis catalyzed by peptidoglycan transpeptidase for cross-linking of two pentapeptide units to give integrity to the bacterial cell wall. B) Action mechanism of penicillin and vancomycin that prevent cross-linking via different action of mechanisms.

45

A B

O H H Nb a b H N −N = 3.3 Å O b c cCOOH N − C = 2.5 Å Na R S Na−cC = 5.4 Å H Penicillins O H Nb Na−Nb = 3.3 Å H3C O H Nb−cC = 2.5 Å cCOOH Na a c Peptidoglycan N − C = 5.7 Å H CH3 N-acyl-D-Ala-D-Ala

Figure 7. Structural similarities between peptidoglycan cell wall termini D-Ala-D-Ala and penicillin. A) Calculated structural overlays of the best conformations of N-Ac-D- Ala-D-Ala and Ampicillin (a β-lactam). Longest RMS distance = 1.70 Å; shortest 0.29 Å B) Distances calculated by Strominger for penicillins and N-acyl D-Ala-D-Ala.258

46 pentapeptide as bait owing to their locked conformation for binding the active site of peptidoglycan transpeptidase enzyme.

Both enantiomer of pentapeptide precursor of bacterial cell wall (D-Ala-L-γ-Glu-D-Lys-

L-Ala-L-Ala) as a free rotating peptide, and enantiomer of cephalosporin as a structurally rigid mimic of L-Ala-L-Ala were synthesized with a polyethyleneglycol (PEG) linker and biotin group attached at the N-termini that can be utilized surface mobilization on streptavidin-coated surface in both biopanning and also for biophysical studies (Figure 8).

Additionally, due to no significant binding difference between vancomycin and UDP-

MurNAc-L-Ala-D-γ-Glu-L-lys-D-Ala-D-Ala, and just pentapeptide L-Ala-D-γ-Glu-L-

Lys-D-Ala-D-Ala, sugar part was omitted in the synthesis of the enantiomer of target molecule to prevent the selection of non-specific binders through phage display experiments (Table 2).261,262

Peptide ligands binding to the bacterial cell wall precursor of vancomycin-resistant

Enterococci (L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Lac) was also investigated through mirror image phage display concept, and hence the enantiomer of the modified pentapeptide precursor, where L-Ala terminal end of pentapeptide was modified to L-Lac, was synthesized and used in the biopanning screenings in Chapter 3.

47

Table 2: Association constants for the binding of vancomycin with various peptides.

Peptide Sequence Ka (L/ mol)

6 Ac-L-Lys-D-Ala-D-Ala 1.5 x 10

4 Ac-D-Ala-D-Ala 2.0 x 10

5 D-Glu-L-Lys-D-Ala-D-Ala 7.6 x 10

5 L-Ala-D-Glu-L-Lys-D-Ala-D-Ala 6.9 x 10

5 UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala 7.2 x 10

48

O NH O H HN H H H O N S H N EA-1-CEP H 12 S O N O

O OH

NH2

H S O H N HN NH O O O H H H NH O 4 N N OH EA-2-ALA O N N H H O O O O OH

NH2

H S O H N HN NH O O O H H H NH O 4 N N OH EA-4-LAC O N O H O O O O OH

Figure 8. The structure of target molecules used in phage display experiments; enantiomer of cephalosporin, EA-1-CEP, enantiomer of bacterial cell wall precursor of S. aureus, EA-2-ALA, and the enantiomer of bacterial cell wall precursor of vancomycin-resistant Enterococci, EA-4-LAC.

49 2.6. Results and Discussions

2.6.1. Control Phage Display Experiments using Streptavidin as a Model Target

Molecule

Streptavidin, a tetrameric protein secreted by Streptomyces avidinii, binds its natural non- peptide ligand biotin (vitamin H) with one of the highest known affinities in the nature, a

-15 Kd of 10 M. Thereby, molecular biology utilizes this non-covalent strong interaction.

Previous studies with phage display peptide library using streptavidin as target molecule revealed that this large protein binds linear and cyclic peptide ligands having His-Pro-Gln

(HPQ) consensus sequences with various of binding constants, 270 nM to 375 µM.263-268

The crystal structure of a heptapeptide FSHPQNT binding to streptavidin have shown that HPQ consensus sequence mimics biotin, and binds through the biotin-binding site of streptavidin.269

A successful phage display screening depends mostly on choice of immobilization technique for target molecule, size and quality of phage display library, nature of target molecule. Even with an excellent selection strategy, the experiment will fail if the desired peptide is not present in the library. Hence, running control experiments by using streptavidin as model target is a good starting point to test efficiency of biopanning specially to decide the immobilization technique. For soluble targets, there are many options for their immobilization. For example, they may be coated directly on a solid surface, such as a polystyrene microplate or petri dish, through non-covalent adsorption onto hydrophobic plastic surface or they may be biotinylated and captured on a streptavidin-coated plate or streptavidin-magnetic beads. In this study three different

50 immobilization strategies, which are solid-phase biopanning by using either a 96-well plate or a petri dish and solution-phase biopanning by using streptavidin-coated magnetic beads, were tested to isolate peptide ligands binding to streptavidin through a linear 12- mer phage display random peptide library.

Results from all biopanning selections (Table 3) showed that randomly picked eight phage clones out of ten from solution-phase biopanning experiment have “HPQ” consensus sequences, whereas only three peptides from solid-phase biopanning using a petri dish have that streptavidin-binding motif. Additionally, no phage clone having

“HPQ” motif was isolated from solid-phase biopanning using 96-well plate set-up, instead randomly isolated phage clones had a W-x-x-W motif for specific to polystyrene binding.270 Therefore, solution-phase biopanning was significantly more effective than solid-phase biopanning using either petri dish or 96-well plate for isolating peptides having “HPQ” consensus sequence specifically binds to streptavidin. There are several advantages of using streptavidin-coated magnetic beads over immobilizing the target on polystyrene surface, such as, increased surface area for target binding, ease of washing and elution steps, preventing denaturation of protein while coating on plastic surface, and less amount of target solution.271,272 The streptavidin-coated magnetic beads provide a larger accessible surface area for target molecule to bind the phage display peptide library in solution. Also magnetic beads can freely rotate in the solution buffer resulting in reduced distance between the target and phage library, thus improving reaction kinetic for the binding. Solution-phase biopanning also eliminates to select peptides that selectively binds polystyrene surface which are typically rich in aromatic residues, Phe, Tyr, Trp,

His.

51

Round Peptide Sequence Abundance 3 D H G L S W L R N H P Q 4 3 I P G I W L F T D H P Q 2 3 H S W S F W L R N H P Q 1 3 M W D H P L D F L H P Q 1 3 S L H W V N L K P V S D 1 3 E S V F Q D Y L A S Y H 1

Round Peptide Sequence Abundance 3 I P G I W L F T D H P Q 1 3 G P A S W L A M H P Q R 1 3 G F L T L I P N D H P Q 1 3 I P L E V A Y S S L I R 1 3 C V S A Y P A C L G H D 1 3 I L P S S F D A W L N R 1 3 I P G I W L G S D H R R 1

Round Peptide Sequence Abundance 3 A L W P P N L H A W V P 4 3 W H W S Y W P G D N R A 2 3 K L W L P N P Q A X X P 1 3 K X W L P N L Q V X V P 1 3 E X W P P N L Q A G V P 1 3 G A S T T W S R M V L D 1 3 T H P S T K V P G T P A 1 3 K X L L A N P L V G A L 1

Table 3. Peptide sequences of randomly picked phage plaques after the third round biopanning against streptavidin as target using linear 12-mer phage display random peptide libraries in solution-phase biopanning by using streptavidin-coated magnetic beads, solid-phase biopanning by using lab-coated streptavidin in 60 × 100 mm petri dish, and 96-well plates. HPQ sequences are highlighted in Rasmol color code.

52

Streptavidin-coated Magnetic Beads Washing away unbound phage

Phage Display Peptide Library Immobilization on Streptavidin-coated beads

Biopanning (4-5 Rounds)

Binding of Target with Phage Display Library Biotinylated Target Molecule Elution

Amplification in E. coli

Phage Titering

ELISA DNA sequencing

Figure 9. The schematic representation of affinity selection in solution-phase biopanning. Target molecule is incubated with phage library, and then immobilized on streptavidin beads. After washing away unbound phage, bound phage is eluted, and amplified in E. coli. After 4-5 rounds of selection, DNA of randomly picked phage is sequenced to reveal peptide sequence that expressed, and subjected to ELISA for binding confirmation.

53 Another advantage of using magnetic beads is that it provides an efficient separation of the bound and unbound phages during the selection procedure via the use of a magnetic stand addition to ease of work during the washing steps.

2.6.2. Linear and Cyclic Peptide Ligands Binding to Bacterial Cell Wall of S. aureus

Linear and cyclic peptide ligands binding to enantiomer of pentapeptide precursor of bacterial cell wall of S. aureus (Figure 10) were identified using commercially available three phage display random peptide libraries, a linear 12-mer, a linear 7-mer and a cyclic

7-mer purchased from New England Biolabs. These phage display libraries are constructed by fusing random peptide sequences to minor coat protein (pIII) of M13 filamentous phage. The displayed peptides are expressed at the N-terminus of pIII protein followed by a short spacer (Gly-Gly-Gly-Ser), and the first residue of the mature protein is the first randomized position (Table 5). Biopanning of each phage display library against each target molecule was carried out by applying solution-phase biopanning strategy where the biotinylated target molecules were first incubated with phage display library, and then immobilized on streptavidin-coated magnetic beads

(Figure 9). The unbound phage were washed away, and bound phage were eluted with low pH buffer, and amplified in host E. coli to use as input for the next round. After sufficient enrichments in favor of target molecules were gained typically in four to five rounds of biopanning, DNA of randomly picked phage clones were sequenced to reveal peptide sequences binding to target molecule.

54 The size of phage display library is one of the crucial parameters in the affinity selection.

All three commercially available libraries have the same diversities, order of 109. This diversity is sufficiently cover the all possible 7-mer peptide sequences, which is equal to

207 = 1.28 × 109 unique 7-mer peptide sequences for both cyclic and linear libraries. On the other hand, in linear 12-mer library only some fraction of possible sequences are covered, 2012 = 4.1 × 1015 possible sequences. Therefore, both libraries have equal diversity but spread over different length as seen in Table 5.

Before starting biopanning experiments, the number of phage clones in each library was determined in order to use 1011 phage as input from naïve library in the initial round according to blue/white plaque assay based on phage tittering method (Table 4). Phage vector in the each library carries the lacZ gene encoding galactosidase, and infected host

E. coli with phage library induce the production of galactosidase in the presence of isopropyl-p-D-thiogalactopyranoside (IPTG). After plating the infected bacteria on medium containing 5-Bromo-4-chloro-3-indolyl-p-D-galactoside (X-gal), galactosidase hydrolyzes the X-gal to yield blue-ink plaques. This allows counting of bacteria infected with phage library, and calculation of the number of phage as in plaque forming unit

(pfu/ml). Since wild-type M13 phage does not contain lacZ gene, it does produce clear plaques instead of blue ones on X-gal/IPTG media. This also allows distinguishing the enrichment of selected phage due to the selectivity for target molecule over an advantageous infectivity. Therefore, blue/white plaque assay is also used as control checking point in each round before and after amplification steps.

55

Table 4. Initial input of each naïve phage display library

Library Phage input (pfu/ml) Linear 12-mer 2.6 x 1013 Linear 7-mer 3.2 x 1013 Cyclic 7-mer 2.7 x 1013

Table 5. Phage display peptide libraries differ in size with the same complexity

Peptide Library Format Library name Library complexity

9 X12$GGGS Linear 12-mer 2.7 x 10

9 X7$GGGS Linear 7-mer 5.2 x 10

ACX7C$GGGS Cyclic 7-mer 3.7 x 109

56

O NH H O NH H H O NH S H NH 12 S O N O

Biotin-PEG12-Cephalosporin O OH EA-1-CEP

NH2

H S O NH NH O NH O O H NH O 4 NH NH OH O NH NH O O O O OH

Biotin-PEG4-D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Ala EA-2-ALA

NH2

H S O NH NH O NH O O H NH O 4 NH NH OH O NH NH O O O

OH O

Biotin-PEG4-D-Ala-L-α-Glu-D-Lys-L-Ala-L-Ala EA-3-ALA

Figure 10. The structure of target molecules used in the biopanning experiments; enantiomer of cephalosporin, EA-1-CEP, enantiomer of pentapeptide precursor of bacterial cell wall of S. aureus, EA-2-ALA, and also enantiomer of pentapeptide precursor of S. aureus with α-Glu instead of γ-Glu that provides extended backbone, EA- 3-ALA.

57 Enantiomer of cephalosporin, EA-1-CEP, was synthesized starting from commercially available 6-aminopenicillanic acid (6-APA) over 10 steps. Both enantiomers of pentapeptide precurcors of bacterial cell wall of S. aureus, EA-2-ALA and EA-3-ALA, were synthesized with standard in-situ neutralization protocol for t-Boc chemistry on 4- hydroxymethyl-phenylacetamidomethyl (PAM) resin. Three target molecules, EA-1-

CEP, EA-2-ALA, and EA-3-ALA, were also coupled with a PEG linker, either PEG4 or

PEG12, and biotin at N-terminus of each molecule. Biotin was used to utilize the strong interactions between streptavidin and biotin to immobilize the target molecule on streptavidin and neutravidin-coated magnetic beads, whereas PEG linker prevents the steric hindrance between the target molecule and biotin, and hence makes the target molecule accessible during the affinity selection for binding.

Tuning the binding interactions between the displayed peptide and target molecule is an important parameter to obtain peptide ligands having high binding affinity and speficificity for the target of interest. Therefore, two different affinity selection strategies were applied in the biopanning: the high yield selection and the more stringent selection.

If binding interactions between a ligand and its target are very tight, and target is presented in high concentration, biopanning results in the selection of tight binding ligands more difficult. On the other hand, if the binding interactions are weaker or moderate, the target concentration should be high enough to boost the chance of finding any interaction. Yield and stringency are two important tuning elements in the selection of the tightest-binders versus moderate-to-high affinity binders. To isolate tight-binders for a target molecule, the more stringent panning conditions are usually applied by decreasing concentration of target molecule in succescive rounds of biopanning,

58 decreasing incubation time between target molecule and phage display library, and increasing the percentage of detergent Tween-20 in the washing steps. The less amount of target molecule leads to a competition between high binding and weak binding ligands during the incubation between target molecule and phage display library. On the other hand, keeping the yield high in the initial round of selection by using high concentration of target molecule also provide selection of peptide ligands found rare in the phage display library. Therefore, utilizing of both biopanning selection strategies might give more information about the peptide ligands specifically bind to target molecule with a different degree of binding.

In the more stringent biopanning strategy, 25 nanograms (about 100 nM) of target molecules, EA-1-CEP, EA-2-ALA, and EA-3-ALA, in each round were subjected to three to five rounds of biopanning. Target molecule EA-3-ALA was used to investigate how the extended backbone of the bacterial cell wall precursor of S. aureus by incorporating of α-Glu instead of γ-Glu in the peptide backbone changes the results of selected peptide ligands. The stringency was also controlled by increasing the percentage of detergent and the washing time in successive rounds. While the washing buffer contains 0.1% Tween-20 in the initial round, it was increased to 0.5% in the successive rounds. As the selection rounds are carried out, phage with slower dissociation rate constants (koff) are usually selected resulting in improved a Kd value by modulating the stringency.

Before DNA sequencing of randomly picked phage clones at the third and the fifth rounds, the phage titers were determined via blue/white plaque assays (from Table 6 to

Table 14). The number of phage titers provides the information of whether or not

59 sufficient numbers of phage were being recovered during the selection process. In a typical biopanning, at least 104 phage clones are needed in order to obtain sufficient phage for further amplification process. Phage titers from the each biopanning showed that more phage were usually recovered after the fourth and fifth rounds of biopanning.

The peptide sequences identified from the more stringent selection are shown in Table 15

- 23 with the frequency in the randomly picked phage clones in each round, and sequence similarities are also highlighted with RasMol color code.

The results from target EA-3-ALA shows that the screening of linear 12-mer, linear 7- mer, and cyclic 7-mer libraries against the target did not yield any consensus sequences, rather repeating peptides were isolated. For instance, 12-mer peptide having sequence of

“SGVYKVAYDWQH” was observed in four randomly picked phage clone out of nine in biopanning of linear 12-mer library. On the other hand, one streptavidin-binding sequences, “HPQ”, were observed out of twelve phage clones after the fifth round.

While biopanning of linear 7-mer library yielded no repeating peptide after the third round, phage clones were enriched in favor of peptide with “SLDKRKK” sequence with additional two rounds. Three repeating peptide sequences were identified through cyclic

7-mer library, “GYSSFNR”, “RGATPMS” and “EGQRWMQ” after third round of biopanning, and after additional two rounds of selection, cyclic 7-mer library yielded dominantly phage clones having “HPQ” consensus sequence and one peptide having

“NWGDRIL”

In the biopanning against target EA-2-ALA, selection of cyclic peptide ligands mostly resulted in streptavidin-binding consensus sequence even though a subtractive selection was applied in the fourth round to eliminate those binders. On the other hand,

60 “MARYMSA” and “RGATPMS” were isolated once among 25 randomly picked phage clones, which were also observed in bioapping against EA-1-CEP and EA-3-ALA.

Also, repeating peptides having sequences of “ADRFQAL”, “PRLPRTR”,

“GKDYMGY”, “MMVLRNQ”, and “AHGRSRG” were isolated after the fourth and the fifth rounds of biopanning of linear 7-mer, and “RKVKRRPRVSNL”,

“ITGLGSGSSTST”, “SGVYKVAYDWQH” from the linear 12-mer library.

The biopanning of three phage display libraries against EA-1-CEP yielded the similar results with other two target molecules, such as, having several repeating peptide sequences instead of one dominant consensus sequences on each isolated peptide.

“RGATPMS” and “NTGSPYE” were repeating two cyclic peptides, while “GYSSFNR” and “SISSLTH” also were isolated once from biopanning of cyclic 7-mer library. More repeating peptides were isolated from the biopanning of linear 7-mer library;

“HYIDFRW”, “QLAVAPS”, “RTYPREK”, “KVKKRPD”, “GASESYL” and

“MIRGTTV”, and “HGGVRLY”.

According to overall results from three target molecules, two identical linear 12-mer peptides, “SGVYKVAYDWQH” and “DRWVARDPASIF”, and two cyclic 7-mer peptides, “MARYMSA” and “RGATPMS”, were isolated from all the biopanning against three different target molecules. Additionally, a linear 7-mer peptide, “HGGVRLY”, and two cyclic 7-mer peptides, “GYSSFNR” and “SISSLTH”, were identified from biopanning against EA-1-CEP and EA-3-ALA. Selection of two identical cyclic peptides from two different target molecules having different structure, one has more rigid and other has a flexible backbone, shows that the biopanning might provide peptide

61 ligands specifically binding to the enantiomer of D-Ala-D-Ala, which also will be further tested by phage ELISA.

Additionally, even though there is no dominant consensus sequences having three/four amino acid residues in the isolated peptides, the some of the identified peptide ligands of

EA-1-CEP, EA-2-ALA, and EA-3-ALA from all three phage libraries share the short motifs that having “G-Q/N-S/T”, “T/S-Q/L”, “ G-S/T”, “S/T-L/V” and “R-L/V”. This also indicates that the biopanning experiments might have resulted in the favor of the selection of target specific peptide ligands.

62

Table 6. Phage titers of target EA-1-CEP with linear 12-mer library

Rounds Input (pfu/mL) Output (pfu/mL)

1 1.0 x 1011 1.7 x 105 2 2.0 x 1011 4.0 x 104 3 1.2 x 1011 4.5 x 105 11 6 4 1.5 x 10 9.6 x 10 5 1.2 x 1011 1.7 x 107

Table 7. Phage titers of target EA-1-CEP with linear 7-mer library

Rounds Input (pfu/mL) Output (pfu/mL) 1 1.0 x 1011 1.5 x 105 11 4 2 1.8 x 10 9.0 x 10 3 2.6 x 1011 2.3 x 105 11 5 4 7.0 x 10 1.9 x 10 5 1.7 x 1011 9.0 x 104

Table 8. Phage titers of target EA-1-CEP with cyclic 7-mer library

Rounds Input (pfu/mL) Output (pfu/mL) 1 1.0 x 1011 6.0 x 104

2 2.1 x 1011 3.0 x 104 3 1.2 x 1011 2.3 x 105 4 2.1 x 1011 1.3 x 105 11 5 5 3.4 x 10 9.2 x 10

63

Table 9. Phage titers of target EA-2-ALA with linear 12-mer library

Rounds Input (pfu/mL) Output (pfu/mL)

1 1.0 x 1011 1.4 x 105 2 1.4 x 1011 3.1 x 105 3 1.5 x 1011 3.2 x 105 11 6 4 4.6 x 10 2.3 x 10 5 4.5 x 1011 5.9 x 107

Table 10. Phage titers of target EA-2-ALA with linear 7-mer library

Rounds Input (pfu/mL) Output (pfu/mL) 1 1.0 x 1011 2.1 x 105 11 4 2 1.8 x 10 8.0 x 10 3 2.6 x 1011 4.7 x 105 11 5 4 4.6 x 10 6.5 x 10 5 2.2 x 1011 2.0 x 107

Table 11. Phage titers of target EA-2-ALA with cyclic 7-mer library

Rounds Input (pfu/mL) Output (pfu/mL) 1 1.0 x 1011 1.5 x 105 2 1.8 x 1011 3.5 x 105 11 5 3 1.4 x 10 2.3 x 10 4 5.3 x 1011 9.6 x 106

5 7.4 x 1011 7.9 x 108

64

Table 12. Phage titers of target EA-3-ALA with linear 12-mer library

Rounds Input (pfu/mL) Output (pfu/mL)

1 1.0 x 1011 7.0 x 104 2 9.5 x 1011 3.0 x 105 3 2.0 x 1011 2.1 x 105 11 4 4 3.1 x 10 6.0 x 10 5 3.0 x 1011 2.4 x 105

Table 13. Phage titers of target EA-3-ALA with linear 7-mer library

Rounds Input (pfu/mL) Output (pfu/mL) 1 1.0 x 1011 6.5 x 105 11 5 2 2.5 x 10 6.7 x 10 3 1.7 x 1012 2.9 x 105 11 5 4 1.9 x 10 1.6 x 10 5 3.2 x 1011 1.0 x 106

Table 14. Phage titers of target EA-3-ALA with cyclic 7-mer library

Rounds Input (pfu/mL) Output (pfu/mL) 1 1.0 x 1011 4.2 x 105

2 1.8 x 1011 1.5 x 106 3 1.7 x 1011 2.5 x 106 4 1.4 x 1011 5.1 x 106 10 7 5 8.9 x 10 7.3 x 10

65

Round Peptide Sequence Abundance 4 A C R G A T P M S C 4 4 A C N T G S P Y E C 2 4 A C G Y S S F N R C 1 4 A C S I S S L T H C 1 4 A C H P V S G Q K C 1 4 A C H S D A N S I C 1 4 A C H N E G N R A C 1 4 A C V P I L E G T C 1 4 A C L W S T G A T C 1 4 A C L K L G E K W C 1 4 A C D K F H E L Q C 1 4 A C D G H D Q S L C 1

5 A C H P Q F L A L C 18

Table 15. Peptide sequences isolated after the fourth and fifth rounds of biopanning with a cyclic 7-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-1-CEP in each round.

66

Round Peptide Sequence Abundance 4 H G G V R L Y 1 4 N T V A N N Y 1 4 R P T A H M A 1 4 G N V G S V R 1 4 W S W G E Q K 1 4 H Y I D F R W 1 4 Q L A V A P S 1

5 H Y I D F R W 4

5 H G G V R L Y 2

5 R T Y P R E K 3 5 R H D I R K T 1 5 R P T A H M A 1 5 K V K K R P D 2 5 Q L A V A P S 2 5 G A S E S Y L 2 5 M I R G T T V 2 5 A V R G Y E W 1

Table 16. Peptide sequences isolated after the fourth and fifth rounds of biopanning with a linear 7-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-1-CEP in each round.

67

Round Peptide Sequence Abundance 4 D R W V A R D P A S I F 1 4 F S P T T W L I N H P Q 4 4 D H G L S W M R N H P Q 3

5 S G V Y K V A Y D W Q H 1 5 G Q S E H H M R V A S F 1 5 T Y T A N D L H L A D L 1 5 F S P T T W L I N H P Q 12 5 D H G L S W L R N H P Q 1

Table 17. Peptide sequences isolated after the fourth and fifth rounds of biopanning with a linear 12-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-1-CEP in each round.

68

Round Peptide Sequence Abundance 4 A C R G A T P M S C 1 4 A C M A R Y M S A C 1 4 A C M A P D S R V C 1 4 A C D N I M T P V C 1 4 A C S H M E Y P R C 1 4 A C S S P F P E F C 1 4 A C H P Q F L A L C 11 4 A C G S F V H P Q C 1

5 A C H P Q F L A L C 13 5 A C G S F V H P Q C 1

Table 18. Peptide sequences isolated after the fourth and fifth rounds of biopanning with a cyclic 7-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-2-ALA in each round.

69

Round Peptide Sequence Abundance 4 A D R F Q A L 5 4 P R L P R T R 4 4 G K D Y M G Y 3 4 G H R V R F P 1 4 M M V L R N Q 2 4 L I Q G T S L 1 4 V Y P G T S L 1

5 A H G R S R G 5 5 P R L P R T R 1 5 A D R F Q A L 3 5 G K D Y M G Y 3 5 M M V L R N Q 1 5 N T A V P L G 1 5 S S D V P Y L 1 5 V Y P G P S Y 1

Table 19. Peptide sequences isolated after the fourth and fifth rounds of biopanning with a linear 7-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-2-ALA in each round.

70

Round Peptide Sequence Abundance 4 R K V K R R P R V S N L 11 4 I T G L G S G S S T S T 8 4 S G V Y K V A Y D W Q H 3 4 Q C F N S V C L H T N P 1

5 I T G L G S G S S T S T 4 5 S G V Y K V A Y D W Q H 3 5 V G S N L R L L H Q W K 1 5 R K V K R R P R V S N L 1 5 D R W V A R D P A S I F 1 5 F S Y G W H P Q G D S T 2 5 D F A G A W L L A H P Q 2 5 F L W D Y K H P Q G D L 1

Table 20. Peptide sequences isolated after the fourth and fifth rounds of biopanning with a linear 12-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-2-ALA in each round.

71

Round Peptide Sequence Abundance 3 A C M A R Y M S A C 1 3 A C R G A T P M S C 2 3 A C E G Q R W M Q C 2 3 A C G Y S S F N R C 2 3 A C G Y K Q E S M C 1 3 A C G V G N A R V C 1 3 A C G T N E T K K C 1 3 A C Q G R N L T Q C 1 3 A C Q N P N Q K F C 1 3 A C E R F N Q G L C 1 3 A C P T F K R H I C 1 3 A C P M D F L N W C 1

5 A C N W G D R I L C 1 5 A C H P Q F L A L C 21

Table 21. Peptide sequences isolated after the third and fifth rounds of biopanning with a cyclic 7-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-3-ALA in each round.

72

Round Peptide Sequence Abundance 3 S S Q S M H Q 1 3 S L L G Q T P 1 3 Q S H S V F R 1 3 M Y V A P S R 1 3 M P A W H F I 1 3 T V I S Q N M 1 3 T P Y M T P K 1 3 Y H M P A L M 1 3 L Y A D S V L 1

5 S L D K R K K 12 5 S S N Q F H Q 1 5 Y E V M H P T 1 5 V E N V H V R 1 5 H G G V R L Y 1 5 W S L S E L H 1 5 Q Y V T P K W 1

Table 22. Peptide sequences isolated after the third and fifth rounds of biopanning with a linear 7-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-3-ALA in each round.

73

Round Peptide Sequence Abundance 3 S G V Y K V A Y D W Q H 4 3 S L C T K E L R H I A E 1 3 G L H T S A T N L Y L H 1 3 G S P L L D W L D T T P 1

5 S G V Y K V A Y D W Q H 9 5 D R W V A R D P A S I F 1 5 F K T H P Q F E T R V I 1

Table 23. Peptide sequences isolated after the third and fifth rounds of biopanning with a linear 12-mer random peptide library in the more stringent selection using 25 ng of target molecule EA-3-ALA in each round.

74 The high yield selection aims to capture as many phages as possible in the initial round of biopanning. It is important to keep the yield high by using high concentration of target molecule to avoid discarding phage found rare in the library or having moderate binding for target. Cysteine is a rare amino acid found in the library and, peptides that having cysteine are usually lost in biopanning selection with high stringent conditions. In the high yield selection, 5 mg of target molecules, EA-1-CEP and EA-2-ALA, were used in the first round, and were decreased to 2 mg, 500 ng, and 100 ng in the successive rounds.

While the stringency of biopanning also was modulated by decreasing the target concentration in the rounds, the stringency of the washing steps was kept as same as the more stringent selection strategy in order to eliminate phage clones binding to contaminant or other components of selection process.

During the high yield selection, negative selections as control checkpoint were also included, where the biopanning was performed only using magnetic beads without target molecules. Thereby, the enrichment factors were calculated in both selections and resulted that the biopannings were completed in favor of selection of target-specific phage clones not background. Enrichment factors were calculated as the number of phage recovered in the presence of target divided by phage recovered in the negative selection. An enrichment ratio greater than one is usually indication of phage binding specifically to the target protein, and phage are typically sequenced when the enrichment ratio has reached a maximum.

Not only streptavidin-coated magnetic beads, also neutravidin-coated magnetic beads were used in the second and fourth round of biopanning to prevent enrichment of

75

Table 24. Phage titers of target EA-1-CEP with linear 12-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 1.7 x 105 - NC-2 - 2.5 x 104 - 2 1.2 x 1011 2.9 x 106 116 NC-3 - 3.1 x 102 - 3 1.5 x 1011 1.3 x 105 419 NC-4 - 2.8 x 102 - 4 8.0 x 1011 4.2 x 105 1500

Table 25. Phage titers of target EA-1-CEP with linear 7-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 1.1 x 105 - NC-2 - 2.2 x 104 - 2 2.0 x 1010 6.5 x 105 30 NC-3 - 1.3 x 102 - 3 1.8 x 1011 8.0 x 104 615 NC-4 - 1.4 x 102 - 4 3.0 x 1011 1.8 x 105 1286

Table 26. Phage titers of target EA-1-CEP with cyclic 7-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 1.0 x 105 - NC-2 - 1.8 x 105 - 2 1.0 x 1011 4.8 x 106 27 NC-3 - 1.4 x 103 - 3 7.7 x 1011 8.2 x 105 586 NC-4 - 3.2 x 101 - 4 1.2 x 1011 1.4 x 104 438

76

Table 27. Phage titers of target EA-2-ALA with linear 12-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 2.0 x 104 - NC-2 - 1.9 x 103 - 2 2.0 x 1010 1.5 x 105 79 NC-3 - 2.2 x 102 - 3 1.7 x 1011 6.1 x 104 277 NC-4 - 2.6 x 102 - 4 7.0 x 10 11 6.5 x 105 2500

Table 28. Phage titers of target EA-2-ALA with linear 7-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 2.3 x 105 - NC-2 - 6.2 x 104 - 2 7.0 x 1010 1.2 x 105 2 NC-3 - 1.8 x 102 - 3 1.6 x 1011 4.0 x 104 222 NC-4 - 2.8 x 102 - 4 1.0 x 1011 3.0 x 105 1071

Table 29. Phage titers of target EA-2-ALA with cyclic 7-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 6.0 x 104 - NC-2 - 4.2 x 104 - 2 2.0 x 1010 2.0 x 105 0.24 NC-3 - 1.3 x 103 - 3 1.2 x 1011 2.2 x 104 20 NC-4 - 3.6 x 102 - 4 7.0 x 1011 1.5 x 105 417

77

Round Peptide Sequence Abundance 3 A C G V G P N R V C 1 3 A C F N M F S R V C 1 3 A C H N R V P L M C 1 3 A C R G A T P M S C 1 3 A C L W S T G A T C 1 3 A C L N S S Q P S C 1 3 A C L K L G E K W C 1 3 A C L H G D V A Y C 1 3 A C E D L T T L S C 1 3 A C S I S S L T H C 1 3 A C M S T G L S S C 1 3 A C S E H N L Q T C 1 3 A C G H S N L S N C 1 3 A C G D G S Q R T C 1 3 A C G Y S S F N R C 1 3 A C S G P G I N L C 1 3 A C S G W Q V R M C 1 3 A C T G K N A P K C 1 3 A C T L R D S P H C 1 3 A C T N T N T A I C 1 3 A C Q H L R G L L C 1 3 A C N T G S P Y E C 1 3 A C N T T E A A S C 1 3 A C Q Y E T P R Y C 1 3 A C N Q N A S H Y C 1 3 A C N E N I V H H C 1 3 A C N A K H H P R C 1 3 A C Q P R N L N N C 1 3 A C V P M Q D H T C 1 3 A C R I N P M S N C 1 3 A C A K S P M N C C 1 3 A C I A A R H M N C 1 3 A C D R T I S N K C 1 3 A C P F W L S G H C 1 3 A C D A M I G K S C 1 3 A C H Y N A H R T C 1

Table 30. Peptide sequences isolated after the third round of biopanning with a cyclic 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, 2 mg, and 500 ng, respectively.

78

Round Peptide Sequence Abundance 4 A C N T G S P Y E C 2 4 A C N R W H H L E C 1 4 A C N M Q I T K G C 1 4 A C N S F G V S M C 1 4 A C M S T G L S S C 1 4 A C K S M M R L N C 1 4 A C R S Q S G S N C 1 4 A C A S K S T H D C 1 4 A C G Y S S F N R C 1 4 A C T P G H T N R C 1 4 A C S T N S H S R C 1 4 A C A G H N R D R C 1 4 A C R G A T P M S C 1 4 A C E G Q R W M Q C 1 4 A C V N L Q K D M C 1 4 A C K T L Q P W T C 1 4 A C E D L T T L S C 1 4 A C P R D L G T D C 1

Table 31. Peptide sequences isolated after the fourth round of biopanning with a cyclic 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, 2 mg, 500 ng, and 100 ng respectively.

79

Round Peptide Sequence Abundance 3 T T L L T V S 5 3 A S L S K Y S 1 3 V T S P Y A F 3 3 K L S M Q H R 1 3 Q L K W Y H A 1 3 H L K H S L L 1 3 A P K P I K L 1 3 L V P S D K L 1 3 L P T G H F L 1 3 Y S G A S T L 1 3 D T A L H S L 1 3 V S R D T P Q 1 3 N S Y D V Q A 1 3 S E V Y P Q K 1 3 G F G Y N V Q 1 3 H V R H Y S D 1 3 D I S R M A T 1

Table 32. Peptide sequences isolated after the third round of biopanning with a linear 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, 2 mg, and 500 ng, respectively.

80

Round Peptide Sequence Abundance 4 T T L L T V S 12 4 S W T A L G P 1 4 H L F T T G V 1 4 H L K H S L L 1 4 H L N Q Q N H 1 4 G V H R E Q I 1 4 G Q S E K H L 1 4 A Q Y V A V G 1 4 I P F S F T G 1 4 L P V R L D W 3 4 W Q W P A R V 1 4 V T S P Y A F 2 4 Y N I S V N K 1 4 Y M L D S T M 1

Table 33. Peptide sequences isolated after the fourth round of biopanning with a linear 7-mer random peptide library in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, 2 mg, 500 ng, and 100 ng, respectively.

81

Round Peptide Sequence Abundance 3 S G V Y K V A Y D W Q H 2 3 R K V K R R P R V S N L 2 3 G Q S E H H M R V A S F 3 3 G S A P L L T V D T S K 1 3 S I N G L L S N S H G S 2 3 G V G L T T N V T R A G 1 3 G M L Q H A L V P K V W 1 3 A K D T H T G R M T N W 1 3 S A G N S A N W L L H W 1 3 V S N F T K P H K P K A 2 3 V P K S V D N T F Y T P 1 3 V F A R G Q Y D A P H P 1 3 L P S D Y R S R W A D P 1 3 K P G D T A M H Y F P P 1 3 Q F D Y M R P A N D T H 1 3 Q V D N T S S I E R L R 1 3 Q D I Q T T P P K F S V 1 3 S L D G A G A A L R T S 1 3 D L M P H K R M I E L R 1 3 I G R T V P H Q D F A R 1 3 F L Y P P N A S Y M S R 1 3 A D W Y H W R S H S S S 1 3 H T S S L W H L F R S T 1 3 M V S V L H S E S E S S 1 3 C P L F D S V R C T S K 1 3 F A P A L S N P P L R D 1

3 T Q A I D D I I I G R I 1 3 A T N A K R T P N T R I 1 3 W H P R Y V V S P L Q Y 1

Table 34. Peptide sequences isolated after the third round of biopanning with a linear 12-mer random peptide library in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, 2 mg, and 500 ng, respectively.

82

Round Peptide Sequence Abundance 4 V S N F T K P H K P K A 10 4 R K V K R R P R V S N L 3 4 S L D G A G A A L R T S 3 4 S G V Y K V A Y D W Q H 2 4 S N S I D K V N R P I N 1 4 G G R R C R I K N C Y A 1 4 G Q S E H H M R V A S F 1 4 E F H P M P D G F R S R 1 4 A T N A K R T P N T R I 1 4 A P I D K T L E R L S K 1 4 Y I G E M D T L P I S T 1

Table 35. Peptide sequences isolated after the fourth round of biopanning with a linear 12-mer random peptide library in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, 2 mg, 500 ng, and 100 ng, respectively.

83

Round Peptide Sequence Abundance 3 A C N T G S P Y E C 2 3 A C S I S S L T H C 2 3 A C G Y S S F N R C 1 3 A C R G A T P M S C 1 3 A C M G F S N M S C 1 3 A C S N H R I M S C 1 3 A C Y V S K N N S C 1 3 A C P V I S N G S C 1 3 A C D Q M W H T S C 1 3 A C S Q L P W Y S C 1 3 A C Q G N P S L R C 1 3 A C Q N W I S R F C 1 3 A C S E G L L N T C 1 3 A C I L L P D K L C 1 3 A C L K L G E K W C 1 3 A C K L T T Q M M C 1 3 A C L R T S N P A C 1 3 A C R S A N I Y T C 1 3 A C H D L N G S M C 1 3 A C D F I M G I T C 1 3 A C N P E H N N H C 1

Table 36. Peptide sequences isolated after the third round of biopanning with a cyclic 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, 2 mg, and 500 ng, respectively.

84

Round Peptide Sequence Abundance 4 A C R G A T P M S C 3 4 A C T N A N H Y F C 2 4 A C G Y S S F N R C 2 4 A C S I S S L T H C 2 4 A C T I L M K I L C 1 4 A C M S W S L Q R C 1 4 A C M E R M S L R C 1 4 A C M A R Y M S A C 1 4 A C V G M Q S N T C 1 4 A C E R Q R W M Q C 1 4 A C N F L Y S W T C 1 4 A C S D A R S P K C 1 4 A C T P S F S K I C 1 4 A C Y V S K N N S C 1

Table 37. Peptide sequences isolated after the fourth round of biopanning with a cyclic 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, 2 mg, 500 ng and 100 ng, respectively.

85

Round Peptide Sequence Abundance 3 A L S Y S R G 2 3 N I V S R E S 2 3 N I G Q D M H 1 3 V L T R C C G 1 3 K C C Y T L P 1 3 Y Q W E L Y S 1 3 Y P W W N T L 1 3 Y P W F I R A 1 3 F P A W F S A 1 3 F P I T Y D F 1 3 M P D M T R Q 1 3 W Q E H R D Q 1 3 G S F W H H N 1 3 G H Y I S A N 1 3 G G G H L S R 1 3 H M G K L N R 1 3 L M P S Y P R 1 3 L P G S E Q R 1 3 E T A L I A A 1 3 T W S L D Y P 1 3 S H G T W T P 1 3 S F V S M P E 1 3 T M Q N I P N 1 3 F S T T H P D 1 3 S T K T L P A 1 3 H V M T K A L 1 3 S L I A H Y Q 1 3 S T V K Y I D 1 3 S Q N F V R E 1

Table 38. Peptide sequences isolated after the third round of biopanning with a linear 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, 2 mg, and 500 ng, respectively.

86

Round Peptide Sequence Abundance 4 M A P T H S I 1 4 Q Q T N W S L 1 4 W S L S E L H 1 4 W D P R V N V 1 4 T G F L V N V 1 4 T T Q V L E A 1 4 T S Q Y L M I 1 4 V S R D T P Q 1 4 H Y I D F R W 1 4 L P V R L D W 1 4 G Q S E K H L 1 4 A L Q P Q K H 1 4 Y G G A A L Q 1 4 V S R A N E G 1 4 S A A W N K S 1 4 N D L M N R A 1

Table 39. Peptide sequences isolated after the fourth round of biopanning with a linear 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, 2 mg, 500 ng, and 100 ng respectively.

87

Round Peptide Sequence Abundance 3 V E A K C C F S M H K T 15 3 G T G L V T L P R L T V 2 3 G G G L G S L H E T S M 1 3 G L V N L M R P W H L L 1 3 G T T W V A T A G K L I 1 3 H H H F V T H P A W V L 1 3 T T V D Y F R K V W V V 1 3 S P D V T R W P Y W V I 1 3 S G V Y K V A Y D W Q H 1 3 T G V Y W T Q L N A D S 1 3 S S S V T P V S A L H G 1 3 N G Y H P P G F L A P E 1 3 Q G R I D L Y G F L S H 1 3 Q L A T L H K L S G P T 1 3 Q I T S S H V W D M G H 1 3 V H W D F R Q W W Q P S 2 3 A N V K A F F S T T Q V 1 3 A S P V N Y R D M F S R 1 3 A T W A R V D A I A R A 1 3 A P F P S V A L K V P L 1 3 F G R M A W T P M A P M 1 3 F H F P L G M H S R D E 1 3 Y A Q V Y S N H G S R I 1 3 H K T D L W M T N T I K 1 3 C L N D S L Y C Q G M P 1 3 L E T R L G T S P G H T 1 3 S F P H F T L R A Y A S 1 3 E G N W F L S F H A S T 1

Table 40. Peptide sequences isolated after the third round of biopanning with a linear 12- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, 2 mg, and 500 ng, respectively.

88

Round Peptide Sequence Abundance 4 V E A K C C F S M H K T 13 4 S S S V T P V S A L H G 3 4 Q L A T L H K L S G P T 1 4 Y S L R S D F L P F A T 1 4 S W F S D W D L E L H A 1 4 G D V S D V W T A A N N 1

Table 41. Peptide sequences isolated after the fourth round of biopanning with a linear 12-mer random peptide library in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, 2 mg, 500 ng and 100 ng, respectively.

89 streptavidin binding peptides. Since the more stringent selection yielded the peptides having “HPQ” motif even with an additional subtractive selection, no such peptide sequences were observed the high yield selection due to changing the magnetic beads from streptavidin to neutravidin in the successive rounds.

According to overall DNA sequence analysis of randomly picked phage clones, the biopannings yielded both the similar short motifs for each target molecule, and also repeating peptide sequences. For instance, more diverse peptide sequences were obtained from the biopanning against target EA-2-ALA with linear 7-mer library and no repeating peptide sequence out of 42 phage clones were iisolated. “VEAKCCFSMHKT” is the dominant peptide isolated from biopanning of linear 12-mer library. And more interestingly, two linear 7-mer peptides, “KCCYTLP” and “VLTRCCG”, share the same motif, “K/R-C-C”, with that dominant linear 12-mer peptide. In the biopanning for target

EA-1-CEP, while affinity selection with the linear 7-mer and the linear 12-mer libraries were enriched in favor of repeating peptides, the isolated cyclic 7-mer peptides had more diverse sequences. Also, the similar short peptide motifs were observed at all the biopannings from both target molecules, for instance, “T-T-L”, “I/L-S/T”, “Q/N-S/T”,

“L-Q”, “G-Q-S”, “G-S”, “G-x-S”, “G-V”, “L-K/R”, “R-V”, “V-L-S”, “N/Q-K/R”.

Moreover, the same peptide sequences from the biopanning against targets EA-1-CEP and EA-2-ALA were isolated in two different selection strategies, the high yield selection and the more stringent selection. For example, “SGVYKVAYDWQH”,

“RKVKRRPRVSNL”, and “GQSEHHMRVASF” from linear 12-mer library,

“HYIDFRW” from linaer 7-mer library, and “NTGSPYE”, “EGQRWMQ”,

90 “GYSSFNR”, “RGATPMS”, “SISSLTH”, “MARYMSA”, and “LKLGEKW” from cyclic 7-mer library.

The major drawback of phage display screening is the selection of false positive and false negative peptide as binders. During the affinity selection, phage library can also bind to the other components of screening system, such as solid support, capturing reagent, contaminants in the target solution, blocking agents as false negative or target-unrelated peptides in the library as false positive. Table 42 shows the common false negative peptide sequences identified from several research groups during biopanning against different target molecules.273-279 None of the repeating peptide sequences observed in the high yield biopanning selection showed similarity to these false negative peptide sequences, whereas only streptavidin-binder “HPQ” was observed in the more stringent selection. Although phage display libraries identify peptide ligands of their specific targets efficiently and rapidly, also false positive binders may appear as repeating peptide sequences. These false positive binders can be recovered due to their faster propagation rates independent from their affinity to target molecule, which is called as target- unrelated peptides. For instance, in M13-based phage libraries, proline is overrepresented while cysteine is underrepresented, and this phenomena is called as sequence-bias and might leads to selection of peptide sequences independent from their affinity.280 Target-unrelated peptides selected during commercially available phage display random peptide libraries can be identified through SAROTUP (Scanner and

Reporter of Target-unrelated Peptide) database. None of peptides identified in both selection methods is found in SAROTUP database, which also shows that these peptides are not target-unrelated peptides.

91

Table 42. Known consensus sequences of target-unrelated peptides

TUP Category TUP Motif

HPQ EPDW(F/Y) Streptavidin binder DVEAW(L/I) GD(F/W)XF PWXWL Neutravidin binder SVP(W/F)(S/T)V VW(R/Q)G Biotin binder WXPPF(K/R) FHQNWPS NRPLYE Bovine Serum Albumin binder HFWNRPL WXXW FHWTWYW Polystyrene binder RAFIASPRIKRP FKFWLYEHVIRG Faster propogation rate HAIYPRH

92 2.6.3. Binding Confirmation of Selected Peptides via Phage-ELISA

To test whether or not the selected phage clones could bind specifically the biotinylated target molecules, phage-ELISA assays were preformed. Once randomly picked phage clones were recovered, the peptide sequence of each phage clone was revelaed by DNA sequencing. After sequencing of each phage clone, either selected phage yielded a short motif appears in some of peptide sequences or more than one copy of peptide ligands were isolated either in the biopanning of the same target or of both target molecules. The binding specificities of phage clones from both selection strategies were confirmed by sandwich phage-ELISA (Figure 11). While all of the phage clones were tested from the more stringent selections, only the phage having the repeating peptide sequences were subjected to phage-ELISA.

Phage-ELISA after biopanning was used to distinguished false true binders from target- specific binding peptides. Briefly, the biotinylated target molecules were immobilized onto the streptavidin-coated 96-well plates. The antibody, horseradish peroxidase conjugated to mouse anti-M13 monoclonal antibody, was then incubated within the wells containing target molecules. This antibody specifically reacts with bacteriophage M13 major coat protein pVIII. After addition of substrate of antibody, ABTS, a green color was produced upon binding and that also read at absorbance of 405 nm. Additionally, the wells coated with streptavidin, blocking agent BSA, and blank wells were used as negative controls. Positive clones containing recombinant phage captured by the antigen produced a visible green color while negative control wells remain nearly colorless.

93

ABTS ABTS-red ABTS ABTS-red ABTS ABTS-red

P P P R R R H H H

Plastic well bottom

Streptavidin

Biotinylated target molecule

P R H Anti-M13 antibody conjugated with HRP

ABTS Substrate for antibody

Figure 11. The schematic representation of sandwich phage-ELISA, where streptavidin- coated wells were incubated with biotinylated target molecule, followed by incubation of the selected phage in the wells having target molecules. pVIII specific mouse anti-M13 monoclonal antibody coupled to horseradish preroxidase was incubated with each well, and then ABTS substrate of the antibody. Reaction of HRP with ABTS substrate produces a green color at absorbance of 405 nm that showing specific binding to target molecule.

94

Target Streptavidin BSA EA-1-CEP Plastic 1.4

1.2

1

0.8

0.6 Absorbance @ 405 nm 0.4

0.2

0 SISSLTH VPILEGT RHDIRKT HSDANSI MIRGTTV HYIDFRW DKFHELQ RTYPREK NTVANNY GASESYL QLAVAPS GYSSFNR KVKKRPD NTGSPYE LWSTGAT RPTAHMA DGHDQSL HGGVRLY HNEGNRA RGATPMS LKLGEKW HPVSGQK GNVGSVR AVRGYEW WSWGEQK TYTANDLHLADL DRWVARDPASIF GQSEHHMRVASF SGVYKVAYDWQH Peptide Sequences

Figure 12. Phage-ELISA results of selected phage cloned from biopanning of target EA- 1-CEP after the more stringent selection with all three libraries, linear 12-mer, linear 7- mer and cyclic 7-mer.

95

1.6 Target Streptavidin BSA 1.4 EA-2-ALA Plastic

1.2

1

0.8

0.6 Absorbance @ 405 nm

0.4

0.2

0 LIQGTSL SSPFPEF SSDVPYL GHRVRFP PRLPRTR DNIMTPV NTAVPLG VYPGPSY ADRFQAL SHMEYPR AHGRSRG GKDYMGY RGATPMS MAPDSRV MMVLRNQ MARYMSA ITGLGSGSSTST QCFNSVCLHTNP VGSNLRLLHQWK RKVKRRPRVSNL Peptide Sequences DRWVARDPASIF HRDPHSALTRSW SGVYKVAYDWQH

Figure 13. Phage-ELISA results of selected phage cloned from biopanning of target EA- 2-ALA after the more stringent selection with all three libraries, linear 12-mer, linear 7- mer and cyclic 7-mer.

96

EA-3-ALA Target 1.4 Streptavidin BSA Plastic 1.2

1 nm

0.8 405 @ 0.6

0.4 Absorbance Absorbance

0.2

0 SISSLTH SLDKRKK NTGSPYE GYSSFNR HGGVRLY NWGDRIL YEVMHPT RGATPMS MYVAPSR MARYMSA DRWVARDPASIF SGVYKVAYDWQH Peptide Sequences

Figure 14. Phage-ELISA results of selected phage cloned from biopanning of target EA- 3-ALA after the more stringent selection with all three libraries, linear 12-mer, linear 7- mer and cyclic 7-mer.

97 Phage-ELISA results from the more stringent biopanning showed that the isolated peptides have highly specific to their target molecules. The peptide sequences selected from the biopanning against three different target molecules, linear 12-mer

“SGVYKVAYDWQH”, “DRWVARDPASIF”, cyclic 7-mer, “MARYMSA”,

“RGATPMS”, “GYSSFNR” “SISSLTH”, and linear 7-mer “HGGVRLY”, have similar binding interactions with each target molecule. Also, one streptavidin-binding peptide was evaluated in phage-ELISA experiments as control (not shown in the graphs), and that peptide only showed specificity for the well just having streptavidin not the target molecules. As a result, 40 peptides were synthesized in D-form (composed of D amino acids) for further antimicrobial activity studies from the more stringent selection experiments.

Also, phage-ELISA was carried out after the high yield biopanning selection for EA-1-

CEP and EA-2-ALA. Phage clones represented more than one time after the fourth round of biopanning were tested for their binding specificities. All the tested phage clones showed the binding specificity to the target molecules from moderate to high rather than negative controls.

For EA-2-ALA, three peptides having “K/R-C-C” motif, VEAKCCFSMHKT,

KCCYTLP and VLTRCCG, showed the higher ELISA signals, hence they have higher binding affinity for EA-2-ALA. This ELISA results also show that this short motif specifically binds to target. On the other hand, a linear 12-mer peptide,

VSNFTKPHKPKA, had the highest ELISA signal for EA-1-CEP, which is also the most abundant peptide isolated from biopanning of linear 12-mer library.

98

1.6 EA-1-CEP

1.4 Target Streptavidin

1.2 BSA Plastic nm 1 405

@ 0.8

0.6 Absorbance Absorbance 0.4

0.2

0 EDLTTLS HLFTTGV TTLLTVS VTSPYAF LPVRLDW VNLQKDM KTLQPWT

Peptide Sequences VSNFTKPHKPKA SLDGAGAALRTS

Figure 15. Phage-ELISA results of selected phage cloned from biopanning of target EA- 1-CEP after the high yield selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer.

99

1.6 EA-2-ALA Target Streptavidin BSA 1.4 Plastic

1.2

nm 1 405 0.8 @

0.6

0.4 Absorbance Absorbance

0.2

0 ILLPDKL NIVSRES TGFLVNV KCCYTLP VLTRCCG YPWFIRA TTQVLEA LPVRLDW ALSYSRG ALQPQKH VSRDTPQ TNANHYF KLTTQMM EGQRWMQ GTGLVTLPRLTV SSSVPPVSALHG VEAKCCFSMHKT

VHWDFRQWWQPS Peptide Sequences

Figure 16. Phage-ELISA results of selected phage cloned from biopanning of target EA- 2-ALA after the high yield selection with all three libraries, linear 12-mer, linear 7-mer and cyclic 7-mer.

100 2.6.4. Bicyclic Peptide Ligands of Bacterial Cell Wall Precursor of S. aureus through

Phage-Coded Peptide Libraries

Bicyclic peptide ligands of target EA-1-CEP and EA-2-ALA also were explored by utilizing of the phage-coded bicyclic peptide libraries. Two bicyclic phage display library constructs of Cys-(Xaa)m-Cys-(Xaa)n-Cys (where Xaa represents random amino acids, m and n = 3, 4, 5 or 6) with different loop lengths, Library A (m and n = 3-5) and

Library B (m and n = 3-6) were obtained from the Heinis Laboratory at EPFL Lausanne.

Although, the diversities of these bicyclic libraries are relatively small compared to other phage libraries used, and mostly on order of 108, these conformationally constrained peptide libraries are likely to suffer a much smaller loss in entropy upon binding of target, and therefore have the potential to provide peptide ligands with tight binding affinities

Table 43.

Compared to traditional phage display selection, bicyclic peptide libraries were subjected to additional steps to generate phage-coded bicyclic peptide libraries from the library construction in the beginning of each round. Figure 17 shows the schematic representation of the selection cycle of bicyclic peptides through biopanning: amplification of phage library, reduction of cysteines with TCEP, generation of bicyclic phage library with TBMB modification, and regular solution-phase biopanning. Phage libraries were constructed with three free cysteine residues, and bicyclic phage libraries were generated by TBMB modification after reduction of possible disulfide bonds with

TCEP formed in the peptide sequences prior to biopanning screening. There is usually a less than one order of magnitude loss in phage titers during the generation of bicyclic libraries

101

Table 43. Phage-coded bicyclic peptide libraries used in the biopanning experiments

Peptide Library Format Library name Library size

ACX CX C$GGSG m n Bicyclic Library A, m, n= 3-5 5.0 x 108

Bicyclic Library B, m, n= 3-6 1.0 x 107 ACXmCXnC$GGSG

Table 44. Library A and Library B with different loop lengths

Library name Loop length A 3 x 4 4 x 3

4 x 4

3 x 5

5 x 3

B 3 x 6 6 x 3

4 x 5

5 x 4

102 especially after PEG precipitation (due to lower recovery of large scale amplification process) and the TBMB modification steps (phage titer data is not shown here)

Also, negative selections were carried out in each round by using just streptavidin-coated magnetic beads in the first round and neutravidin-coated magnetic beads in the second round without any target molecules to quantify the number of phage clones that bound specifically to target molecule. In the second round sufficient enrichments in favor of target molecules were achieved compared to solely magnetic beads, therefore DNA of randomly picked phage clones were sequenced to reveal the peptide sequences without further rounds of biopanning (Table 45).

Biopanning experiments were performed by using the high yield selection strategy, where

5 mg of target molecule was used in the first round and was decreased to 2 mg in the second round. Table 50 and Table 51 show the identified peptides from the biopanning of Library A and Library B against target EA-2-ALA. While some of the bicyclic peptides with different loop lengths were isolated more than once in each selection, the same short peptide motifs, “T-T-S”, “Q-L”, “Q-R/K”, “S-Q-L”, “V-S-Q-L”, “G-Q-V”,

“S-V”, were also observed. The similar results were obtained from the target EA-1-CEP

(Table 52 and Table 53) where short peptide motifs, “G-Q-V”, “Q-L”, “S-S-V”, “T/P-Q”,

“Y-Q-S/L”, “Q-R-L” were abundant in addition to the repeating bicyclic peptides. On the other hand, these short peptide motifs are similar to those obtained from commercially available phage display peptide libraries, T-T-L”, “I/L-S/T”, “Q/N-S/T”,

“L-Q”, “G-Q-S”, “G-S”, “G-x-S”, “G-V”, “L-K/R”, “R-V”, “V-L-S”, “N/Q-K/R”.

103 Overall biopanning results of these short peptide motifs from both commercially available linear and mono-cyclic phage display libraries and bicyclic phage display libraries indicates that identified peptides specifically bind to target molecules EA-1-

CEP and EA-2-ALA addition to the phage-ELISA results.

Generation of each selected bicyclic phage in small quantity would yield less than the required amount due to loss of phage titers during the TBMB modification, and PEG purification. Therefore, phage-ELISA was not carried out for bicyclic phage display biopanning. Instead, bicyclic peptides for further studies were chosen according to their structural similarities within the linear and cyclic peptides from the previous section.

104

Table 45. Selected bicyclic peptides for EA-1-CEP and EA-2-ALA

Peptide Name Peptide Sequence

EA-BC1-84 ACREVTCHHLQ

EA-BC1-85 ACQTDVCQRTI

EA-BC2-86 ACYESCRVQSAL

EA-BC2-87 ACETRGCYQRFR

EA-BC2-88 ACDQGDCHQKIN

EA-BC1-89 ACGQVCNQKV

EA-BC1-90 ACYAQRCGVTG

EA-BC2-91 ACFKQNCSQSRS

EA-BC2-92 ACIPIKRCNDQL

EA-BC2-93 ACIEQPACPNIF

EA-BC1-94 ACSTLDRCYQL

105

Reducon of Cysteines Cyclizaon with TBMB

S Br Br SH HS S HS SH

Br

2-3 Rounds of Phage Selecon Affinity Selecon via Biopanning

Amplificaon of Phage Library DNA sequencing

Synthesis of Bicyclic Pepdes

Characteizaon of Bicyclic Pepdes

Figure 17. Schematic representation of biopanning experiments with bicyclic phage display peptide libraries. First, phage library is produced. Then partially oxidized cysteines are reduced by TCEP reduction, and bicyclized with TBMB modification. Phage-coded bicyclic phage library is subject to biopanning experiments for 2-3 rounds. DNA of isolated phage is sequenced, and translated into peptide sequences. Further characterization of bicyclic peptides is done after chemical synthesis.

106

Table 46. Phage titers of EA-1-CEP and negative controls after each round of biopanning with Library A and enrichment factors

Round 1 Round 2 Enrichment factor

Control (No target) 2.8 x 104 1.6 x 104 -

4 7 EA-1-CEP 1.9 x 10 3.6 x 10 1.9 x 103

Table 47. Phage titers of EA-2-ALA and negative controls after each round of biopanning with Library A and enrichment factors

Round 1 Round 2 Enrichment factor

Control (No target) 1.8 x 103 1.1 x 102 -

EA-2-ALA 4.2 x 104 3.0 x 105 2.5 x 103

107

Table 48. Phage titers of EA-1-CEP and negative controls after each round of biopanning with Library B and enrichment factors

Round 1 Round 2 Enrichment factor

Control (No target) 1.30 x 104 3.50 x 103 -

5 6 EA-1-CEP 2.75 x 10 1.25 x 10 3.6 x 102

Table 49. Phage titers of EA-2-ALA and negative controls after each round of biopanning with Library A and enrichment factors

Round 1 Round 2 Enrichment factor

Control (No target) 2.6 x 104 1.10 x 103

3 EA-2-ALA 2.0 x 105 1.75 x 105 1.6 x 10

108

Library Loop length Peptide Sequence Abundance A 3 X 5 A C P E S C L D L Q W C 1 A 3 X 5 A C T T S C V K S S I C 1 A 3 X 5 A C F Q L C P S V D F C 1

A 4 X 4 A C R E V T C H H L Q C 3 A 4 X 4 A C R P Q E C A Q H V C 1 A 4 X 4 A C Q T D V C Q R T I C 2

A 4 X 3 A C Q D G Q C P R N C 1

Table 50. Peptide sequences isolated after the second round of biopanning of bicyclic phage display peptide library A, Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3-5, in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, and 2 mg, respectively.

109 Library Loop length Peptide Sequence Abundance B 4 X 5 A C L S S G C S A Q D L C 1 B 4 X 5 A C Q S L E C A M R A H C 1 B 4 X 5 A C R Q A Y C S N L L L C 1 B 4 X 5 A C P M A L C S Q G A T C 1 B 4 X 5 A C E T R G C Y Q R F R C 2 B 4 X 5 A C Q I S H C Q N M I I C 1 B 4 X 5 A C P T S A C M Q Q S G C 1 B 4 X 5 A C V Y T S C V Q S L T C 1 B 4 X 5 A C V Q L T C E Y L Y A C 1 B 4 X 5 A C D Q G D C H Q K I N C 2 B 4 X 5 A C D Q E L C R E L T S C 1 B 4 X 5 A C Q L I S C T G G L Q C 1 B 4 X 5 A C L T Q P C N N P R P C 1 B 4 X 5 A C S M G M C A L P W Q C 1 B 4 X 5 A C T L E I C R S Q L G C 1

B 5 X 4 A C V G Q E P C L S Y T C 1 B 5 X 4 A C Q L I N L C H D F L C 1 B 5 X 4 A C S L I T Q C G G V G C 1 B 5 X 4 A C Q Q F G Q C S Q F S C 1 B 5 X 4 A C P Q P Q P C L R T S C 1 B 5 X 4 A C Q S Q D H C F H K D C 1 B 5 X 4 A C S K Q H T C V S P V C 1 B 5 X 4 A C V P S Q W C Y A Q R C 1 B 5 X 4 A C P P Q R R C T A F A C 1 B 5 X 4 A C S S R E Q C M I T V C 1 B 5 X 4 A C Y H V R P C S S Q L C 1 B 5 X 4 A C Q S P S L C M G L P C 1 B 5 X 4 A C Q A G V I C L Q Q V C 1

B 3 X 6 A C S Y L C E P A Q H V C 1 B 3 X 6 A C Q L L C V Q S S S E C 1 B 3 X 6 A C Y E S C R V Q S A L C 2 B 3 X 6 A C S H Q C R S S E L L C 1 B 3 X 6 A C I S K C T S V A Q S C 1

B 6 X 3 A C N D V T K L C S Q F C 1 B 6 X 3 A C Y N Q R S S C A M S C 1 B 6 X 3 A C N Q R P P F C L V R C 1 Table 51. Peptide sequences isolated after the second round of biopanning of bicyclic phage display peptide library B, in the high yield selection using decreasing amount of target molecule EA-2-ALA in each round, 5 mg, and 2 mg, respectively.

110

Library Loop length Peptide Sequence Abundance A 3 X 4 A C G Q V C N Q K V C 4

A 3 X 4 A C Q A V C Q L G P C 3

A 3 X 4 A C V N S C S S L K C 1

A 3 X 4 A C A R A C Q F G A C 1

A 3 X 4 A C D H Q C G D H L C 1

A 3 X 4 A C T Q Q C P S S V C 1

A 3 X 4 A C W R S C P K G Y C 1

A 3 X 4 A C R A A C N P F I C 1

A 4 X 4 A C Y A Q R C G V T G C 3 A 4 X 4 A C A E Q S C I F N L C 1 A 4 X 4 A C Y Q S P C P S G L C 1

A 4 X 3 A C W E Q A C S Q E C 1

A 4 X 3 A C S Q S N C V K A C 1

A 3 X 5 A C M R A C V M Q F D C 1 A 3 X 5 A C D V P C V A Q Y I C 1 A 3 X 5 A C Y S G C G N L Q G C 1 A 3 X 5 A C T P L C T P Q H V C 1

A 5 X 3 A C S T L D R C Y Q L C 1 A 5 X 3 A C Q Q R Y S C F T N C 1 A 5 X 3 A C Q P T P T C G W T C 1

Table 52. Peptide sequences isolated after the second round of biopanning of bicyclic phage display peptide library A, Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3-5, in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, and 2 mg, respectively.

111

Library Loop length Peptide Sequence Abundance B 4 X 5 A C F K Q N C S Q S R S C 2 B 4 X 5 A C L T A H C P Q S I S C 1 B 4 X 5 A C N L S P C L L P P Q C 1 B 4 X 5 A C P V P Q C D P K K L C 1 B 4 X 5 A C S W Y K C F N Q P S C 1 B 4 X 5 A C T V P T C S Q S L R C 1 B 4 X 5 A C Q L N M C T S A N N C 1 B 4 X 5 A C Q P R H C I H S T V C 1 B 4 X 5 A C V N S L C T L P S Q C 1 B 4 X 5 A C R S T P C Q N Q L E C 1 B 4 X 5 A C Q S F S C G Q R L S C 1 B 4 X 5 A C Q S P W C Q R L D L C 1 B A C V W Q G C A L N W R C 1

B 5 X 4 A C I P I K R C N D Q L C 2 B 5 X 4 A C I E Q P A C P N I F C 2 B 5 X 4 A C P T M T V C Q H P R C 1 B 5 X 4 A C I S R V G C Q N P M C 1 B 5 X 4 A C A L L I D C Q Y P L C 1 B 5 X 4 A C Q L Q L F C Q T R T C 1 B 5 X 4 A C Q P S T S C L I Q R C 1

B 3 X 6 A C Q H P C K S T V P N C 1 B 3 X 6 A C L R N C D Y V Q P P C 1 B 3 X 6 A C P D T C Q A A F F L C 1 B 3 X 6 A C T L N C N S G F Q R C 1 B 3 X 6 A C N L S C T S Q T L E C 1

B 6 X 3 A C I S L Q Q L C I R A C 1

Table 53. Peptide sequences isolated after the second round of biopanning of bicyclic phage display peptide library B, Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3-6, in the high yield selection using decreasing amount of target molecule EA-1-CEP in each round, 5 mg, and 2 mg, respectively.

112 2.6.5. Antibacterial Activity Assays of Synthesized D-peptides

The enantiomers (D-peptides) of identified peptides from the biopanning screenings were synthesized manually with standard Fmoc-chemistry on Rink-amide resin to have C- terminus amide and a short linker at C-terminus, Gly-Gly-Gly-Ser for linear and cyclic peptides derived from commercially available libraries and Gly-Gly-Ser-Gly for bicyclic peptide libraries for further studies.281

Minimum inhibitory concentrations (MIC) are considered the “gold standard” for determining the susceptibility of bacteria to antimicrobials, and are therefore used to judge the performance of all other methods of susceptibility testing. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent, to give a definitive answer when a borderline result is obtained by other methods of testing, and also to monitor the activity of new antimicrobial agents.

Antibiotic susceptibility of synthesized D-peptides on several Gram-positive and Gram- negative strains were tested according to broth micro-dilution protocol described by

NCCLS with the slight modifications done by Wiegan et al for specifically peptide antibiotics.282-286 Briefly, in broth micro-dilution method, bacteria are inoculated into a liquid growth medium in the presence of increasing two-fold concentrations of D- peptides. After 16-20 h at 37 °C, the bacterial growth was assessed by visual inspection of turbidity in each well and by measuring the absorbance of each well at 600 nm using a plate reader. MIC was defined as the lowest concentration in µg/ml of antimicrobial agent that prevents visible growth of a microorganism under defined conditions. Each

113 test was conducted in triplicate in three different days to ensure the reproducibility of the results, and two known antibiotics, vancomycin and melittin, were also used as control antibiotics at each time.

MIC determination is the one of the first steps to evaluate the antimicrobial potential of promising new compounds. However, MIC values do not represent an absolute value.

The “true” MIC is somewhere between the lowest test concentration that inhibits the organism's growth (that is, the MIC reading) and the next lower test concentration. For instance, the two-fold dilutions yielded a MIC value of 16 µg/mL, the “true” MIC would be between 16 and 8 µg/mL.

The MIC values of unoptimized antibacterials from target-based screenings are up to 16-

32 µg/mL have been suggested to be sufficient for primary hit compounds. Thus, MIC values of ≤ 32 µg/mL were considered as high reactivity, while = 64 - 256 µg/mL as moderate reactivity and ≥ 256 µg/mL no antibacterial activity for the tested bacterial strains.

Some of D-peptides (linear 12-mer, cyclic 7-mer, and bicyclic) showed higher activity,

MIC values in the range 8 – 32 µg/mL, some of them had moderate activity, MIC values of 64-128 µg/mL, and some showed no antibacterial activity, that is MIC value of ≥

256 µg/mL, and only against Gram-positive strains as we expected. The most potent D- peptide on S. aures and MRSA, was bicyclic peptide EA-BC1-85 isolated from the biopanning of EA-2-ALA, having MIC value of 8 µg/mL on both strains. Not only those two strains, this bicyclic peptide was also showed high antibacterial activity on E. faecalis.

114

Table 54. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

Vancomycin >256 0.5 0.5 0.5 1 >256 32

Melittin 32 2 2 4 8 32 32

EA-L12-01 >256 >256 >256 >256 >256 >256 >256

EA-L12-02 >256 >256 >256 >256 >256 >256 >256

EA-L12-03 >256 >256 >256 >256 >256 >256 >256

EA-L12-04 >256 32 32 64 32 >256 >256

EA-L12-05 >256 >256 >256 >256 >256 >256 >256

EA-L12-06 >256 >256 >256 >256 >256 >256 >256

EA-L12-07 >256 >256 >256 >256 >256 >256 >256

EA-L12-08 >256 >256 >256 >256 >256 >256 >256

EA-L12-09 >256 >256 >256 >256 >256 >256 >256

EA-L12-10 >256 >256 >256 >256 >256 >256 >256

115

Table 54 cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-L12-11 >256 >256 >256 >256 >256 >256 >256

EA-L12-12 >256 >256 >256 >256 >256 >256 >256

EA-L12-13 >256 32 32 64 32 >256 128

EA-L12-14 >256 >256 >256 >256 >256 >256 >256

EA-L7-15 >256 >256 >256 >256 >256 >256 >256

EA-L7-16 >256 >256 >256 >256 >256 >256 >256

EA-L7-17 >256 >256 >256 >256 >256 >256 >256

EA-L7-18 >256 128 128 >256 >256 >256 >256

EA-L7-19 >256 128 128 >256 >256 >256 >256

EA-L7-20 >256 >256 >256 >256 >256 >256 >256

EA-L7-21 >256 >256 >256 >256 >256 >256 >256

EA-L7-22 >256 32 64 >256 >256 >256 >256

116 Table 54 cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-L7-23 >256 >256 >256 >256 >256 >256 >256

EA-L7-24 >256 >256 >256 >256 >256 >256 >256

EA-L7-25 >256 >256 >256 >256 >256 >256 >256

EA-L7-26 >256 >256 >256 >256 >256 >256 >256

EA-L7-27 >256 >256 >256 >256 >256 >256 >256

EA-L7-28 >256 >256 >256 >256 >256 >256 >256

EA-L7-29 >256 >256 >256 >256 >256 >256 >256

EA-L7-30 >256 >256 >256 >256 >256 >256 >256

EA-L7-31 >256 >256 >256 >256 >256 >256 >256

EA-L7-32 >256 >256 >256 >256 >256 >256 >256

EA-L7-33 >256 >256 >256 >256 >256 >256 >256

EA-L7-34 >256 32 32 64 64 >256 >256

117

Table 54 cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-L7-35 >256 >256 >256 >256 >256 >256 >256

EA-L7-36 >256 >256 >256 >256 >256 >256 >256

EA-L7-37 >256 >256 >256 >256 >256 >256 >256

EA-L7-38 >256 >256 >256 >256 >256 >256 >256

EA-L7-39 >256 >256 >256 >256 >256 >256 >256

EA-L7-40 >256 >256 >256 >256 >256 >256 >256

EA-L7-41 >256 >256 >256 >256 >256 >256 >256

EA-L7-42 >256 >256 >256 >256 >256 >256 >256

EA-L7-43 >256 >256 >256 >256 >256 >256 >256

EA-L7-44 >256 >256 >256 >256 >256 >256 >256

EA-L7-45 >256 >256 >256 >256 >256 >256 >256

EA-L7-46 >256 >256 >256 >256 >256 >256 >256

118

Table 54 cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-L7-47 >256 >256 >256 >256 >256 >256 >256

EA-C7C-48 >256 >256 >256 >256 >256 >256 >256

EA-C7C-49 >256 32 64 64 64 >256 >256

EA-C7C-50 >256 >256 >256 >256 >256 >256 >256

EA-C7C-51 >256 128 128 128 256 >256 >256

EA-C7C-52 >256 >256 >256 >256 >256 >256 >256

EA-C7C-53 >256 32 32 64 32 >256 64

EA-C7C-54 >256 >256 >256 >256 >256 >256 >256

EA-C7C-55 >256 >256 >256 >256 >256 >256 >256

EA-C7C-56 >256 >256 >256 >256 >256 >256 >256

EA-C7C-57 >256 >256 >256 >256 >256 >256 >256

EA-C7C-58 >256 >256 >256 >256 >256 >256 >256

119 Table 54 cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-C7C-59 >256 >256 >256 >256 >256 >256 >256

EA-C7C-60 >256 64 64 64 128 >256 >256

EA-C7C-61 >256 >256 >256 >256 >256 >256 >256

EA-C7C-62 >256 >256 >256 >256 >256 >256 >256

EA-C7C-63 >256 64 64 64 128 >256 >256

EA-C7C-64 >256 >256 >256 >256 >256 >256 >256

EA-C7C-65 >256 >256 >256 >256 >256 >256 >256

EA-C7C-66 >256 >256 >256 >256 >256 >256 >256

EA-BC1-84 >256 >256 >256 >256 >256 >256 >256

EA-BC1-85 >256 8 8 32 32 128 32

EA-BC2-86 >256 >256 >256 >256 >256 >256 >256

EA-BC2-87 >256 >256 >256 >256 >256 >256 >256

120 Table 54 cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-BC2-88 >256 >256 >256 >256 >256 >256 >256

EA-BC1-89 >256 >256 >256 >256 >256 >256 >256

EA-BC1-90 >256 >256 >256 >256 >256 >256 >256

EA-BC2-91 >256 >256 >256 >256 >256 >256 >256

EA-BC2-92 >256 >256 >256 >256 >256 >256 >256

EA-BC2-93 >256 >256 >256 >256 >256 >256 >256

EA-BC1-94 >256 >256 >256 >256 >256 >256 >256

121 Antibiotics can exert their activity in either a bactericidal manner by actively killing bacterial cells or a bacteriostatic manner by simply inhibiting their growth. It is necessary to determine the nature of antibacterial action against relevant bacterial strains, not only to define a compound’s treatment potential. By definition, antibiotics are bactericidal against a specific bacterial strain if they kill 99.9% of a bacterial population after 18-24 hours of incubation. There are two methods by which antibacterial compounds can be assessed for this activity. While the determination of a compound’s killing kinetics is the most commonly used method, many describe the determination of minimum bactericidal concentrations (MBCs) as the most reliable way to assess bactericidal activity. MBC values of potent D-peptides were easily determined immediately following broth microdilution method of MIC. After 20 h incubation of the

MIC plate, 10 µl from each well with the lowest MIC value was spread onto nutrient-rich agar plates after subjecting to 10-fold serial dilutions. The agar plates were grown for 24 h, and the number of viable cells was determined. The MBC for a test compound is the lowest concentration that allows the growth of only 0.1% of the number of cells in the treated control. When using this method, bactericidal activity, highly desirable mode of action, is indicated by a ratio of MBC to MIC no greater than 4. The most potent D- peptides on S. aures strain were subjected to MBC test, and results are shown in Table

55.

122

Table 55. Minimum bactericidal concentration (MBC) of potent D-peptides on S. aureus

Peptide Name Peptide Sequence MIC (µg/ml) MBC (µg/ml)

EA-L12-04 VGSNLRLLHQWK 32 64

EA-L12-13 VHWDFRQWWQPS 32 32

EA-L7-18 KCCYTLP 128 >256

EA-L7-19 VLTRCCG 128 >256

EA-L7-22 TGFLVNV 64 128

EA-L7-34 HYIDFRW 32 128

EA-C7C-49 LKLGEKW 64 128

EA-C7C-51 RGATPMS 128 >256

EA-C7C-53 SISSLTH 32 64

EA-C7C-60 LWSTGAT 64 >256

EA-C7C-63 DNIMTPV 64 >256

EA-BC1-85 ACQTDVCQRTI 8 32

123

Chapter 3

Overcoming Vancomycin Resistance by Phage-derived D-peptide Antibiotics

124 3.1. “The Antibiotic of Last Resort”: Vancomycin

Vancomycin stands as last line of defense against an increasing number of resistant microbial pathogens. It is a glycopeptide antibiotic with a high level of activity against S. aureus with minimal human toxicity.287,288 It was discovered in 1956 as a secondary metabolite of Streptomyces orientalis found in the soil in the island of Borneo.289

Vancomycin is only effective against Gram-positive bacteria, since the outer lipid membrane of Gram-negative strains prevents access to the cell wall, crucial in its mode of action.

The main structural feature of vancomycin is the rigid heptapeptide backbone, consisting of five amino acids with aromatic side chains and two with aliphatic groups. N-methyl leucine at position 1; substituted phenylglycines at positions 2, 4, 5, 6, and 7; and an asparagine at position 3 with configurations of R, R, S, R, R, S, and S at the alpha carbons define the compound. These amino acids are of paramount importance in the mechanism of action employed by vancomycin. The fourth amino acid residue contains a phenolic ether linkage to an amino sugar disaccharide, L-vancosaminyl-β-D-glucose.

The function of the disaccharide moiety is still not certain, and removal of the sugar residue does not result in significant change in the conformation of the vancomycin:ligand complex, however it is believed to increase solubility and possibly help facilitate back to back intermolecular dimerization between two vancomycin molecules. It has been proposed that the sugar residues of glycopeptides contribute to the cooperativity of ligand binding, thereby increasing ligand affinity and enhancing antimicrobial activity. There is also emerging evidence to suggest that the disaccharide

125 may increase activity by interfering with surrounding transglycosylase enzymes.290,291

Vancomycin targets the final step in bacterial cell wall biosynthesis, and inhibits the cross-linking reaction by binding the D-Alanyl-D-Alanine terminal end of the growing pentapeptide unit of peptidoglycan non-covalently (Figure 18). The binding of vancomycin to D-Ala-D-Ala represents one of the smallest peptide-peptide interaction in nature, and is facilitated by five hydrogen bonds with a reasonable strong binding affinity

292 characterized by a Kd of 1 µM. Binding of vancomycin to the L-Lys-D-Ala-D-Ala precursor is very fast, estimated to be on the order of 1010 s-1 M-1, a number pointing to a diffusion controlled on rate, and upon binding it undergoes a subtle conformational change.293 Hydrophobic interactions and a strong ionic interaction between the N- terminal cationic amine of vancomycin and the carboxylate anion of L-Lys-D-Ala-D-Ala allow vancomycin to form a cap around the growing cell wall prior to the crucial cross- linking step.

The binding interaction is bacteriostatic since the last step of bacterial cell wall biosynthesis is interrupted, and the terminal D-Alanine residue cannot depart the scene, and consequently cell wall monomers are unable to cross-link. This ultimately results in bacterial cell lysis as the cell wall strength is weakened and osmotic pressure eventually bursts the nascent cell.

126

OH O

NH3 HO O O HO O Cl HO O O

HO

OH Cl H

O O O H N O N N N N NH H H H O O H2N OOC O CH3

NH2

OH HO OH

O O H N N-acyl-D-Ala-D-Ala R N O H O

Figure 18. The structure of vancomycin is composed of a rigid heptapeptide backbone and is adorned by a disaccharide sugar unit linked to the fourth amino acid residue. Vancomycin binds to the L-Lys-D-Ala-D-Ala terminal end of the growing bacterial cell wall through five hydrogen bonds and other non-covalent interactions.

127 3.2. Bacterial Resistance to Vancomycin

For more than 30 years, vancomycin has been a reliable antibiotic in the treatment of antibiotic-resistant staphylococcal and enterococcal infections due to its unique mode of action. Since the first isolation of vancomycin-resistant Staphylococcus aureus (VRSA), resistance to vancomycin is a clear and present danger. This emergence of vancomycin resistant enterococci (VRE) is significant, and has the potential to cause death in immune-compromised patients such as those suffering from Acquired Immune

Deficiency Syndrome (AIDS) and organ transplant patients. More concerning is the possibility of vancomycin resistance spreading to multi-drug resistant pathogenic bacteria, such as methicillin resistant Staphylococcus aureus (MRSA).

Bacteria exhibiting vancomycin resistance have a modified cell wall precursor pentapeptide, where some of all of the terminal D-alanine residues are substituted with D- lactate. This substitution results in an estimated 1000-fold reduced binding affinity for vancomycin due to the loss of one hydrogen bond and introduction of a repulsive interaction.

3.3. Resistance Mechanism of Vancomycin-resistant Bacteria

Resistance mechanisms to vancomycin occur in two major ways: replacement of terminal

D-Alanine residue to D-Lactate through vanA, vanB, or vanD genes or to D-Serine through vanC, vanE or vanG genes. Modification of D-Ala to D-Lac is the more common mechanism causing a 1000-fold decrease in binding affinity whereas the

128 A OH O

NH3 HO O O HO O Cl HO O O

HO

OH Cl H

O O O H N O N N N N NH H H H O O H2N OOC O CH3

NH2

OH HO OH

O O H N N-acyl-D-Ala-D-Ala R N O H O

O O N-acyl-D-Ala-D-Lac O R N O H O

B vanR vanS vanH vanA vanX

O NH3 O NH3 O O H HO O O N O O O

Figure 19. Vancomycin resistance: A) an elegant antibiotic invasion mechanism is engendered due to the presence of the D-Ala-D-Lac depsipeptide and it is dominant in resistant bacteria through the aegis of a five gene cluster. All five genes are required for inducible vancomycin resistance. A two-component regulatory system, VanR–VanS positively regulates the expression of VanH, VanA and VanX in response to antibiotic exposure. VanH catalyzes the synthesis of D-Lactate and VanA ligates this D-Lactate to D-Alanine. VanX is a peptidase specific for D-Ala-D-Ala that continues to be synthesized in vivo by the endogenous D-Ala-D-Ala ligase.

129 D-Ser modification is of lesser concern as it causes a seven-fold decrease in binding affinity.294-296

High-level resistance in Enterococcus faecium and Enterococcus faecalis is characteristic of the vanA, vanB, and vanD types. VanA type strains (about 70%) are resistant to high concentrations of all clinically used variants of vancomycin, while vanB resistant strains

(about 20%) are less common and still susceptible to the clinical glycopeptide teicoplanin.297,298

The resistance mechanism characterized by D-Lactate modification of the terminal D-

Alanine residue requires a two-component regulatory system, VanR and VanS, and three enzymes, VanH, VanA and VanX (Figure 19).299 In the presence of vancomycin, the two-component system activates the expression of the vanH, vanA and vanX genes.

VanH is an α-ketoacid reductase that converts pyruvate to D-lactate. Although VanA is a homologue of the essential D-Ala-D-Ala ligases that produce the cell wall precursor, it prefers the D-Ala-isostere, D-Lactate, as substrate, and generates the depsipeptide D-

Alanine-D-Lactate. Finally, the highly specific Zn+2 dependent dipeptidase VanX deplets

D-Ala-D-Ala constitutively produced by normal bacterial cell wall biosynthesis.

Consequence, bacteria become resistant to vancomycin by incorporating the depsipeptide

D-Ala-D-Lac rather than D-Ala-D-Ala. This apparently minor change from nitrogen to oxygen results in the loss of a hydrogen bond donor and electronic repulsion between acyl-D-Ala-D-Lac and vancomycin contributing to a ca. 1000-fold decrease in the affinity between the antibiotic and its binding partner. A common feature of VISA is a thickened bacterial cell wall as a result of an elevated cell wall metabolism.

130 The low-level of resistance mechanism involving replacement of D-Ala-D-Ala by D-Ala-

D-Ser is utilized by the intrinsically glycopeptide-resistant Enterococcus gallinarum,

Enterococcus casseliflavus, and Enterococcus flavescens (VanC type) and is present in the VanE and VanG types in E. faecalis. This results in a seven-fold decrease in binding affinity that stems mainly from the increased bulk of the hydroxymethyl group of serine relative to the methyl group resident in the alanine residue. VanC-type resistant bacteria are susceptible to teicoplanin with an MIC value of 0.5–1.0 µg/ml, whereas the MIC for vancomycin is higher at 2-32 µg/ml.

3.4. What Has Been Done to Overcome Vancomycin Resistance?

There are a number of reports detailing the design and testing of vancomycin-inspired synthetic molecules containing an aromatic template with one or more cationic peptide chains mimicking the action mechanism of vancomycin. These molecules can be classified as semi-synthetic glycopeptide derivatives and non-glycopeptide antibiotics.

One of early studies showed that small cationic cyclic systems binding to the D-Ala-D-

Ala ligand with a Kd of 19 mM.300 Another approach involves conserving the carboxylate binding pocket of vancomycin, and extending the peptide backbone with a tripeptide to mimic the peptide binding region of vancomycin to eliminate the repulsive interaction between the oxygen of the lactate ester in D-Ala-D-Lac.301 These simple structures allow free rotation of the tripeptide unit participating in binding and make them more accessible than in vancomycin for ligand binding. Although a significant increase in binding efficiency over vancomycin was observed, there have been no further follow-

131 up reports indicating that there may be other issue with the constructs or that this is the maximal level of binding efficiency possible with the approach.

Many semi-synthetic strategies have been used by various groups to develop new glycopeptide analogues.302-315 Haldar and co-workers reported the development of lipophilic cationic vancomycin analogues possessing 1000-fold increased antibacterial activity against vancomycin-resistant Enterococci (VRE).316 These compounds exerted their bactericidal activity at lower concentrations than vancomycin. Later, studies on optimizing the same compounds showed higher bacterial activity against a methicillin- resistant Staphylococcus aureus infected mouse model without any apparent toxicity.

Designing macrocycles is yet another synthetic approach to overcome resistance problems of VRE strains and one such study was carried out by Ma et al.317 The rigid heptapeptide backbone of vancomycin was modified by replacement of the carbonyl group of the fourth amino acid residue with -CHNHCOR to restore the missing hydrogen bond and prevent unfavorable electronic repulsion between vancomycin and the D-Ala-

D-Lac precursor of the growing bacterial cell wall in resistant strains. Resulting macrocyclic compounds were active against both vancomycin-sensitive and vancomycin- resistant strains. Additionally, one of the compounds was more active against VRE than vancomycin and teicoplanin.

Semisynthetic glycopeptides such as oritavancin, dalbavancin, and telavancin containing hydrophobic groups have been shown to exhibit potent antibacterial properties against resistant strains with excellent pharmacological profiles.318 It has also been shown that oritavancin and telavancin have an additional mechanism of action as bacterial membrane

132 disruption agents.319,320 Another semi-synthetic derivative, teicoplanin, that has an acyl- linked aliphatic moiety, showed a higher in vivo activity against VRE, whereas it was less active against coagulase-negative staphylococci.321

Boger and co-workers have synthesized a vancomycin aglycon amidine showing potent antibacterial activity with an MIC value of 0.31 µg/mL against vancomycin-resistant enterococci strains.322,323

The modifications of glycopeptides, such as deglycosylation, acylation and alkylation, have resulted in the most promising semisynthetic compound LY-333328 with the increased potency against VRE strains with 0.5–1 µg/ml MIC, and also high potency against MRSA.324-326 LY-333328 has a chlorinated biphenyl, and an extra vancosamine sugar different than vancomycin. Since LY-333328 has bactericidal activity rather than bacteriostatic as seen in vancomycin, it has been assumed that the modified disaccharide moiety with the additional vancosamine sugar of LY-333328 may inhibit the transglycosidation step of cell wall biosynthesis, and thereby be responsible for the observed bactericidal activity. Some candidates of this origin have also entered clinical phase studies. On the other hand, the modification of the peptide backbone of these compounds did not affect the activity against vancomycin-resistant and sensitive strains, however similar modifications to vancomycin result in a loss of antibacterial activity.

Süssmuth and coworkers have elucidated the cyclization reaction mechanism of linear peptide precursors; and were able to manipulate steps to replace the 3-chloro-β- hydroxytyrosine residue with 3-fluoro-β-hydroxytyrosine generating a new glycopeptide with enchanced activity against VRE.327

133 Xu and coworkers used the combinatorial libraries to identify synthetic receptors binding

D-Ala-D-Lac of vancomycin-resistant bacteria with a 5-fold higher affinity than vancomycin.328

Liskamp and co-workers used a 2917-member combinatorial tripodal library based on cyclotriveratrylene scaffold (CTV) to identify peptide receptors binding to D-Ala-D-Ala and D-Ala-D-Lac precursors.329 Each arm of the tripodal peptide library has one unique peptide sequence. According to the results obtained from libraries screened against each target molecule, peptide ligands having at least one basic amino acid, predominantly a lysine residue, were shown to bind to D-Ala-D-Ala, whereas ligands binding to D-Ala-D-

Lac had a significant number of polar amino acids, mostly glutamine and serine residues.

Another combinatorial approach was employed by Chiosis et al. by screening tri and tetrapeptide libraries.330 The small nucleophilic compounds called SProlinol derivatives were capable of cleaving the terminal D-Ala-D-Lac to re-sensitize the strains to vancomycin. These small molecules ensure that only growing cell wall substrates having

D-Ala-D-Ala are present in the growing peptidoglycan, resulting in the reactivation of vancomycin. Although administration of vancomycin and SPro5 together reduced MIC of VanA-resistant E. faecium by 8 to 16-fold, neither vancomycin nor SProC5 were bactericidal when administered alone. A similar strategy was attempted by using catalytic antibodies that hydrolyse the D-Ala-D-Lac bond. However, they were limited to in vitro assays, and their in vivo applications would be presumably limited by accessibility of antibody to the interior crevasses of the cell wall.331

Additionally, the dimerization of vancomycin has been investigated as a potential

134 therapeutic modality.332 It has been shown that dimerization of vancomycin covalently by a carboxamide tether significantly increased in vitro activity on VRE strains having 3- to 10-times more stronger binding to D-Ala-D-Lac than vancomycin. The only drawback of this study was that these compounds did not show potent antibacterial activity to have therapeutic value. Whitesides has examined the dimerization effect on binding properties of vancomycin. The binding of substrate Ac2-L-Lys-D-Ala-D-Ala to monomeric vancomycin is 33 kJ/mole, whereas dimeric ligand binding to a dimeric vancomycin has a binding affinity of 50 kJ/mole, for an increase of 17 kJ/mole corresponding to a second binding event.333 Also a trivalent system of vancomycin was studied earlier.302

Alternatively new glycopeptide derivatives, glycylcyclines, ramoplanin, daptomycin and

D-L-α-peptides are attractive alternatives to vancomycin due to their differential bactericidal activities.

As non-semisynthetic approaches got monoclonal antibody catalysis (mAb) hydrolyzing he ester bond of D-Ala-D-Lac depsipeptide was designed as new immunotherapy.334

Potential bactericidal activities of such new antibodies are still being investigated.

The strategies targeting to D-Ala-D-Lac substrate rather than the protein synthesis mechanism has advantages, since it reduces the chance of developing a new bacterial resistance related to the slight modification in biosynthesis. Bacteria would not need to acquire a new pathway to overcome resistance, and would have to use secondary alternative pathways with non-transferable genes vanC and vanE limiting the development of bacterial resistance.

135 3.5. Results and Discussions

3.5.1. Peptide Ligands of Vancomycin-resistant Strains by Screening Linear and

Cyclic Phage Display Peptide Libraries

Linear and cyclic peptide ligands binding to the enantiomer of pentapeptide precursor of bacterial cell wall of vancomycin-resistant Enterococci, (Figure 20) were identified by biopanning of commercially available a linear 12-mer, a linear 7-mer and a cyclic 7-mer phage display peptide libraries (purchased from New England Biolabs). Target EA-4-

LAC were synthesized manually by solid-phase peptide synthesis using Fmoc-chemistry on 2-chlorotrityl chloride resin linker as described in literature.335 It also has a biotin tag to utilize streptavidin-biotin interactions during affinity selection and a PEG4 spacer as stated in previous section.

NH2

H S O H N HN NH O O O H H H NH O 4 N N OH O N O H O O O O OH

Biotin-PEG4-D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Lac

Figure 20. The structure target molecule EA-4-LAC, the enantiomer of pentapeptide precursor of bacterial cell wall of vancomycin-resistant enterococci (VRE).

136 Biopanning experiments were performed by applying the high yield selection strategy thorugh four rounds of selection, and the amount of target molecule EA-4-LAC was decreased to control the stringency, 5 mg, 2 mg, 500 ng, and 100 ng, in the successive rounds. A negative selection was also included after the second round of biopanning to determine the enrichment factor (Table 56-58). The magnetic beads were changed in the alternating rounds from streptavidin-coated magnetic beads to neutravidin-coated magnetic beads to avoid selecting binders of streptavidin and neutravidin. Additionally, the stringency was controlled by raising the concentration of Tween-20 in the washing and the binding steps after first round to 0.5% from 0.1%.

After the third and the fourth round of affinity selection, DNA of randomly picked 42 phage clones were sequenced to reveal the displayed peptide sequences. From biopanning of the linear 7-mer library no repeating peptide sequences were isolated after the fourth round of biopanning, whereas one peptide with sequence of “KFYAHLD” was isolated twice. Within cyclic 7-mer library selection, six repeating cyclic peptides with sequences of “TNANHYF”, “NQTAARV”, “GLKALKE”, “GYSSFNR”, “KHLLGEN”,

“NTGSPYE” were isolated aftert both the third and the fourth rounds. Furthermore, three peptides, “RGATPMS”, “MARYMSA”, and “LKLGEKW”, were observed once that were also isolated from the biopanning against EA-1-CEP and EA-2-ALA in the previous chapter. In the biopanning of linear 12-mer library, the enrichments were achieved in favor of three peptides, “RKVKRRPRVSNL”, “GLHTSATNLYLH” and

“FIPFDPMSMRWE”. However, some of the isolated peptides were also identified from the biopanning against EA-1-CEP and EA-2-ALA, cyclic 7-mer, “RGATPMS”,

“MARYMSA”, “GYSSFNR”, “NTGSPYE”, “LKLGEKW”, “EGQRWMQ”, and

137 “TNANHYF”, and linear 7-mer, “HYIDFRW”, “LPVRLDW” and “HGGVRLY”, and linear 12-mer, “SGVYKVAYDWQH”, “RKVKRRPRVSNL”, “GLHTSATNLYLH” in the previous chapter. Addition to the repeating peptides, also the similar short peptide motif trend was observed in these biopannings, “T-T-L”, “I/L-S/T”, “Q/N-S/T”, “L-Q”,

“G-Q-S”, “G-S”, “G-x-S”, “G-V”, “L-K/R”, “R-V”, “V-L-S”, “N/Q-K/R”.

The binding specificies of twenty-two phage clones isolated as the repeating peptide sequences were tested for target molecule EA-4-LAC by phage-ELISA. The same sandwish phage-ELISA protocol was carried out with previous chapter. ELISA signals that are shown in Figure 21 indicate that the selected phage clones have higher binding affinity for target EA-4-LAC rather than negative controls. Since all isolated peptides specifically bind to target EA-4-LAC, the enantiomers (D-peptiddes) of these twenty-two peptides were synthesized for futher studies.

138

Table 56. Phage titers of target EA-4-LAC with linear 12-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 1.9 x 105 - NC-2 4.7 x 104 - 2 6.0 x 1010 9.4 x 104 2 NC-3 3.6 x 103 - 3 3.8 x 1011 4.0 x 104 1.1 x 101 NC-4 1.8 x 102 - 4 1.0 x 1011 3.8 x 104 2.11 x 102

Table 57. Phage titers of target EA-4-LAC with linear 7-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 2.8 x 105 - NC-2 1.1 x 104 - 2 2.0 x 1011 1.6 x 105 1.5 x 101 NC-3 2.6 x 103 - 3 2.5 x 1011 1.7 x 105 6.5 x 101 NC-4 1.2 x 102 - 4 1.2 x 1011 1.4 x 104 1.16 x 102

Table 58. Phage titers of target EA-4-LAC with cyclic 7-mer library

Rounds Phage input Phage output Enrichment factor

1 1.5 x 1011 9.0 x 104 - NC-2 2.0 x 104 - 2 6.0 x 1010 1.5 x 105 7.5 NC-3 1.3 x 104 - 3 6.2 x 1011 2.8 x 105 2.2 x 101 NC-4 6.5 x 102 - 4 3.0 x 1011 3.0 x 104 4.6 x 101

139

Round Peptide Sequence Abundance 3 A C K M S M L H N C 1 3 A C M A R Y M S A C 2 3 A C R S A T H S A C 1 3 A C R G A T P M S C 2 3 A C Q M Q L R S A C 1 3 A C T V R T S A D C 1 3 A C A S K S T H D C 1 3 A C T E R T S T E C 1 3 A C N T G S P Y E C 2 3 A C G Y S S F N R C 2 3 A C G N S S L N R C 1 3 A C K D H V T R V C 1 3 A C K A A L T R W C 1 3 A C L V S Q H T D C 2 3 A C L K N Q S D Q C 1 3 A C F G Q G T L Q C 1 3 A C E G Q R W M Q C 1 3 A C S V G T N F Q C 1 3 A C L K L G E K W C 1 3 A C G L K A L K E C 2 3 A C Y G N V T N T C 1 3 A C N H D A T H T C 1 3 A C D H T Y T N K C 1 3 A C Y A F N Y P H C 1 3 A C T Q M N D S F C 1 3 A C N I I H H Q T C 1 3 A C L D I F S S S C 1

Table 59. Peptide sequences isolated after the third round of biopanning with a cyclic 7- mer random peptide library in high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, 2 mg, and 500 ng, respectively.

140

Round Peptide Sequence Abundance 4 A C N T G S P Y E C 3 4 A C N Q T A A R V C 2 4 A C N P T H Y R S C 1 4 A C T N A N H Y F C 3 4 A C T V R T S A D C 1 4 A C T K S L A H T C 1 4 A C T Q S S A M S C 1 4 A C G Y S S F N R C 2 4 A C G L K A L K E C 7 4 A C L K L G E K W C 1 4 A C K H L L G E N C 2 4 A C Q M Q L R S A C 1 4 A C M A R Y M S A C 1 4 A C R G A T P M S C 1 4 A C V Q M P A H S C 1 4 A C P V A L S T K C 1 4 A C S S V T D R W C 1 4 A C G G G P L Y M C 1 4 A C D H P H K Q Q C 1

Table 60. Peptide sequences isolated after the fourth round of biopanning with a cyclic 7-mer random peptide library in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, 2 mg, 500 ng and 100 ng, respectively.

141 Round Peptide Sequence Abundance 3 F V R I H D V 1 3 A G K P F H F 1 3 L T L G L P Y 1 3 V H P L K L I 1 3 L P N S A Y V 1 3 G M W H L P Q 1 3 Y P W F I R A 1 3 Y P F F S S M 1 3 S F F E Q V H 1 3 F S Y S F Q H 1 3 S F S Q N L H 1 3 K F Y A H L D 2 3 K P P P T L D 1 3 T Q T V L G D 1 3 L V M H S E N 1 3 L A Q S S I Q 1 3 S S L R I P V 1 3 N L R L P Y I 1 3 I S T P Y I G 1 3 G V M N H T F 1 3 S N M S H A T 1 3 D S V E T K P 1 3 V D S R Y H P 1 3 I R I A E P M 1 3 A P T P G N V 1 3 A H T D W F N 1 3 A I D F A R N 1 3 N D R L H T R 1 3 W P T H Y L V 1 3 T D E I K L L 1 3 T V N F K L Y 1 3 N S I Y Q A W 1 3 D V M M P R H 1 3 V T N T P W P 1 3 S E H N G T Q 1 3 K T A L A L E 1 3 V P V W A L T 1

Table 61. Peptide sequences isolated after the third round of biopanning with a linear 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, 2 mg, and 500 ng, respectively.

142

Round Peptide Sequence Abundance 4 V S G F R I D 1 4 V S Q R T E P 1 4 V P I Y H L T 1 4 Y P W F I R A 1 4 Y Q W E L Y S 1 4 Y Y N T T P N 1 4 Y S E P A V T 1 4 S H E N F T S 1 4 S S N Q F H Q 1 4 S F R I G P A 1 4 S P W Q Y T N 1 4 L P K M Y S Q 1 4 L P V R L D W 1 4 H Y I D F R W 1 4 H I A R L S Y 1 4 H G G V R L Y 1 4 G S P D S E F 1 4 W G R I S H V 1 4 S A D Y S A R 1 4 A Y D D W F W 1

Table 63. Peptide sequences isolated after the fourth round of biopanning with a linear 7- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, 2 mg, 500 ng, and 100 ng respectively.

143

Round Peptide Sequence Abundance 3 G L H T S A T N L Y L H 2 3 G L P H A R E D Y L D L 1 3 G D S G L V E S H R N V 1 3 M D L G Y V E G S A R V 1 3 I P L G R D G G S Y Q R 1 3 Y P D K I T W Q A P W L 1 3 V H W D F R Q W W Q P S 1 3 S W H H G D G P I W Y G 2 3 R K V K R R P R V S N L 2 3 F I P F D P M S M R W E 2 3 N H L S T P V W S I T G 1 3 S V L S Y S V A Y S D S 1 3 H V V T S S K T A G P A 1 3 Q S H Y D S H L A M L V 2 3 V S H R S T A N F I G S 1

Table 64. Peptide sequences isolated after the third round of biopanning with a linear 12- mer random peptide library in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, 2 mg, and 500 ng, respectively.

144

Round Peptide Sequence Abundance 4 R K V K R R P R V S N L 6 4 G L H T S A T N L Y L H 4 4 K H F P L G M E Y L V T 1 4 F I P F D P M S M R W E 2 4 H K H W S T P E F L S S 1 4 V H W D F R Q W W Q P S 1 4 S G V Y K V A Y D W Q H 1 4 S W P A G V N S R V N G 1

Table 65. Peptide sequences isolated after the fourth round of biopanning with a linear 12-mer random peptide library in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, 2 mg, 500 ng and 100 ng, respectively.

145

EA-4-LAC Target 1.4 Streptavidin BSA Plastic 1.2

1 405 0.8 @

0.6 Absorbance

0.4

0.2

0 VPIYHLT KHLLGEN HYIDFRW GLKALKE LKLGEKW GYSSFNR LVSQHTD YPWFIRA NTGSPYE KFYAHLD LPVRLDW TNANHYF QMQLRSA NQTAARV RGATPMS MARYMSA GLHTSATNLYLH HKHWSTPEFLSS RKVKRRPRVSNL FIPFDPMSMRWE QSHYDSHLAMLV SGVYKVAYDWQH Peptide Sequences

Figure 21. Phage-ELISA results of selected phage clones from biopanning of all three commercially available libraries, linear 12-mer, linear 7-mer and cyclic 7-mer, against target EA-4-LAC after the high yield selection.

146 3.5.2. Bicyclic Peptide Ligands for Bacterial Cell Wall Precursor of Vancomycin- resistant Enterococci

Bicyclic peptide ligands of target EA-4-LAC were also investigated by utilizing two phage-coded bicyclic peptide libraries, Library A and Library B. Bicyclic phage display libraries were generated with the same procedure that applied in the previous chapter, amplification of linear phage display libraries, reduction of cysteine residues with TCEP, production of bicyclic phage display peptide libraries with reaction of TBMB with linear phage display libraries, and solution-phase biopanning of these two libraries against target EA-4-LAC. Biopanning experiments were carried out via the high yield selection strategy, where 5 mg of target molecule was used in the first round and was decreased to

2 mg in the second round. The stringency of affinity selection was controlled by decreasing the amount of target molecule and increasing the concentration detergent

Tween-20 in the successive rounds. Table 68 and Table 69 show the peptide sequences isolated from the biopanning of Library A and Library B against target EA-4-LAC.

While some of bicyclic peptides with different loop lengths were isolated more than once, each library yielded the same and similar short peptide motifs, “G-Q-S-V”, “S-S-V”, “G-

Q-S”, “V-T/S”, “Q-L-S”, “D-Q”, “L-S-Q”, “S/P-Q-L”, “Q-L-S”, and “G-V-Q-S”. These short peptide motifs are also similar those observed in the biopanning of targets EA-2-

ALA and EA-1-CEP.

To conduct antibacterial activity assays, five of those bicyclic peptides that have the similar peptide motifs isolated from the linear and cyclic phage display peptide libraries were synthesized in enantiomeric form (D-peptides) (Table 70).

147

Table 66. Phage titers of EA-4-LAC and negative controls after each round of biopanning with Library A and enrichment factor after the second round.

Round 1 Round 2 Enrichment factor

Control (No target) 1.3 x 104 1.1 x 104 -

EA-4-LAC 5.6 x 104 4.6 x 106 4.1 x 102

Table 67. Phage titers of EA-4-LAC and negative controls after each round of biopanning with Library B and enrichment factor after the second round.

Round 1 Round 2 Enrichment factor

Control (No target) 3.1 x 103 1.6 x 104 -

EA-4-LAC 1.7 x 104 3.9 x 106 2.4 x 102

148

Library Loop length Peptide Sequence Abundance A 3 X 5 A C S N I C L A K P H C 1 A 3 X 5 A C T R D C P S Q A H C 1 A 3 X 5 A C L P S C Q H A E I C 1 A 3 X 5 A C Y M P C G Q S V V C 1 A 3 X 5 A C T G N C V S S V G C 1 A 3 X 5 A C A L S C H Q V S L C 1

A 3 X 4 A C T D S C P P Q S C 2

A 3 X 4 A C P D Q C Q F S S C 1

A 3 X 4 A C V P T C S R S G C 1

A 3 X 4 A C V K S C G Q S V C 1

A 3 X 4 A C Q D L C G Q M V C 1

A 3 X 4 A C L V P C T Q Y V C 1

A 4 X 4 A C H R Q L C S P S E C 1 A 4 X 4 A C G G G I C R T H N C 2 A 4 X 4 A C R S E T C A Y Q D C 1 A 4 X 4 A C Q Y N D C D M L H C 2

A 4 X 3 A C L S Q F C V I D C 3

A 5 X 3 A C A A H Q Y C W S T C 2 A 5 X 3 A C Q F H I G C Y S N C 3 A 5 X 3 A C T A P G N C S Q L C 1 A 5 X 3 A C L R E S A C S K Q C 1

Table 68. Peptide sequences isolated after the second round of biopanning with bicyclic phage display peptide library A, Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3-5, in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, and 2 mg, respectively.

149

Library Loop length Peptide Sequence Abundance B 4 X 5 A C P Q L S C P S G G S C 3 B 4 X 5 A C P Q S S C Q G L R L C 2 B 4 X 5 A C Q Q Y N C V P V G R C 1 B 4 X 5 A C E K K Y C T Q Q L P C 1 B 4 X 5 A C T Q V P C T P Y Q G C 1 B 4 X 5 A C H R T P C S L P T T C 1 B 4 X 5 A C G A Q G C F G V Q S C 1 B 4 X 5 A C I A R D C W Q G F S C 1 B 4 X 5 A C T S S L C Q L S V L C 1 B 4 X 5 A C T L T Q C S L S K A C 1

B 5 X 4 A C S S H V I C N S N S C 1 B 5 X 4 A C E M L Q S C Q Q D W C 1 B 5 X 4 A C N P P H I C Q N P K C 1 B 5 X 4 A C P A V L S C T A E Q C 1

B 3 X 6 A C A P L C G H R V P Q C 1 B 3 X 6 A C T Q L C C T A S P F C 1 B 3 X 6 A C T R V C S S S Q L Y C 1

Table 69. Peptide sequences isolated after the second round of biopanning with bicyclic phage display peptide library B, Cys-(Xaa)m-Cys-(Xaa)n-Cys (Xaa are random amino acids, m and n = 3-6, in the high yield selection using decreasing amount of target molecule EA-4-LAC at each round, 5 mg, and 2 mg, respectively.

150

Table 70. Synthesized bicyclic peptides for antimicrobial activity assays

Peptide name Peptide Sequence

EA-BC1-78 ACVKSCGQSV

EA-BC1-79 ACQYNDCDMLH

EA-BC1-80 ACLSQFCVID

EA-BC2-81 ACPQSSCQGLRL

EA-BC2-82 ACTRVCSSSQLY

EA-BC1-83 ACQFHIGCYSN

151 3.5.3. Antibacterial Activity Assays of D-Peptides againts Vancomycin-resistant

Strains

Peptides that showed the high binding affinity in phage-ELISA from linear and cyclic phage display libraries and also ones having sequence similarity from bicyclic peptides libraries were synthesized manually in D-form using standard Fmoc chemistry on Rink- amide resin to have C-terminus amide and a short linker at C-terminus, Gly-Gly-Gly-Ser for linear and cyclic peptides derived from commercially available libraries and Gly-Gly-

Ser-Gly for bicyclic peptide libraries for further studies for further studies.281

Synthesized D-peptides were tested for their antibacterial activities against both Gram- positive (B. subtilis, S. aureus, E. faecalis, MRSA, vanA type VRE, vanB type VRE,) and Gram-negative (E. coli) bacteria addition to two control antibiotics in each test, vancomycin and melittin.

None of these D-peptides showed high or moderate antibacterial activity for vancomycin- resistant bacteria, vanA and vanB type, whereas six of them had moderate activity against

S. aureus, MRSA, and E. faecalis strains with MIC values ranging from 32 to 128 µg/ml as seen in Table 71. Since three of them are bicyclic peptides isolated only from the biopanning of EA-4-LAC, other identified bicyclic peptides will also be tested in further studies.

152 Table 71. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

Vancomycin >256 0.5 0.5 0.5 1 >256 32

Melittin 32 2 2 4 8 32 32

EA-L12-01 >256 >256 >256 >256 >256 >256 >256

EA-L12-08 >256 >256 >256 >256 >256 >256 >256

EA-L12-67 >256 >256 >256 >256 >256 >256 >256

EA-L12-68 >256 >256 >256 >256 >256 >256 >256

EA-L12-69 >256 >256 >256 >256 >256 >256 >256

EA-L12-70 >256 >256 >256 >256 >256 >256 >256

EA-L7-21 >256 >256 >256 >256 >256 >256 >256

EA-L7-71 >256 >256 >256 >256 >256 >256 >256

EA-L7-72 >256 >256 >256 >256 >256 >256 >256

EA-L7-73 >256 >256 >256 >256 >256 >256 >256

153

Table 71. cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-L7-34 >256 32 32 64 64 >256 >256

EA-C7C-74 >256 >256 >256 >256 >256 >256 >256

EA-C7C-75 >256 >256 >256 >256 >256 >256 >256

EA-C7C-76 >256 >256 >256 >256 >256 >256 >256

EA-C7C-77 >256 >256 >256 >256 >256 >256 >256

EA-C7C-78 >256 >256 >256 >256 >256 >256 >256

EA-C7C-52 >256 >256 >256 >256 >256 >256 >256

EA-C7C-54 >256 >256 >256 >256 >256 >256 >256

EA-C7C-51 >256 128 128 128 256 >256 >256

EA-C7C-50 >256 >256 >256 >256 >256 >256 >256

EA-C7C-49 >256 32 64 64 64 >256 >256

EA-C7C-61 >256 >256 >256 >256 >256 >256 >256

154

Table 71. cont. Minimum inhibitory concentrations (MIC) in µg/ml of D-peptide antibiotics on several bacterial strains

E. coli B. subtilis S. aureus MRSA E. faecalis VRE (vanA-type) VRE (vanB-type) Peptide (ATCC 25992) (ATCC 6633) (ATCC 6538) (ATCC 43300) (ATCC 29212) (ATCC 51559) (ATCC 51299)

EA-BC1-78 >256 >256 >256 >256 >256 >256 >256

EA-BC1-79 >256 64 64 128 128 >256 >256

EA-BC1-80 >256 128 128 >256 >256 >256 >256

EA-BC2-81 >256 >256 >256 >256 >256 >256 >256

EA-BC2-82 >256 128 128 >256 >256 >256 >256

EA-BC1-83 >256 >256 >256 >256 >256 >256 >256

155

Chapter 4

Experimental Procedures

156 4.1.Synthesis of Target Molecules

4.1.1. Synthesis of Enantiomer of Cephalosporin

O H N H H O 2 S O Na2CO3, H2O, RT H H BnBr, Et3N, DMF, RT N N N S O O 45 % O 60 % COOH N O O COOH 6-APA (1)

O O SO2Cl2, CCl4, 0 °C DBU, CH2Cl2 H H N H H N S S 30 min O 64 % O N N O O O O O O

(2) (3)

O O

N H Cl N H H SCl SnCl2, THF, reflux S O3, Me2CO, - 78 °C O O N 30 % over 2 steps N quant. O O O O O O

(trans:cis = 4:1) (5) (4)

O O 1M N H in THF pTSA, DMF, 100 °C H H 2 4 H H O N S N -78 °C, 30 min S 52 % O O N 40 % N O O O O O O (7) (6)

O H H H2N S NH O NHS-PEG12-Biotin HN H H H H N S N O DIPEA, DMF, 0 °C H N O H 12 52 % S O N O O O O O (8) (9)

O AlCl3, PhOMe, DCM, NO2Me NH O HN H H H H 0 °C to RT, 8h O N S H N 28 % H 12 S O N O (10) O OH EA-1-CEP Scheme 1. Synthesis scheme of the enantiomer of cephalosporin ((6S)-Cephalosporin) starting from 6-Aminopenicillanic acid (6-APA) over 10 steps.

157 (2S,5R,6R)-6-(1,3-dioxoisoindolin-2-yl)-3,3-dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylic acid (1)

O H N H H O 2 S O Na2CO3, H2O, RT H H N N N S O O 45 % O COOH N O O COOH 6-APA (1)

To a vigorously stirred solution of 6-aminopenicillanic acid (3.00 g, 13.9 mmol) and

Na2CO3 (1.47 g, 13.9 mmol) in water (20 ml) was added finely ground N- carboethoxyphthalimide (3.05 g, 13.9 mmol). The mixture was stirred at room temperature for 2 h and then washed with CH2Cl2 (3 x 5 ml). The aqueous layer was mixed with a fresh portion of CH2Cl2 (20 ml) and acidified during vigorous stirring with

1 M HCl (40 mL). Phases were separated and the extraction was completed with two additional portions of CH2Cl2 (2 x 5 ml). The combined organic extracts were washed with water (2 x 10 ml) and satd. brine (5 ml). The organic layer was dried over MgSO4, and evaporated in vacuo to afford compound 1 (2.30 g, 45 %) as an off-white solid which

1 was used in the next step without further purification. H NMR (CDCl3, 500 MHz) δ 7.89

(dddd, 2 H, J = 8.5, 5.5, 3.0, 1.0 Hz), 7.77 (dddd, 2 H, J = 8.5, 5.5, 3.0, 1.0 Hz), 5.69 (dd,

1 H, J = 4.0, 1.0 Hz), 5.59 (dd, 1 H, J = 4.0, 1.0 Hz), 4.71 (s, 1 H), 1.85 (s, 3 H), 1.62 (s,

3 H).

158 (2S,5R,6R)-benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,3dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylate (2)

O O H H BnBr, Et3N, DMF, RT N H H S N S 60 % O N O N O O O COOH O (1) (2)

To a stirred solution of compound 1 (2.3 g, 76.64 mmol) in DMF (12 ml) was added triethylamine (0.926 ml, 6.64 mmol) followed by benzyl bromide (0.987 ml, 8.3 mmol).

The mixture was stirred at room temperature for 6 h and then poured into vigorously stirred ice water (55 ml). The resulting suspension was extracted with CHCl3 (3 x 25 ml) and the combined organic layers were washed with satd. NaHCO3 (3 x 15 ml), water (3 x

15 ml), and saturated brine (15 ml). The organic phase was dried over MgSO4 and evaporated in vacuo to obtain the crude compound as light yellow oil which was purified by flash chromatography on SiO2 (60% hexanes:EtOAc) to afford 2 (1.87 g, 65%) as a

1 white solid. H NMR (CDCl3, 300 MHz) δ 7.88 (dd, 2 H, J = 5.4, 3.0 Hz), 7.77 (dd, 2 H,

J = 5.4, 3.0 Hz), 7.42-7.37 (m, 5 H), 5.68 (d, 1 H, J = 3.9 Hz), 5.59 (d, 1 H, J = 3.9 Hz),

4.70 (s, 1 H), 4.51 (s, 2 H), 1.80 (s, 3 H), 1.44 (s, 3 H).

159 (2S,5R,6S)-benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,3-dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylate (3)

O O DBU, CH2Cl2 H H N H H N S S O 64 % O N N O O O O O O

(2) (3)

To a solution of ester 2 (1.87 g, 4.28 mmol) in CH2Cl2 (16 ml) was added 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) (0.042 ml, 0.285 mmol) and the mixture was stirred at room temperature for 90 min. The solution was washed with 1 M NH4Cl (2 x 10 ml), water (3 x 10 ml), and satd. brine (5 ml). The organic phase was dried over MgSO4 and evaporated in vacuo to afford a white foam which was purified by flash chromatography on SiO2 (60 % Hexanes:EtOAc) to afford 3 (1.87 g, 64 %) as a white

1 foam. H NMR (CDCl3, 500 MHz) δ 7.90 (dd, 2 H, J = 5.5, 3.0 Hz), 7.77 (dd, 2 H, J =

5.5, 3.0 Hz), 7.41-7.34 (m, 5 H), 5.57 (d, 1 H, J = 2.0 Hz), 5.40 (d, 1 H, J = 2.0 Hz), 5.22

(d, 2 H, J = 2.0 Hz), 4.66 (s, 1 H), 1.64 (s, 3 H), 1.43 (s, 3 H).

160 (2S,5S,6S)-benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylate (5)

O O O SO Cl , CCl , 0 °C 2 2 4 SnCl2, THF, reflux H H H N H H N Cl N S 30 min SCl 30 % over 2 steps S O O O N N N O O O O O O O O O

(trans:cis = 4:1) (3) (5) (4)

A solution of compound 3 (0.050 g, 0.114 mmol) in CCl4 (2 ml) was treated with equimolar amount of sulfuryl chloride (0.114 mmol, 1M solution in CCl4) and stirred at 0

°C for 30 min. The solvents were evaporated in vacuo to give a mixture of trans ds, cis ds

(desired) and SM in a ratio of ca. 3:1:1.2 as a yellow foam. The foam was dissolved in

THF (2 ml) and treated with anhydrous SnCl2 (0.0167 mg, 0.088 mmol). The mixture was stirred at room temperature for 2 h and then the solvent was evaporated in vacuo.

The residual oil was dissolved in EtOAc (5 ml) and washed with water (3 x 5 ml) and satd. brine (5 ml). The organic phase was dried over MgSO4 and evaporated in vacuo to give a white foam which was purified by flash chromatography on SiO2 (100% CH2Cl2 -

1 94% CH2Cl2 : EtOAc) to afford 5 (0.005 g, 30% over two steps) as a white foam. H

NMR (CDCl3, 300 MHz) δ 7.88 (dd, 2 H, J = 5.4, 3.0 Hz), 7.75 (dd, 2 H, J = 5.4, 3.0

Hz), 7.48-7.34 (m, 5 H), 5.62 (d, 1 H, J = 3.9 Hz), 5.31 (d, 2 H, J = 6.0 Hz), 5.26 (d, 2 H,

J = 3.9 Hz), 4.02 (s, 1 H), 1.70 (s, 3 H), 1.69 (s, 3 H).

161 Benzyl(2S,4S,5S,6S)-3,3-dimethyl-7-oxo-6-phthalimido-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylate 4-oxide (6)

O O H H O N H H N S O3, Me2CO, - 78 °C S O O N quant. N O O O O O O

(5) (6)

Compound 5 (0.115 g, 0.263 mmol) was dissolved in acetone (5.0 ml), cooled to -78 °C, and treated with a stream of ozone. The solvent was evaporated in vacuo to give a white foam compound 6 (0.132 mg, quant. yield) that was used without purification. 1H NMR

(CDCl3, 500 MHz) δ 7.91 (dd, 2 H, J = 5.5, 3.0 Hz), 7.80 (dd, 2 H, J = 5.5, 3.0 Hz), 7.48-

7.35 (m, 5 H), 5.92 (d, 1 H, J = 4.8 Hz), 5.37 and 5.30 (ABq, 2 x 1 H, J = 12.0 Hz), 4.56

(d, 1 H, J = 4.8 Hz), 4.30 (s, 1 H), 1.61 (s, 3 H), 1.55 (s, 3 H).

162 (6S,7S)-benzyl-7-(1,3-dioxoisoindolin-2-yl)-3-methyl-8-oxo-5-thia-1- azabicyclo[4.2.0]oct-2-ene-2-carboxylate (7)

O O pTSA, DMF, 100 °C H H O N N H H S 52 % S O O N N O O O O O O (6) (7)

A stirred solution of sulfoxide compound 6 (0.132 g, 0.292 mmol) and anhydrous p- toluenesulfonic acid (pTSA, 0.0068 g, 0.0394 mmol) in THF (15 ml) was heated at 100

°C for 2 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (5 ml) and washed with water (4 x 5 ml), satd. NaHCO3 (3 x 5 ml), and satd. brine (2 ml).

The organic layer was dried over MgSO4 and evaporated in vacuo to yield a brown foam which was column purification (100% CH2Cl2 – 98% CH2Cl2 : EtOAc) to afford

1 compound 7 (0.059 g, 52%) as a white foam. H NMR (CDCl3, 300 MHz) δ 7.89 (dd, 2

H, J = 5.5, 3.0 Hz), 7.78 (dd, 2 H, J = 5.5, 3.0 Hz), 7.46-7.32 (m, 5 H), 5.73 (d, 1 H, J =

4.3 Hz), 5.31-5.21 (m, 2 H), 5.11 (d, 1 H, J = 4.3 Hz), 3.73 and 3.01 (q, 2 x 1 H, J = 15.5

Hz) , 2.33 (s, 3 H).

163 (6S,7S)-benzyl-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2- carboxylate (8)

O H H H2N H H 1M N2H4 in THF S N S O -78 °C, 30 min N N O O 40 % O O O O (8) (7)

To a solution of compound 7 (0.0478 g, 0.110 mmol) at -78 °C in THF (2.0 ml) was added 1 M N2H4 solution in THF (0.220 ml, 0.220 mmol). The reaction mixture stirred at

-78 °C for 30 min. The phthalhydrazide complex was decomposed by addition of 1 M

HCl (0.28 ml) and the solvents were evaporated in vacuo. The residual oil was dissolved, with vigorous stirring, in water (2.0 ml) and the insoluble phthalhydrazide was removed by filtration. The aqueous filtrate was layered with EtOAc (2.0 ml) and basified with satd. NaHCO3 to pH 8. Phases were separated and the extraction completed with two additional portions of EtOAc (2 x 2 ml). The combined organic extracts were washed with water (3 x 2 ml) and satd. brine (1 ml), dried over MgSO4, and evaporated in vacuo to give a thick yellow oil which was purified by flash chromatography on SiO2 (100%

CH2Cl2 – 70% CH2Cl2 : EtOAc) to afford compound 8 (0.021 g, 40 %) as a white foam.

1 H NMR (CDCl3, 500 MHz) δ 7.43-7.30 (m, 5 H), 5.27 (d, 2 H, J = 4.1 Hz), 4.91 (d, 1 H,

J = 4.9 Hz), 4.70 (d, 1 H, J = 4.9 Hz), 3.52 and 3.21 (ABq, 2x1 H, J = 18.4 Hz) , 2.11 (s,

3 H).

164 (6S,7S)-benzyl-7-(poly(ethylene glycol)ether-2-(biotinylamino)ethane)-3-methyl-8- oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate (9)

O H H H2N S NH O NHS-PEG12-Biotin HN H H H H O N S N DIPEA, DMF, 0 °C H N O H 12 65 % S O N O O O O O (9) (8)

To a solution of compound 8 (0.007 g, 0.023 mmol) and NHS-PEG12biotin (0.025 g,

0.026 mmol) at 0 °C in DMF (0.5 ml) was added DIPEA (12 ml, 0.069 mmol). The reaction mixture was stirred at 0 °C for 24 h and the solvents were evaporated in vacuo to give a thick yellow oil which was purified four times by RP-HPLC (rt = 16.5 min, C18 semi-prep column, 30-40% B; A = 98 % H2O:acetonitrile; B = 99 % acetonitrile:H2O) to

1 afford 9 as a white solid. H NMR (CDCl3, 500 MHz) δ 7.43-7.35 (m, 5 H), 6.72 (bs, 1

H), 5.81 (q, 1 H, J = 4.5 Hz), 5.28 (dd, 2 H, J = 20.0, 12.5 Hz), 4.98 (d, 1 H, J = 5.0 Hz),

4.55-4.50 (m, 1 H), 4.32-4.36 (m, 1 H), 3.77 (dd, 2 H, J = 11.5, 5.0 Hz), 3.68-3.65 (m, 47

H), 3.59-3.57 (m, 2 H), 3.50 and 3.25 (ABq, 2x1 H, J = 18.5 Hz), 3.47-3.44 (m, 2 H),

3.19-3.15 (m, 1 H), 2.93 (dd, 1 H, J = 12.5, 5.0 Hz), 2.74 (d, 1 H, J = 12.5 Hz), 2.62-2.57

(m, 1 H), 2.26-2.22 (m, 2 H), 2.16 (s, 3 H), 1.71-1.64 (m, 4 H), 1.50-1.43 (m, 2 H).

165 (6S,7S)-benzyl-7-(poly(ethylene glycol)ether-2-(biotinylamino)ethane)-3-methyl-8- oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid (10) EA-1-CEP

O O NH H O H NH HN H H AlCl , PhOMe, DCM, NO Me O O N S 3 2 HN H H H H N H N O S H 12 0 °C to RT, 8h H N S O N H 12 O 28 % S O N O O O (10) (9) O OH EA-1-CEP

To an ice-bath cooled solution of compound 9 (0.172 gr, 0.152 mmol) and anisole (198

µL, 1.82 mmol) in CH2Cl2 (3 ml) was added a cold solution of AlCl3 (0.122 gr, 0.91 mmol) in MeNO2. The ice-bath was removed, and the reaction mixture was stirred for 8 h in room temperature. The reaction mixture was diluted with EtOAc (9 ml), and washed with 1 M HCl (3 x 9 ml) and sat. brine (10 ml). The combined aqueous extracts were washed with Et2O (10 ml), acidified to pH 1 with 1 M HCI, and re-extracted with EtOAc

(3 x 10 ml). The combined organic layers were washed with satd. Brine (10 ml), dried over MgSO4, and evaporated in vacuo to give compound 10 (EA-1-CEP) The compound

10 was purified by RP-HPLC (rt = 16.5min, C18 semiprep column, 30-40% B; A = 98%

1 H2O:acetonitrile; B = 99% acetonitrile:H2O). H NMR (CDCl3, 500 MHz) δ 5.81 (q, 1 H,

J = 4.5 Hz), 5.49 (dd, 2 H, J = 20.0, 12.5 Hz), 4.98 (d, 1 H, J = 5.0 Hz), 4.51-4.448 (m, 1

H), 4.29-4.33 (m, 1 H), 3.77 (dd, 2 H, J = 11.5, 5.0 Hz), 3.62-3.58 (m, 47 H), 3.59-3.57

(m, 2 H), 3.50 and 3.25 (ABq, 2x1 H, J = 18.5 Hz), 3.46-3.43 (m, 2 H), 3.24-3.19 (m, 1

H), 2.89 (dd, 1 H, J = 12.5, 5.0 Hz), 2.68 (d, 1 H, J = 12.5 Hz), 2.57-2.5 (m, 1 H), 2.2-

2.19 (m, 2 H), 1.8 (s, 3 H), 1.65-1.5 (m, 4 H), 1.33-1.28 (m, 2 H).

166 4.1.2. Synthesis of Enantiomer of Pentapeptide Precursor of Bacterial Cell

Wall of S. aureus

1. Boc-Xaa-OH (0.8 mmol), HBTU (0.72 mmol), O HO DIEA (1.2 mmol) in DMF EA-2-ALA CH2 C NH CH2 2. TFA (neat), 1 min and 3. Biotin-PEG -NHS (2 eq), DIEA (4 eq) 4 EA-3-ALA PAM resin overnight 4. HF/anisole (9:1 v/v, 0 °C, 2 h)

NH2

H S O H N HN NH O O O H H H NH O 4 N N OH O N N H H O O O O OH

Biotin-PEG4-D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Ala

EA-2-ALA

NH2

H S O H N HN NH O O O H H H NH O 4 N N OH O N N H H O O O

HO O

Biotin-PEG4-D-Ala-L-α-Glu-D-Lys-L-Ala-L-Ala EA-3-ALA

Scheme 2: Solid-phase peptide synthesis scheme of enantiomer of biotinylated pentapeptide cell wall precursor D-Ala-L-γ−Glu-D-Lys-L-Ala-L-Ala, of S. aureus with Boc-chemistry on PAM resin.

167 The enantiomer of bacterial cell wall precursor, D-Ala-L-γ−Glu-D-Lys-L-Ala-L-Ala, and its control peptide with modified Glu residue, D-Ala-L-α−Glu-D-Lys-L-Ala-L-Ala were synthesized manually using the in-situ neutralization protocol for t-Boc chemistry on 4-

Hydroxymethyl-phenylacetamidomethyl (PAM) resin (0.2 mmol scale, 0.85 mmol/g).

Boc-protected amino acids (0.8 mmol) were coupled in 3.2 ml DMF with HBTU (0.72 mmol) and DIEA (1.2 mmol) for 15 min. N-Boc group were removed by treatment of neat TFA for 2 x 1 min followed by a 30 seconds wash with DMF three times. After coupling of the last amino acid residue, Biotin-PEG4-NHS (2 eq.) linker was coupled to both pentapeptides with overnight coupling after 30 sec of DIEA (4 eq.) activation.

Following day, the completion of biotin linker coupling was checked with the detection of free amine via Kaiser test. The peptide-resin was washed with DMF, DCM, and diethyl ether twice, and transferred into HF-cleavage apparatus to dry overnight in high vacuum. Peptides were cleaved from PAM-resin by treatment with HF/anisole (9:1 v/v) at 0 °C for 2 h, and then precipitated with cold diethyl ether. Lyophilized crude peptides were purified by semi-prep RP–HPLC [Vydac C18, 10 µm, 10 mm × 250 mm] with gradient of 5% to 30% B. The purities of peptides were confirmed by analytical RP–

HPLC [Vydac C18, 5 µm, 4 mm × 250 mm]. The molar masses of peptides were determined by MALDI-TOF MS: EA-2-ALA, calcd [M], 962.46, obsd [M+H]+, 962.223;

EA-3-ALA, calcd [M], 962.46, obsd [M+H]+, 962.223.

168 4.1.3. Synthesis of Enantiomer of Bacterial Cell Wall Precursor of Vancomycin-

resistant Enterococci

O 1.Fmoc-L-Ala-OH (1.5 mmol) Cl HO Cl DIC (1.5 mmol) and DMAP (0.08 mmol) DMSO:DCM (1:1) in DMF, overnight OHLi Cl O overnight, RT 2. 20 % piperidine/DMF OH O (1.5 mmol)

1. Fmoc-D-Lys(Boc)-OH (0.8 mmol) DIEA (1.2 mmol), HBTU (0.72mmol) in DMF 45 min 2. 20 % piperidine in DMF (5 and 15 min) 3. Fmoc-L-Glu(OtBu)-OH (0.8 mmol) O Cl DIEA (1.2 mmol), HBTU (0.72 mmol) in DMF 45 min TFA:TIPS:H2O (95:2.5:2.5) H2N O O 4. 20 % piperidine in DMF O 5. Fmoc-D-Ala-OH (0.8 mmol) DIEA (1.2 mmol), HBTU (0.72 mmol) in DMF 45 min 6. 20 % piperidine in DMF 7. Biotin-PEG4-NHS (0.8 mmol), DIEA (1.6 mmol) in DMF, overnight

NH2

H S O H N HN NH O O O H H H NH O 4 N N OH O N O H O O O O OH

Biotin-PEG4-D-Ala-L-γ-Glu-D-Lys-L-Ala-L-Lac EA-4-LAC

Scheme 3. Synthesis scheme of the enantiomer of cell-wall analogue of vancomycin- resistant enterococci (VRE) starting with L-Lac lithium salt on 2-chlorotrityl chloride resin by standard solid-phase peptide synthesis with Fmoc-chemistry.

169 The enantiomer of the bacterial cell wall found in vancomycin-resistant bacteria (VRE),

D-Ala-L-γ−Glu-D-Lys-L-Ala-L-Lac, was synthesized manually by solid-phase peptide synthesis using Fmoc-chemistry on 2-chlorotrityl chloride resin (0.2 mmol scale, 1.6 mmol/g). 1.5 mmol L-Lac lithium salt was coupled directly to 2-chlorotritiyl chloride resin in 5 ml of DMSO:DCM (1:1) overnight. Following day, peptide-resin was washed with DMSO, DMF and DCM twice. The next residue Fmoc-L-Ala-OH (1.5 mmol) in 2 ml DMF was added to resin-complex, mixture was cooled to 0 °C. DIC (1.5 mmol) and catalytic amount of DMAP (0.08 mmol) in 2 ml DMF were added to resin-complex drop- wise over 30 minutes, and left overnight for coupling. Following day, N-Fmoc protecting group was removed with 20% piperidine in DMF for 5 and 15 min. treatment. The following amino acids, Fmoc-D-Lys(Boc)-OH, Fmoc-L-Glu(tBu)-OH and Fmoc-D-Ala-

OH, were coupled using standard Fmoc-chemistry, where 0.8 mmol amino acid in 3.2 ml

DMF was activated by HBTU (0.72 mmol) and DIEA (1.2 mmol) for 30 sec, and coupled to peptide-resin for 45 min. After coupling of the last amino acid, Fmoc-D-Ala-OH, N-

Fmoc group was removed with 20 % piperidine in DMF, and 0.8 mmol Biotin-PEG4-

NHS linker in 3.2 ml DMF with 1.6 mmol DIEA was coupled to peptide-resin overnight.

After checking Kaiser test, the peptide-resin was washed with DMF, DCM, and diethyl ether twice to dry overnight in high vacuum. Biotinylated peptide was cleaved from resin with an acidic cleavage cocktail containing Trifluoroacetic acid (TFA)/Tri- isopropylsilane (TIPS)/H2O (95:2.5:2.5) for 3 hours. TFA was evaporated under flow of nitrogen, and the cleaved peptide was precipitated with ice-cold diethyl ether three times.

Lyophilized crude peptide was purified by semi-prep C18 RP-HPLC [Vydac C18, 10 µm,

10 mm × 250 mm] using a gradient of 10% to 45% solvent B over 40 min. The purity of

170 peptide was confirmed by analytical RP–HPLC [Vydac C18, 5 µm, 4 mm × 250 mm].

Final pure peptide gave MALDI-TOF-MS data as calcd [M], 963.46, obsd [M+H]+,

963.115.

4.2. Phage Display Screening with Linear and Cyclic Phage Display Peptide

Libraries

4.2.1. Materials and Buffers

Phage Display Peptide Libraries: Linear 12-mer, linear 7-mer, and cyclic 7-mer libraries purchased from New England Biolabs.

Streptavidin-coated Magnetic Beads: Purchased from New England Biolabs (# S1420S)

Neutravidin-coated Magnetic Beads: Purchased from GE Healtcare (#78152104011150)

Luria-Bertani (LB) Medium: 10 g Bacto-tryptone, 5 g Yeast extract, 5 g NaCl, in 1 L dH2O, autoclaved.

LB Agar: 10 g Bacto-tryptone, 5 g yeast extract, 5 g NaCl, 7.5 g Bacto-agar in 1L dH2O, autoclaved.

Top-Agar: 10 g Bacto-tryptone, 5 g yeast extract, 5 g NaCl, 3.5 g Bacto-agar in 1L dH2O, autoclaved.

171 LB/ IPTG/X-gal Plate: 1% Bacto-tryptone, 0.5% Yeast extract, 1.0% NaCl, 1.5% Bacto-

Agar, 60 µg/ml X-gal, 0.1 mM IPTG.

LB-Agar Plate: LB Agar supplied with 10 µg/ml of tetracycline.

Tetracycline Stock Solution: 20 mg/ml tetracycline in 1:1 ethanol:water

Streptavidin Stock Solution: 1.5 mg/ml in 1 ml 10 mM PBS (pH 7.2)

Binding Buffer: 0.1 M NaHCO3 (pH 8.6)

Blocking Buffer: 0.1 M NaHCO3 (pH 8.6), 5 mg/ml BSA

TBS: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl in 1 L dH2O, autoclaved

Washing Buffer-1: 500 ml TBS with 0.1% (v/v) Tween-20

Washing Buffer-2: 500 ml TBS with 0.5% (v/v) Tween-20

Elution Buffer: 0.2 M Glycine-HCl (pH 2.2), 0.1% (w /v) BSA

Neutralization Buffer: 1.0 M Tris–HCl (pH 9.1)

PEG Purification Buffer: 20% (w /v) polyethylene glycol-8000, 2.5 M NaCl

Bacterial Strain: E. coli host strain ER2738 (F ́ proA+B+ lacIq (lacZ)M15 zzf::Tn10

(TetR)/fhuA2 glnV (lac-proAB) (hsdMS-mcrB)5 [rk– mk–McrBC–]) was provided by phage display kits (New England Biolabs)

172 4.2.2. Maintenance of E. coli ER2738 Host Cells

E. coli host strain ER2738 (New England Biolabs) has a mini-transposon that confers tetracycline resistance, and hence E. coli cells were streaked out on LB-agar plate containing 10 µg/ml of tetracycline. 1 µl from glycerol stock of E. coli ER2738 was streaked-out on LB-agar (10 µg/ml Tet) plates overnight at 37 °C. Following day one well-separated colony from streaked-out plate was re-streaked-out on LB-agar (10 µg/ml

Tet) plate overnight 37 °C to ensure each colony has a single DNA. The re-streaked-out plates were wrapped with parafilm and stored at 4 °C in the dark for a week.

4.2.3. Checking Phage Titers of Linear and Cyclic Phage Display Libraries

The number of phage clone in each phage display peptide library (New England Biolabs), linear 12-mer, linear 7-mer and cyclic 7-mer, were confirmed by checking the number of phage titers upon received. Single colony from re-streaked out host cell E. coli ER2738 was inoculated in 10 ml of LB medium (10 µg/ml Tet) at 37 °C by shaking at 250 rpm till cells were grown to mid-log phase, OD600 of 0.5. Meanwhile, serial ten-fold dilutions of each library were prepared in 200 µl LB medium from 102 to 1010. Top-agar was melted, and dispended of 3 ml into the sterilized test tube (BD-Falcon 352003) for each serial dilution, and kept them in hot water bath at 45 °C till use. LB medium/X-gal/IPTG plates (Teknova, San Diego) for each dilution were pre-warmed in incubator at 37 °C at least one hour. When the bacterial culture reached OD600 of 0.5, 10 µl of serial dilutions

173 of each library were infected with 200 µl of E. coli culture by incubating for 3 min at room temperature. Infected library dilutions were mixed with top-agar by quick vortexing, and immediately spread onto LB medium/X-gal/IPTG plates. After cooling in room temperature for 5 min, the plates were incubated at 37 °C overnight. Following day, the blue plaques were counted and phage titers were calculated as plaque forming unit (pfu/ml).

4.2.4. Control Biopanning Experiment with Streptavidin as Model Target

4.2.4.1. Solid-phase Biopanning in 96-well plate

One well of 96-well plate was coated with 150 µL of 100 µg/mL of streptavidin in 0.1 M

NaHCO3 (pH 8.6) at 4 °C in a humidified box with constant shaking overnight.

Following day, streptavidin solution was discarded, and the coated well was blocked with blocking buffer containing 0.1 µg/ml streptavidin to complex any free biotin in BSA by filling the well completely for 2 h at 4 °C. The blocked well was washed with TBST (0.1

% Tween-20) six times by inverting on clean paper towel each time. Linear 12-mer

Phage Display Library (PH.D.-12, New England Biolabs) were diluted 100-fold to get

1011 pfu/ml phage in 100 µl of TBST (0.1 % Tween-20), and incubated in streptavidin- coated well for 30 min by constant shaking at room temperature. Meanwhile single colony from re-streaked out plate of E. coli ER2738 was inoculated in 20 ml of LB

174 medium at 37 °C by shaking at 250 rpm till cells grow to early-log phase, OD600 of 0.1 for amplification step. Unbound phage were removed, and the well was washed ten times with TBST (0.1 % Tween-20) by vigorous shaking for 2 min at each time. Then, bound phage were eluted by incubating with 100 µL of 0.1 mM biotin solution for 1 h. 1 µL of eluted phage was used to determine the number of phage covered during screening with phage tittering method as described above. Remaining eluted phage were amplified by adding into E. coli culture at OD600 of 0.05 for 4.5 h shaking at 37 °C at 250 rpm. Then, the culture was transferred into a 50 ml polypropylene tube (BD-Falcon 352070) and centrifuged at 12,000 g at 4 °C for 10 minutes with Sorvall SA-600 rotor. The supernatant was transferred into a new 50 ml tube, and spun again for another 10 minutes. 80% of the upper supernatant was transferred into a fresh 50 ml Falcon tube that was added 1/6 volume of 20% (v/v) PEG/2.5 M NaCl to precipitate the phage overnight in the fridge. The next day, the precipitated culture was spun down at 12,000 g at 4 °C for 15 minutes. The supernatant was discarded, and the precipitated phage was re- spun to completely remove the residual supernatant for another 3 min. The precipitated pellet was suspended in 1 ml of TBS for 1 hour till completely dissolved, and transferred into a sterile 1.5 ml microcentrifuge tube. The suspended phage was centrifuged for 5 minutes at 4 °C, and the 80% of the upper supernatant were transferred into a new sterile microcentrifuge tube. The phage was precipitated on ice by adding 1/6 volume of 20%

(v/v) PEG/2.5 M for one hour. The precipitated phage were centrifuged at 14,000 rpm at

4 °C for 10 minutes. The supernatant was discarded, and the tube was re-spun for another 2 min to pellet any insoluble contaminants. After removing the residual supernatant with sterile pipet, the white pellet was suspended in 200 µL of TBS for 1 h.

175 This is the amplified eluate, and 10 µL of the amplified eluate was tittered to determine the input titer for the subsequent rounds of biopanning. Biopanning was repeated for three rounds, and in subsequent rounds; Tween-20 concentration was raised from 0.1% to

0.5%. The second round was carried out using of the first-round amplified eluate as input phage, and the second-round amplified eluate was used as phage input in the third round selection.

4.2.4.2. Solid-phase Biopanning in 60 × 100 mm petri-dish

60 × 100 mm petri dish was coated with 1.5 ml of 100 µg/ml streptavidin in 0.1 M

NaHCO3, pH 8.6 at 4 °C with gentle agitation in a sealed humid box overnight.

Following day, streptavidin solution was discarded, and the streptavidin-coated petri dish was blocked with blocking buffer containing 0.1 µg/ml streptavidin by filling till the top for 2 h at 4 °C. The blocked well was washed with TBST (0.1% Tween-20) six times by inverting on clean paper towel each time. Linear 12-mer Phage Display Library (PH.D. -

12, New England Biolabs) were diluted 100-fold to get 1011 pfu/ml phage in 100 µl of

TBST (0.1% Tween-20), and incubated in streptavidin-coated well for 30 min by constant shaking at room temperature. Meanwhile single colony from re-streaked out plate of E. coli ER2738 was inoculated in 20 ml of LB medium at 37 °C by shaking at

250 rpm till cells grew early-log phase, OD600 of 0.05 for amplification step. Unbound phage were removed, and the well was washed ten times with TBST (0.1 % Tween-20) by vigorous shaking for 2 min at each time. Then, bound phage were eluted by

176 incubating with 1 ml of 0.1 mM biotin solution for 1 h. 1 µL of eluted phage was used to determine the number of phage covered during screening with phage tittering method as described above. Remaining eluted phage were amplified by adding into E. coli culture at OD600 of 0.1 for 4.5 h shaking at 37 °C at 250 rpm. Then, the culture was transferred into a 50 ml polypropylene tube (BD-Falcon 352070) and centrifuged at 12,000 g at 4 °C for 10 minutes with Sorvall SA-600 rotor. The supernatant was transferred into a new 50 ml tube, and spun again for another 10 minutes. 80% of the upper supernatant was transferred into a fresh 50 ml Falcon tube that was added 1/6 volume of 20% (v/v)

PEG/2.5 M NaCl to precipitate the phage overnight in the fridge. The next day, the precipitated culture was spun down at 12,000 g at 4 °C for 15 minutes. The supernatant was discarded, and the precipitated phage was re-spun to completely remove the residual supernatant for another 3 min. The precipitated pellet was suspended in 1 ml of TBS for

1 hour till completely dissolved, and transferred into a sterile 1.5 ml microcentrifuge tube. The suspended phage was centrifuged for 5 minutes at 4 °C, and the 80% of the upper supernatant were transferred into a new sterile microcentrifuge tube. The phage was precipitated on ice by adding 1/6 volume of 20% (v/v) PEG/2.5 M for one hour. The precipitated phage were centrifuged at 14,000 rpm at 4 °C for 10 minutes. The supernatant was discarded, and the tube was re-spun for another 2 min to pellet any insoluble contaminants. After removing the residual supernatant with sterile pipet, the white pellet was suspended in 200 µL of TBS for 1 h. This is the amplified eluate, and

10 µL of the amplified eluate was tittered to determine the input titer for the subsequent rounds of biopanning. Biopanning was repeated for three rounds, and in subsequent rounds; Tween-20 concentration was raised from 0.1% to 0.5%. The second round was

177 carried out using of the first-round amplified eluate as input phage, and the second-round amplified eluate was used as phage input in the third round selection.

4.2.4.3.Solution-phase Biopanning with Streptavidin-coated Magnetic Beads

50 µL of Streptavidin-coated (SA-coated) magnetic beads were transferred into a sterile

1.5 ml microcentrifuge tube, and washed with TBST (0.1% Tween-20) twice to remove any contaminants by pelleting for 2 min with magnetic capture. Beads were blocked with

1 ml of blocking buffer containing 0.1 µg/ml streptavidin for 2 h at 4 °C. Then, SA- coated beads were washed with four times TBST (0.1 % Tween-20) by pelleting for 1 minute each time. Linear 12-mer phage library was diluted 100-fold to get 1011 pfu/ml in

200 µl TBST (0.1% Tween-20), and added into suspended SA-coated beads to incubate for 15 min by gentle shaking at room temperature. After incubation, the unbound phage were pipette out, and the non-specifically bound phages were washed away by ten times

TBST (0.1% Tween-20) by pelleting for 1 minute each time. The specifically bound phage were recovered through competitive elution with 0.1 mM biotin solution for 1 h at room temperature. The eluate was amplified by infecting of host E. coli ER2738. A single colony from host E. coli ER2738 strain was grown in 20 ml LB medium till OD600 is 0.05. Eluates from each round were added into grown E. coli culture, and incubated for

4.5 h at 37 °C at 250 rpm for infection process. The culture was transferred into a 50 ml

Falcon tube, and centrifuged at 12,000 g at 4 °C for 10 minutes. The supernatant was transferred into a new sterile tube, and spun for another 10 minutes. 80% of the upper

178 supernatant was transferred into a fresh falcon tube that was filled with 1/6 volume of

20% (v/v) PEG/2.5 M NaCl to precipitate the phages overnight in the fridge. The next day, the precipitated culture was centrifuged at 12,000 g, at 4 °C for 15 minutes. The supernatant was discarded, and the precipitated phage was spun for 3 minutes to completely remove the residual supernatant. The precipitated pellet was observed one side of Falcon tube as white smear. It was suspended in 1 ml of TBS for 1 h, and transferred to a sterile 1.5 ml microcentrifuge tube. The suspended phage was centrifuged for 5 min, and the 80% of the upper supernatant was transferred into a new sterile microcentrifuge tube. The phage were precipitated by adding 150 µL of 1/6 volume of 20% (v/v) PEG/2.5 M NaCl on ice for 1 h, followed by centrifugation at

12,000 g at 4 °C for 10 minutes. The supernatant was discarded, and the tube was re- spun for another 3 min to pellet any insoluble contaminants. After removing the residual supernatant, the white precipitated pellet was suspended in 200 µL of TBS for 1 h to dissolve completely. This is the amplified eluate. 10 µL of the amplified eluate was tittered to determine the number of phage and phage input for the next round. Biopanning was repeated for three rounds, and in subsequent rounds; Tween-20 concentration was raised from 0.1% to 0.5%.

4.2.5. DNA sequencing and Analysis

After the third round of each biopanning, 102 - 104 dilutions of unamplified eluate were spotted on LB/Xgal/IPTG plate with phage tittering protocol described above. Randomly

179 picked 15 plaques from each experiment with these plates were sent to Genewiz with -96 gIII sequencing primer 5 ́-OHCCC TCA TAG TTA GCG TAA CG –3 ́, without further application. The reverse complement sequences of the sequenced oligonucleotides were then translated into peptide sequences using ExPASy Translate Tool

(http://web.expasy.org/translate/).

4.2.6. Biopanning Experiments with Target Molecules using Linear and Cyclic

Phage Display Peptide Libraries

4.2.6.1.The More Stringent Biopanning Selection

50 µL of Streptavidin-coated magnetic beads were transferred into 1.5 ml microcentrifuge tube, and washed with TBST (0.1% Tween-20) twice to remove any contaminants by pelleting for 2 min with magnetic capture. SA-coated beads were then blocked with 1 ml of blocking buffer containing 0.1 µg/ml streptavidin for 2 h at 4 °C without agitation. During the blocking of streptavidin-beads, 25 nanograms of target molecules were incubated with 100-fold diluted (1011 pfu/ml) phage display peptide libraries in 200 µl TBST (0.1% Tween-20) for 30 min by agitation in room temperature.

Then, blocking buffer was washed away with 1 ml of TBST (0.1 % Tween-20) six times by pelleting for 1 min each time. The phage/target molecule solution was added into the blocked streptavidin-coated magnetic beads, and incubated for 30 min at room temperature by gentle agitation. After incubation, unbound phage were removed by

180 pelleting on magnetic rack, and the solution was washed with 1 ml of TBST (0.1%

Tween-20) eight times by pelleting for 1 min each time. The bound phage were eluted by incubating with 1 ml of elution buffer for 15 min, and then immedidately neutralized with

150 µl of neutralization buffer. This is the eluted phage to be used for further amplification. 1 µl of eluted phage were used to run blue/white phage tittering assays to determine the number of phage recovered during the selection. The rest was amplified by infecting host cell E. coli ER2738.

4.2.6.2.Phage Amplification and Purification

A single colony from host E. coli ER2738 strain was inoculated in 20 ml LB medium till

OD600 reached to 0.05. The eluate from the first round was amplified by infecting of host

E. coli ER2738 by incubating for 4.5 h at 37 °C at 250 rpm. The culture was transferred into a 50 ml Falcon tube, and centrifuged at 12,000 g at 4 °C for 10 minutes. The supernatant was transferred into a new sterile tube, and spun for another 10 minutes.

80% of the upper supernatant was transferred into a new sterile 50 ml Falcon tube. The phage were added 1/6 volume of 20% (v/v) PEG/2.5 M NaCl, and precipitated overnight in the fridge. The next day, the precipitated culture was centrifuged at 12,000 g, at 4 °C for 15 minutes. The supernatant was discarded, and the precipitated phage were re-spun for 3 min to completely remove the residual supernatant. The white precipitated pellet was observed on one side of Falcon tube. The phage pellet was suspended with 1 ml of

TBS for 1 h, and transferred to a sterile 1.5 ml microcentrifuge tube. The suspended

181 phage was centrifuged for 5 min, and the 80% of the upper supernatant was transferred into a new sterile 1.5 ml microcentrifuge tube. The phage were precipitated again by adding 150 µL of 1/6 volume of 20% (v/v) PEG/2.5 M NaCl on ice for 1 h, followed by centrifugation at 12,000 g at 4 °C for 10 minutes. The supernatant was discarded, and the tube was re-spun for another 3 min to pellet any insoluble contaminants. After removing the residual supernatant, the white precipitated pellet was suspended in 200 µL of TBS for 1 h to dissolve completely. This is the amplified eluate. 10 µL of the amplified eluate was tittered to determine the number of phage recovered and also phage input for the next round.

4.2.6.3. Additional Rounds

Additional four more rounds of biopanning were carried out for each target molecule. In each round, the same amount of target molecules (25 nanograms) was used during the selection. The stringency was control by raising the concentration of Tween-20 in the washing and the binding steps after first round to 0.5% from 0.1%. 40 phage from the fourth and the fifth rounds of biopanning were randomly selected for DNA sequencing.

Also, in the fourth round of biopanning, subtractive screening was utilized by first incubation the phage input from the third round of biopanning with streptavidin-coated magnetic beads to eliminate the streptavidin-specific peptide binders. Then, the unbound phage were transferred into a new microcentrifuge tube, and used as library stock in the incubation with target molecule to accomplish the biopanning.

182 4.2.6.4.The High Yield Biopanning Selection

50 µL of streptavidin-coated magnetic beads were transferred into 1.5 ml microcentrifuge tube, and washed with TBST (0.1 % Tween-20) twice to remove any contaminants by pelleting for 2 min with magnetic capture. Streptavidin-coated magnetic beads were then blocked with 1 ml of blocking buffer containing 0.1 µg/ml streptavidin for 2 h at 4 °C without agitation. During the blocking of streptavidin-beads, 5 mg of target molecules were incubated with 100-fold diluted (1011 pfu/ml) phage display peptide libraries in 200

µl TBST (0.1% Tween-20) for 1 h by agitation in the room temperature. Then, blocking buffer was washed away with 1 ml of TBST (0.1% Tween-20) six times by pelleting for

1 min each time. The phage/target molecule solution was added into the blocked streptavidin-coated magnetic beads, and incubated for 30 min at room temperature by agitation. After incubation, unbound phage were removed by pelleting on magnetic rack, and the solution was washed with 1 ml of TBST (0.1% Tween-20) eight times by pelleting for 1 min each time. The bound phage were eluted by incubating with 1 ml of elution buffer for 15 min, and then immedidately neutralized with 150 µl of neutralization buffer. This is the eluted phage to be used for further amplification. 1 µl of eluted phage were used to do blue/white phage tittering assays to determine the number of phage recovered during the selection. The rest of the eluate was amplified by infecting host cell E. coli ER2738.

183 4.2.6.5. Phage Amplification and Purification

A single colony of E. coli ER2738 strain was inoculated in 20 ml LB medium till OD600 reached to 0.05. The eluate from the first round was amplified by infecting of host E. coli

ER2738 by incubating for 4.5 h at 37 °C at 250 rpm. The culture was transferred into a

50 ml Falcon tube, and centrifuged at 12,000 g at 4 °C for 10 minutes. The supernatant was transferred into a new sterile tube, and spun for another 10 minutes. 80% of the upper supernatant was transferred into a new sterile 50 ml Falcon tube. The phage were added 1/6 volume of 20% (v/v) PEG/2.5 M NaCl, and precipitated overnight in the fridge. The next day, the precipitated culture was centrifuged at 12,000 g, at 4 °C for 15 minutes. The supernatant was discarded, and the precipitated phage were re-spun for 3 min to completely remove the residual supernatant. The white precipitated pellet was observed on one side of Falcon tube. The phage pellet was suspended with 1 ml of TBS for 1 h, and transferred to a sterile 1.5 ml microcentrifuge tube. The suspended phage was centrifuged for 5 min, and the 80% of the upper supernatant was transferred into a new sterile 1.5 ml microcentrifuge tube. The phage were precipitated again by adding

150 µL of 1/6 volume of 20% (v/v) PEG/2.5 M NaCl on ice for 1 h, followed by centrifugation at 12,000 g at 4 °C for 10 minutes. The supernatant was discarded, and the tube was re-spun for another 3 min to pellet any insoluble contaminants. After removing the residual supernatant, the white precipitated pellet was suspended in 200 µL of TBS for 1 h to dissolve completely. This is the amplified eluate. 10 µL of the amplified eluate was tittered to determine the number of phage recovered and also phage input for the next round.

184 4.2.6.6.Additional Rounds

Additional three more rounds of biopanning were carried out. In each round of selection, the amount of target molecule used in screening was decreased from the first round to the fourth round as in the trend of 5 mg, 2 mg, 500 ng and 100 ng, respectively. Instead of a subtractive screening method, the magnetic beads were changed in the alternating rounds from streptavidin-coated magnetic beads to neutravidin-coated magnetic beads.

Additionally, the stringency was controlled by raising the concentration of Tween-20 in the washing and the binding steps after first round to 0.5% from 0.1%. Randomly selected phage clones from the unamplified elutes of third and the fourth rounds of selection were picked for DNA sequencing.

4.2.6.7.Negative Selection for Both Biopanning

Negative selection was also run at the second, the third and the fourth rounds of biopanning to check the enrichment factors of each round. The same biopanning procedure was carried out in negative selection by using solely neutravidin-coated magnetic beads incubated the phage library without target molecule in the second and the fourth rounds, and solely streptavidin-coated magnetic beads incubated the phage library without target molecule in the third round. After biopanning selection, blue/white plaque assays were carried out to determine the number of phage recovered that bound to magnetic beads to calculate enrichment factors.

185 4.2.6.8. Blue/White Plaque Assays for Phage Titering

After each round of biopanning, the number of phage in each unamplified and amplified eluate was determined by blue/white plaque assay as in a number of plaque forming units. Single colony from re-streaked out host cell E. coli ER2738 was inoculated in 10 ml of LB (10 µg/ml tetracycline) medium at 37 °C by shaking at 250 rpm till cells grew mid-log phase, OD600 of 0.5. Meanwhile, serial ten-fold dilutions of each library were prepared in 200 µl LB medium from 102 to 1010. 102 to 104 serial dilutions were prepared for unamplified eluates, and 108 to 1011 serial dilutions for amplified eluates after each round. Top-agar was melted, and dispended 3 ml for each serial dilution into sterilized test tube (BD-Falcon 352003), and top-agar was kept in hot water bath at 45 °C till use.

LB medium/X-gal/IPTG plates (Teknova, San Diego) for each dilution were pre-warmed in incubator at 37 °C at least one hour. When the bacterial culture reached OD600 of 0.5,

10 µl of serial dilutions of each library were infected with 200 µl of E. coli culture by incubating for 3 min at room temperature. Infected library dilutions were mixed with top-agar by a quick vortexing, and immediately spread onto LB medium/X-gal/IPTG plates. After cooling at room temperature for 5 min, the plates were incubated at 37 °C overnight. Following day, the blue plaques are appeared as a result of cleavage of XGal by β-galactosidase encoded by lacZ gene in M13 phage in the presence of inducer IPTG on LB medium/X-gal/IPTG plates, and were counted and phage titers were calculated as plaque forming unit (pfu/ml).

186 4.2.6.9.Plaque Purification for DNA sequencing and Phage ELISA

Single colony of E. coli ER2537 was inoculated overnight in 10 mL of LB (10 µg/ml Tet) medium at 37 °C at 250 rpm. Overnight culture of E. coli was diluted to 1:100 in LB medium, and 1 ml of diluted culture was transferred into a sterile test tube for each plaques to be tested. Randomly picked single well-separated blue plaques from

LB/IPTG/Xgal plates were transferred into 1 ml of diluted E. coli culture by stabbing on the agar then slowly aspirated over the plaque with a sterile pipette. Then, the phage plaques were amplified by incubating for 4.5 h at 37 °C at 250 rpm. After incubation, the amplified phage plaques culture was transferred into 1.5 ml microcentrifuge tube, and centrifuged at 14,000 rpm for 1 min at room temperature. The supernatant containing the amplified phage were transferred to a new sterile tube, and re-spun for another 1 min.

The upper 600 µl of the supernatant was then mixed with 600 µl of 50% glycerol in a new microcentrifuge tube. Individual phage stocks were kept at -20 °C till further use for

DNA purification and phage-ELISA.

4.2.6.10. DNA Sequencing and Analysis

After plaque purification of each phage clones of interest, 50 µl of each amplified phage supernatant was sent Genewiz, Cambrige, MA with -96 gIII sequencing primer 5 ́-

OH CCC TCA TAG TTA GCG TAA CG –3 ́ for DNA sequencing. The reverse complement sequences of the sequenced oligonucleotides were then translated into

187 peptide sequences using ExPASy Translate Tool (http://web.expasy.org/translate/). The peptides were examined for their isoelectric point (pI) and net charge through Innovagen

Peptide Property Calculator (http://www.innovagen.se/custom-peptide-synthesis/peptide- property-calculat or/peptide-property-calculator.asp). The hydrophobicity of peptides was predicted using Antimicrobial Peptide Predictor (http://aps.unmc.edu/AP/ prediction/prediction_main.php). Also, the peptide sequences were blasted for similar antibacterial peptide sequence through Antimicrobial Peptide Database

(http://aps.unmc.edu/AP/database/query_input.php).

4.3. Phage ELISA for Binding Confirmations to Target Molecules

4.3.1. Buffers

TBS: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl in 1 L dH2O, autoclaved.

Binding Buffer: 0.1 M NaHCO3 (pH 8.6)

Blocking Buffer: 0.1 M NaHCO3 (pH 8.6), 5 mg/ml BSA

Washing Buffer: TBS with 0.05 % Tween-20

0.05 M Citric Acid: 10.507 gr of Citrate monohydrate in 1 L of dH2O, pH is adjusted to 4 with 10 N NaOH, sterilized.

188 ABTS Stock Solution: 0.22 mg of azino-bis(3-ethylben-zothiazole) sulfonic acid (ABTS) in 1 ml of 0.05 M Citric Acid.

ABTS Substrate Solution: 36 ml of 30% H2O2 in 21 ml of ABTS stock solution for each

96-well plate.

4.3.2. Phage ELISA via Sandwich ELISA Protocol

The maxisorp 96-well ELISA plates (Nunc Maxisorp #44-2404-21, Fisher Scientific) were coated with 50 µl of 100 µg/ml streptavidin solution overnight at 4 °C in a humidifier box. Following day, 96-well plates were washed with TBST (0.5% Tween-

20) four times. Then, the wells were blocked with blocking buffer for 2 hour in fridge, and washed six times with TBST (0.5% Tween-20). 200 pmol of biotinylated target molecules in binding buffer was added into each well, and allowed to bind for 2 h at room temperature by shaking at 200 rpm. After washing six times with TBST (0.5%

Tween-20), diluted amplified phage to 1010 pfu/ml in 100 µl washing buffer was added into each well and incubated for 2 h at room temperature. After incubation, the wells were washed ten times with TBST (0.5% Tween-20), and 1:5000 diluted Horseradish

Peroxidase-conjugated anti-M13 monoclonal antibody (GE Healthcare, #27-9421-01) in

200 µl blocking buffer to each well, followed by incubation for 1 with agitation at room temperature. After washing the wells six times with TBST (0.5% Tween-20), freshly prepared 200 µl ABTS substrate solution, pH 4, (Sigma Aldrich, #A-1888) with 30%

H O from stock solution was added into each well, and incubate for 1 h at room 2 2

189 temperature with gentle agitation. After color development of ABTS substrate, the absorbance of each well was recorded at 405 nm by Tecan microplate reader. Also, wells coated with streptavidin, bovine serum albumin and empty well were used as negative controls for binding specificity by carrying out the same protocols without using any target molecule.

4.7. Biopanning Experiments with Bicyclic Phage Display Peptide Libraries

4.7.1. Buffers and Materials

Bicyclic Phage Display Library: Glycerol stocks of Library A and Library B constructs provided by Prof. Christian Heinis Group, EPFL Lausanne.

Streptavidin-coated Magnetic Beads: Purchased from New England Biolabs (# S1420S)

Neutravidin-coated Magnetic Beads: Purchased from GE Healtcare (#78152104011150)

2 x YT medium: 16 g Bacto-tryptone, 10 g yeast extract, and 5 g NaCl in 1 L dH2O, autoclaved.

2 x YT medium / 20% Glycerol: 2 x YT medium with 230 ml of glycerol in 1 L dH2O, autoclaved.

2 x YT- Agar: 2 × YT medium with 15 g of Bacto-agar in 1 L dH2O, autoclaved.

190 2 x YT- Agar Plates 1: 2 × YT agar with 30 µg/ml chloramphenicol

2 x YT- Agar Plates 2: 2 × YT agar

Chloramphenicol stock solution: 30 mg/ml of chloramphenicol in ethanol.

Reaction buffer: 20 mM NH4HCO3, 5 mM EDTA, pH 8.0

Binding buffer: 10 mM TBS, 150 mM NaCl, pH 7.4

Blocking buffer: TBS buffer with 1% (w/v) BSA and 0.1% (v/v) Tween-20

Washing buffer 1: TBS buffer with 0.1% (v/v) Tween-20

Elution buffer: 0.2 M Glycine-HCl (pH 2.2)

Neutralization buffer: 1.0 M Tris–HCl (pH 8.0)

PEG Purification Buffer: 20% (w /v) polyethylene glycol-8000, 2.5 M NaCl

TCEP stock solution: 20 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma Aldrich,

#C4706) in dH2O.

TBMB stock solution: 50 mM 1,3,5- tris(bromomethyl)benzene (TBMB) (Sigma Aldrich,

# 140066), stock in acetonitrile.

Host bacterial cell: E. coli TG1 (Lucigen, #60502) [F' traD36 proAB lacIqZ ΔM15] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK - mK -)

191 4.7.2. Maintenance of Host E. coli TG1 cells

1 µl aliquot from glycerol stock of E. coli TG1 host strain were streaked out on 2 x YT agar plate without antibiotic, and incubated overnight at 30 °C. Following day one well- separated colony from streaked-out plate was re-streaked-out on 2 x YT agar plate overnight 30 °C to ensure each colony has a single DNA. The re-streaked-out plates were wrapped with parafilm and stored at 4 °C in the dark for a week.

4.7.3. Bicyclic Phage Library Production and Amplification

In 2 L erlenmeyer flasks containing 500 ml of 2 × YT/chloramphenicol for each library and one control for each target molecule were inoculated with the library glycerol stocks to reach an OD600 of 0.1 overnight by vigorous shaking at 250 rpm at 30 °C. Following day, the number of inoculated cells was determined by taking 50 µL aliquot from each flask and, plated serial 10-fold dilutions (from 100 to 106) on 2 × YT/30 µg/ml chloramphenicol agar plates.

192 4.7.4. PEG purification of Amplified Phage Library

The overnight amplified phage library was transferred into 1 L centrifuge bottle and centrifuged at 7500 rpm at 4 °C with Sorvall Lynx 4000 for 30 min. The phage- containing supernatant is transferred into a new sterile 1 L centrifuge bottle. The phage were purified by precipitation with 1/4 volume of ice-cold 20% (w/v) PEG8000/2.5 M

NaCl on ice-bath for 1 h by inverting several times. After PEG precipitation, purified phage were centrifuged at 8500 rpm for 45 min at 4 °C to obtain greyish white phage pellet on one side of the centrifuge bottle. The supernatant was discarded, and the phage pellet is suspended in 10 ml freshly degassed Reaction Buffer for 30 min. After dissolving the phage pellet completely, the phage containing solution was transferred to a sterile 50 ml Falcon tube. The brownish pellets were obtained by centrifugation at 4000 rpm for 15 min at 4 °C, and the supernatant containing phage is carefully transferred to a new 50 ml falcon tube, and 50 µL aliquot from PEG supernatant was used to determine the number of phage recovered.

4.7.5. Reduction of Cysteine Residues in Phage Library

To the 10 ml PEG-purified phage solution, 1 ml of freshly prepared and degassed 20 mM

TCEP was added to get 1 mM final concentration of TCEP to reduce the cysteine residues on phage. The tubes were mixed well by inverting several times and incubated for 1 h in a hot water bath at 42 °C. The reduced solution was cooled by incubation for 5

193 min on ice-bath, then transferred to a Vivaspin-20 100,000 PES filter tubes (Sartorius

Stedim, #VS2042) to centrifuge at 4000 rpm at 4 °C until the volume is reduced to around 1 ml. 10 ml of ice-cold degassed reaction buffer was added into filter tube, and spun again until the volume is reduced to around 1 ml. It was repeated one more time to ensure to recovered all reduced phage and also washed away excess TCEP solution. The phage were pipette into a sterile 50 ml Falcon tube and the volume was adjusted to 8 ml by adding ice-cold degassed reaction buffer. 50 µL aliquot from TCEP reduction supernatant was used to determine the number of phage recovered.

4.7.6. Bicyclization of Phage Library

To the 8 ml of TCEP-reduced phage solution, 2 ml 100 µM TBMB in acetonitrile was added by preparing prior to use by diluting the 50 mM stock in acetonitrile. The phage containing solution was mixed by inverting the Falcon tube several times, and incubated for 1 h in a hot water bath at 30 °C. Then, the phage were precipitated by adding 2.5 ml

(1/4 volume) ice-cold 20% (w/v) PEG8000/2.5 M NaCl solution on ice-bath for 30 min.

The phage were pelleted by centrifugation at 4000 rpm for 30 min at 4 °C, and white smear-sized pellet were collected on one side of the Falcon tube. The supernatant was discarded without disturbing the phage pellets, and the phage pellet was suspended in 1 ml blocking buffer at 4 °C until it is used for the biopanning. 50 µL aliquot from TBMB modification supernatant was used to determine the number of phage recovered.

194 4.7.7. Phage Titering

One colony of E. coli TG1 cells from re-streaked out agar plates were inoculated in 5 ml

2 × YT medium till OD600 reached to 0.4. Meanwhile, eight 10-fold dilutions in 2 × YT medium were prepared for each sample in 96-well plate. 20 µl from each dilution was added to 180 µl of E. coli TG1 cells in exponential growth (OD600 of 0.4) to allow infection for 90 min at 37 °C. After incubation, 20 µl of each dilution was spotted on 2 ×

YT/30 µg/ml chloramphenicol agar plates, and incubated overnight at 37 °C. Following day, the number of phage at each dilution were counted and calculated as in transducing unit (TU).

4.7.8. Biopanning

The day before biopanning selection, 5 ml of 2 x YT medium was inoculated with a single colony of E. coli TG1 overnight is inoculated overnight. 50 µl Streptavidin-coated magnetic beads for each biopanning for target molecule and negative controls were transferred to a 1.5 ml centrifuge tube, washed twice with 1 ml binding buffer by pelleting 1 min each. SA-beads were suspended in 50 µl of binding buffer, and 5 mg of biotinylated target molecule in 50 µl binding buffer was added into SA-beads to incubate for 30 min by slow agitation at room temperature. After incubation, excess biotinylated target molecule was washed away there times pelleting for 2 min on magnetic rack.

Target-coupled SA-beads were blocked with 1 ml of blocking buffer for 1 h at room

195 temperature. After pipetting out the blocking buffer from microcentrifuge tube, SA- beads were washed three times with washing buffer, and then 500 µl of overnight pre- blocked phage library were added to SA-beads to incubate for 1 h at room temperature by slow agitation. Meanwhile, 1 ml of overnight-grown E. coli TG1 culture was inoculated in 100 ml of 2 × YT medium by shaking at 37 °C at 250 rpm till OD600 reached to 0.4.

The unbound phage were washed away with 1 ml washing buffer eight times by pelleting

2 min, and transferred to a new 1.5 ml sterile microcentrifuge tube, and washed with 1 ml of binding buffer twice. After the last washing, the washing buffer was removed completely, and the bound phage were eluted with 100 µl elution buffer for not more than

10 min. After 10 min incubation, the supernatant containing phage was neutralized in a new sterile microcentrifuge tube containing 100 µl neutralization buffer. When E. coli

TG1 culture reached an OD600 of 0.4, 30 ml of the culture were transferred to 50 ml

Falcon tube, 200 µl the eluted phage were added to the culture to amplify at 37 °C for 90 min without shaking. After amplification of phage library, the cells were pelleted by centrifugation at 4000 rpm for 5 min at 4 °C, and the pellets were re-suspended in 1 ml 2

× YT medium, plated on two large (150 x 100 mm) 2 × YT/30 µg/ml chloramphenicol agar plates at 37 °C overnight. The following day, cells were scraped from the agar plates and were harvested with 4 ml 2 × YT medium/ 20% Glycerol per plate, and aliquots of 0.5 ml stored at - 80 °C. To start the next round of selection, phage were produced using these glycerol stocks. Also, 50 µL aliquot from amplified phage was used to determine the number of phage recovered.

196 4.7.9. Additional Rounds

The second round of panning were performed with the same procedure described above by using neutravidin-coated magnetic beads instead of streptavidin in order to prevent the enrichment of streptavidin-specific peptide binders. Also the amount of target molecule used decreased to 2 mg from 5 mg to control stringency.

4.7.10. Negative Selections as Control Biopanning

Negative selections were also run at the first and the second rounds of biopanning to check the enrichment factors after each round. The same biopanning procedure was carried out in negative selection by using solely streptavidin-coated magnetic beads incubated the phage library without target molecule in the first round, and solely neutravidin-coated magnetic beads incubated the phage library without target molecule in the second round. After biopanning selection, 50 µL aliquots from amplified phage were used to determine the number of phage recovered against the magnetic beads by plating on 2 × YT medium/30 µg/ml chloramphenicol agar plates, and the rest were discarded.

197 4.7.11. DNA Sequencing and Analysis

After the second round of each biopanning, 50 µL aliquots from 2 × YT medium/ 20%

Glycerol stocks for each target molecule were plated on 2 × YT/30 µg/ml chloramphenicol agar plates to pick randomly single phage clones expressing the peptides bound to each target according to phage tittering protocol. Following day, the randomly picked 40 plaques from each experiment on agar plates were sent to Genewiz with 21seqba primer, 5’-TAA TTG CTC GAC CTC CTC TC-3’, without further application. The reverse complement sequences of the oligonucleotides were then translated into peptide sequences using ExPASy Translate Tool

(http://web.expasy.org/translate/).

4.8. Synthesis of Enantiomer of Identified L-peptides: D-peptides

4.8.1.Synthesis of Linear D-peptides

Selected linear D-peptides were synthesized manually by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on Rink-4-(2',4'- dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink Amide) resin (0.1 mmol scale, 0.33 mmol/g). Each amino acid were coupled using standard protocol, where 0.8 mmol amino acid in 3.2 ml DMF was activated by HBTU (0.72 mmol) and DIEA (1.2

198 mmol) for 30 sec, and coupled to peptide-resin for 45 min. N-Fmoc group was removed with the treatment of 20% piperidine in DMF twice, 5 and 15 min, and wash twice with

DMF to couple the next residue. After coupling of the last amino acid, N-Fmoc group was removed, and the peptide-resin was washed with DMF, DCM, and diethyl ether twice to dry overnight in high vacuum. Each peptide was cleaved from resin with a cleavage cocktail containing Trifluoroacetic acid (TFA)/Tri-isopropylsilane (TIPS)/H2O

(95:2.5:2.5) for 3 hours. TFA was evaporated under flow of nitrogen, and the cleaved peptide was precipitated in dry-ice cold diethylether three times, and centrifuged. Crude peptides were dried under high vacuum, dissolve in 50% acetonitrile/water and lyophilized. Lyophilized crude peptide was purified by semi-prep C18 RP-HPLC [Vydac

C18, 10 µm, 10 mm × 250 mm] using a gradient of 10 % to 45 % solvent B over 40 min.

The purity of peptides was confirmed by analytical RP–HPLC [Vydac C18, 5 µm, 4 mm

× 250 mm], and mass was checked with MALDI-TOF MS. (see appendix for MALDI-

TOF MS data)

4.8.2.Synthesis of Cyclic D-peptides

Selected cyclic D-peptides were synthesized manually by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on Rink Amide resin (0.1 mmol scale, 0.33 mmol/g). Each amino acid were coupled using standard protocol, where 0.8 mmol amino acid in 3.2 ml DMF was activated by HBTU (0.72 mmol) and

DIEA (1.2 mmol) for 30 sec, and coupled to peptide-resin for 45 min. N-Fmoc group

199 was removed with the treatment of 20% piperidine in DMF twice, 5 and 15 min, and wash twice with DMF to couple the next residue. After coupling of the last amino acid,

N-Fmoc group was removed, and the peptide-resin was washed with DMF, DCM, and diethyl ether twice to dry overnight in high vacuum. Each peptide was cleaved from the resin with reducing acidic cleavage cocktail containing trifluoroacetic acid (TFA) (90%, v/v), thioanisole (2.5%, v/v), phenol (2.5%, w/v), 1,2-ethanedithiol (2.5%, v/v) and H2O

(2.5%, v/v) for 3 hours. TFA was evaporated under flow of nitrogen, and the cleaved peptide was precipitated in dry-ice cold diethylether three times, and centrifuged. Crude peptides were dried under high vacuum, dissolve in 50% acetonitrile/water and lyophilized. Lyophilized crude peptide was purified by semi-prep C18 RP-HPLC [Vydac

C18, 10 µm, 10 mm × 250 mm] using a gradient of 10% to 45% solvent B over 40 min.

The purity of peptides was confirmed by analytical RP–HPLC [Vydac C18, 5 µm, 4 mm

× 250 mm], and mass was checked with MALDI-TOF MS. Cyclization of peptides was achieved by air-oxidation forming disulfide bond formation. 5 mg of purified peptide was dissolved in 5 ml of oxidation buffer (20 mM NH4CO3, pH 8) and incubated open-air overnight. Following day, the oxidized peptides were lyophilized and purity checked by analytical RP–HPLC [Vydac C18, 5 µm, 4 mm × 250 mm]. (see appendix for MALDI-

TOF MS data)

200 4.8.3. Synthesis of Bicyclic D-peptides

Linear D-peptides having three cysteines were synthesized by standard Fmoc-chemistry on Rink Amide resin (0.1 mmol scale, 0.33 mmol/g). Each amino acid were coupled using standard protocol, where 0.8 mmol amino acid in 3.2 ml DMF was activated by

HBTU (0.72 mmol) and DIEA (1.2 mmol) for 30 sec, and coupled to peptide-resin for 45 min. N-Fmoc group was removed with the treatment of 20% piperidine in DMF twice, 5 and 15 min, and wash twice with DMF to couple the next residue. After coupling of the last amino acid, N-Fmoc group was removed, and the peptide-resin was washed with

DMF, DCM, and diethyl ether twice to dry overnight in high vacuum. Peptides were cleaved from with reducing acidic cleavage cocktail containing trifluoroacetic acid (TFA)

(90%, v/v), thioanisole (2.5%, v/v), phenol (2.5%, w/v), 1,2-ethanedithiol (2.5%, v/v) and

H2O (2.5%, v/v). TFA was evaporated under flow of nitrogen, and the cleaved peptide was precipitated in dry-ice cold diethylether three times, and centrifuged. Crude peptides were dried under high vacuum, dissolve in 50% acetonitrile/water and lyophilized. The lyophilized 1 mM crude peptides in 0.1 M NH4HCO3 (pH 8) were reacted with 1.2 mM

TBMB in acetonitrile for 1 h in hot water bath at 30 °C. Lyophilized crude peptide was purified by semi-prep C18 RP-HPLC [Vydac C18, 10 µm, 10 mm × 250 mm] using a gradient of 10 % to 45 % solvent B over 40 min. The purity of peptides was confirmed by analytical RP–HPLC [Vydac C18, 5 µm, 4 mm × 250 mm], and mass was checked with MALDI-TOF MS that showed +114 as a bicyclic product. (see appendix for

MALDI-TOF MS data)

201 4.9. Antibacterial Activity Assays of Synthesized D-Peptides

4.9.1. Bacterial Strains and Medium

Bacteria Strain Medium

E. coli (ATCC 25922) Gram-negative control Medium 18

B. subtilis (ATCC 6633) Gram-positive control Medium 18

S. aureus (ATCC 6538) Methicillin-sensitive Medium 18

MRSA (ATCC 43300) Methicillin-resistant Medium 18

E. faecalis (ATCC 29212) Vancomycin-sensitive Medium 44

VRE (ATCC 51559) vanA type resistant Medium 44

VRE (ATCC 51299) vanB type resistant Medium 2174

Medium 18: Tryptic soy medium (BD-236950)

Medium 44: Brain heart infusion medium (BD-237500)

Medium 2174: Brain heart Infusion medium (BD-237500) with 4 mcg/ml vancomycin

202 Muller-Hinton Broth (MHB) 2 g Beef extract, 17.5 g acid digest of casein, 1.5 g starch in

1 L dH2O (BD-275730).

Muller-Hinton Agar Plate: 21 g MH broth and 15 g Bacto-Agar in 1 L dH2O

4.9.2. Determination of Minimum Inhibitory Concentration (MIC) with Broth

Micro-dilution Method

The antimicrobial activities of synthesized D-peptides were tested using broth micro- dilution method to determine the peptides minimal inhibitory concentration (MIC) against the different bacterial strains. The MIC assays were essentially done according to protocol outlined by the Clinical and Laboratory Standards Institute with modifications for peptide antibiotics reported by Wiegand et al. Two-fold serial peptide antimicrobial dilutions were prepared from 2560 µg/ml to 5 µg/ml in 0.2% bovine serum albumin

(BSA) and 0.01% acetic acid buffer, which helped to prevent high concentrations of peptides from aggregating in the broth or attaching to the wells, in sterile 96-well polypropylene plates (COSTAR, #3879). Bacterial cultures of different bacterial strains were prepared by inoculating 5 colonies from each bacterial strain in 5 ml of Mueller

Hinton (MH) broth at 37 °C with shaking. Bacterial cultures were allowed to grow until

0.5 McFarland standard had been reached. Finally, a 1:200 dilution of bacterial suspensions was made to obtain a final inoculum concentration of 8 x 105 CFU/ml. 10 µl of two-fold serial dilution of peptide antibiotics inoculated with 90 µl of bacterial

203 inoculum to a final volume of 100 µl. The microtiter plate was sealed with parafilm, placed in a humidified container and incubated without shaking at 37 °C for 18 hours.

Following day, MIC was determined visually on the basis of turbidity as the lowest concentration inhibiting bacterial growth. Also, MH broth without antibiotic and bacterial suspension was used as a growth control. Additionally, vancomycin and melittin were used as control peptide antibiotics in each assay. Each assay was repeated for each bacterial strain in triplicate in three different days.

4.9.3. Determination of Minimum Bactericidal Concentration (MBC)

The antibacterial activity assasys to determine the minimum bactericidal concentration

(MBC) were carried out after MIC assays. After 16 h of incubation of MIC assays, 10 µl aliquots from the wells having complete inhibition were taken and subjected to 10-fold serial dilutions. 10 µl aliquots from each dilution were plated on MHB agar plates, and incubated at 37 °C for 24 h. Bacterial growth on agar was observed and the concentration which has a colony count of less than 10 was considered as the MBC value. The MBC value was defined as the lowest concentration of antimicrobial agent that can kill > 99% of the microorganism population where there is no visible growth on MHB agar plate.

204

Chapter 5

Conclusions and Future Directions

205 The emergence of drug resistant bacterial strains presents a clear need for new antibiotics.

The increasing drug resistance among Gram-positive bacteria is a significant problem because they are responsible for one third of nosocomial infections. Antibiotic resistance in Gram-positive organisms, such as, staphylococci, pneumococci, enterococci, has increased in the last 15 years.336,337 Methicillin-resistant S. aureus (MRSA) is one of the most frequently reported nosocomial pathogens in developed countries. MRSA infections are responsible for more deaths (20,000 deaths per year) in the U.S. each year than AIDS.338 Therefore developing new antibacterial agents to interfere with unexploited bacterial molecular targets is an important and underserved need.

Peptide based therapeutics afford a high level of specificity. However, the use of these compounds is limited because of hydrolytic cleavage by proteases, possible induction of a vigorous humoral immune response and administration mostly as injectables.228-230

Increased protease stability would result in lower dosing, reduced cost, and shortened hospital stays. Short D-peptides (<12 residues) have been known to be absorbed through the gut making them viable oral drugs.226,227 These features make D-peptides an excellent starting point for the design of a new class of antimicrobial compounds. In this study we aimed to discover stable, protease resistant D-peptides as antibiotics binding D- alanyl-D-alanine termini of vancomycin-sensitive bacteria and D-alanyl-D-lactate termini of vancomycin-resistant bacteria with a mechanism action similar to that of vancomycin based on the mirror-image phage display concept.

In chapter 2, we explored peptide antibiotics composed of D-amino acids active against

S. aureus and methicillin-resistant S. aures (MRSA) applying the mirror-image phage display concept.232 The goal is accomplished by screening phage display random peptide

206 libraries, linear, cyclic and bicyclic of different lengths ranging from 7 to 12 amino acid residue, against the enantiomer of the β-lactam antibiotic cephalosporin mimicking the

D-Ala-D-Ala terminus of peptidoglycan structures in bacterial cell walls, which is crucial for crosslinking and providing structural integrity to the cell. The mechanism action of penicillins and cephalosporins has been ascribed to their mimicking the conformation of the terminal D-Ala-D-Ala terminus in a high-energy state during the crosslinking by the transpeptidase. 252-257 Because of the conformational and structural similarity of the β- lactams to the terminal end D-Ala-D-Ala recognized by the transpeptidase enzyme, the enantiomer of cephalosporin (EA-1-CEP) serves as a more structurally rigid analog of L-

Ala-L-Ala during the phage display screening. In addition, the enantiomer of the pentapeptide precursor of the nascent growing bacterial cell wall of S. aureus, D-Ala-L-

γ-Glu-D-Lys-L-Ala-L-Ala (EA-2-ALA) was also screened as well as its control peptide

(EA-3-ALA) with α-Glu instead of γ-Glu that provides extended backbone. We initially utilized three commercially available phage display libraries, a linear 12-mer, a linear 7- mer and a cyclic 7-mer purchased from New England Biolabs, in two different biopanning strategies: the high yield screening and the more stringent screening. A fraction of the peptide sequences identified from both the high yield screening and the more stringent screening against both enantiomers of the cephalosporin and pentapeptide precursor targets were identical, suggesting that β-lactams make viable target surrogates.

We also utilized two different bicyclic libraries having a construction of Cys-(Xaa)m-Cys-

(Xaa)n-Cys (where Xaa represents random amino acids, m and n = 3, 4, 5 or 6) with different loop lengths, Library A (m and n = 3-5) and Library B (m and n = 3-6)

207

Table 72. Common peptide sequences isolated from all the biopanning of three commercially available phage display peptide libraries against target molecules EA-1- CEP, EA-2-ALA, EA-3-ALA and EA-4-LAC.

Peptide sequences Phage Library Target Molecules

SGVYKVAYDWQH Linear 12-mer EA-1-CEP, EA-2-ALA, EA-3-ALA, EA-4-LAC

DRWVARDPASIF Linear 12-mer EA-1-CEP, EA-2-ALA, EA-3-ALA

RKVKRRPRVSNL Linear 12-mer EA-2-ALA, EA-4-LAC

HGGVRLY Linear 7-mer EA-1-CEP, EA-3-ALA

YPWFIRA Linear 7-mer EA-2-ALA, EA-4-LAC

LPVRLDW Linear 7-mer EA-4-LAC, EA-2-ALA

HYIDFRW Linear 7-mer EA-4-LAC, EA-2-ALA

GYSSFNR Cyclic 7-mer EA-1-CEP, EA-2-ALA, EA-3-ALA, EA-4-LAC

RGATPMS Cyclic 7-mer EA-1-CEP, EA-2-ALA, EA-3-ALA, EA-4-LAC

MARYMSA Cyclic 7-mer EA-1-CEP, EA-2-ALA, EA-3-ALA, EA-4-LAC

SISSLTH Cyclic 7-mer EA-1-CEP, EA-3-ALA

NTGSPYE Cyclic 7-mer EA-1-CEP, EA-3-ALA

TNANHYF Cyclic 7-mer EA-2-ALA, EA-4-LAC

LKLGEKW Cyclic 7-mer EA-4-LAC, EA-2-ALA, EA-1-CEP

NTGSPYE Cyclic 7-mer EA-4-LAC, EA-2-ALA

EGQRWMQ Cyclic 7-mer EA-4-LAC, EA-2-ALA, EA-3-ALA

208 developed by Prof. Heinis Laboratory at EPFL Lausanne. These conformationally constrained peptide libraries are likely to suffer a much smaller loss in entropy upon binding of target, and therefore have the potential to provide peptide ligands with tight binding affinities.163 Table 72 shows common peptide sequences isolated from all the biopanning of three commercially available phage display peptide libraries against target molecules EA-1-CEP, EA-2-ALA, EA-3-ALA and EA-4-LAC. Isolated three peptides from the biopanning of linear 12-mer and linear 7-mer phage display libraries against

EA-2-ALA, “VEAKCCFSMHKT”, “KCCYTLP” and “VLTRCCG”, share the same

“K/R-C-C” motif and have moderate activity for S. aureus which also showed that biopanning successfully resulted in the selection of target-specific peptides. Surprisingly, the peptides identified from two different bicyclic libraries have similar short motifs that also found in some of the peptides identified through commercially available phage display libraries, for instance, “T-T-L”, “I/L-S/T”, “Q/N-S/T”, “L-Q”, “G-Q-S”, “G-S”,

“G-x-S”, “G-V”, “L-K/R”, “R-V”, “V-L-S”, “N/Q-K/R”.

The binding specificities of all the identified peptides to target molecules were confirmed by conducting phage-ELISA assays. According to ELISA results and negative controls, selected peptides selectively bind to their target molecules rather than negative controls, such as streptavidin, BSA, plastic surface. Peptides giving higher ELISA signals were synthesized as in D enantiomeric for called D-peptides for further studies. Lastly, the antimicrobial activities of synthesized D-peptides (total of 94) were assessed to determine the minimum inhibitory concentrations (MIC) against both Gram-positive (B. subtilis, S. aureus, E. faecalis, MRSA, vanA type VRE and vanB type VRE) and Gram-negative (E. coli) bacteria. Some of D-peptides showed higher activity, MIC values in the range 8 –

209 32 µg/mL, some of them had moderate activity, MIC values of 64-128 µg/mL, and some showed no antibacterial activity, that is MIC value of ≥ 256 µg/mL, and only against Gram-positive strains as we expected. While five of synthesized D-peptides were found to be potent showin high activity against S. aureus, MRSA and E. faecalis, seven

D-peptides showed moderate activity. The most potent D-peptide, EA-BC1-85, was isolated from biopanning of bicyclic phage display Library A against EA-2-ALA. The bicyclic D-peptide showed antibacterial activity within a factor of 10 of vancomycin against S. aureus and MRSA and also active against resistant bacteria.

In chapter 3, the same approach was utilized to develop D-antibiotics that would be similar to vancomycin in their mode of action active against vancomycin-resistant

Enterococci. Vancomycin, an antibiotic of last resort especially in hospital infections after surgery caused by Gram-positive bacteria, functions by binding this terminal epitope. The enantiomer of the bacterial cell wall precursor of vancomycin-resistant enterococci, L-Ala-D- γ-Glu-L-Lys-D-Ala-D-Lac (EA-4-LAC), was synthesized to use as bait in phage display screening of both commercially available linear and cyclic libraries and bicyclic peptide libraries. A similar experimental design scheme used in chapter 2 was followed. While the same repeating peptide sequences and the similar short motifs were isolated in the biopanning against EA-4-LAC, none of the D-peptides showed antibacterial activity against vancomycin-resistant bacteria. On the other hand two D- peptides, one cyclic and one bicyclic, isolated from biopanning of EA-2-ALA were active against vanB type vancomycin-resistant bacteria.

210 A survey of the peptide sequences and their antibacterial activities shows that the identified potent D-peptides in both chapter 2 and chapter 3 serve as leads for further modification using a medicinal chemistry approach, and may also result in direct first-hit compounds that are both protease resistant and potent antimicrobials, rivaling and/or exceeding vancomycin activity.

For further studies, since the most potent D-peptide antibiotic was identified through bicyclic peptide libraries, we would like to test all identified bicyclic peptides to evaluate their antibacterial activities against both Gram-positive and Gram-negative strains. The more stringent screening would be carried out with bicyclic phage display libraries to isolate high binding peptide ligands. For the most potent D-peptides, we will evaluate proteolytic stability using both bacterial protease mixtures and also crude intestinal protease preparations even though we do not expect protease activity on D-versions of the identified sequences. Sequences that are active but have protease susceptibility will be re-evaluated. In addition, when appropriate, we will substitute unnatural amino acids

(fluorinated, others) and peptide bond isosteres (triazole) into the sequence.339,340

Additionally, hemolysis will be measured against fresh human red blood cells (hRBCs) to check toxicity of the most potent D-peptides. For D-peptides having high antibacterial activity and low toxicity that lead to higher therapeutic index, we will measure the binding ability to bacterial cell wall precursors of S. aureus and vancomycin-resistant enteroccoci by surface plasmon resonance (SPR).

We believe that the overall promising results in developing new D-peptide antibiotics will pave the way to develop new antibacterials to win the war of bacterial resistance between human beings and bacteria.

211

Chapter 6

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255

Chapter 7

Appendix

256 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm 1.09 1.03 2.28 2.77 6.98 7.84 0.77 6.58 3.24 3.00 2.99 5.74 6.46 3.91 6.03 13.08 151.21

Figure A 1. 1H NMR of EA-1-CEP

257 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure A 2. 13C NMR of EA-1-CEP

258

Figure A 3. RP-HPLC Chromatogram of compound EA-1-CEP. Gradient is 20% to 40% solvent B over 30 min.

259

Figure A 4. MALDI TOF-MS of pure compound EA-1-CEP. (Calculated mass = 1040.63 [M], observed mass = 1063.40 [M+Na]+).

260

Figure A 5. RP-HPLC Chromatogram of compound EA-2-ALA. Gradient is 5% to 20 % solvent B over 30 min.

261

2500 962.121 Intens. [a.u.]

2000

1500

1000 1000.004 984.133

500

500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Figure A 6. MALDI TOF-MS of pure compound EA-2-ALA. (Calculated mass = 962.46 [M], observed mass = 962.223 [M+H]+).

262

Figure A 7. RP-HPLC Chromatogram of compound EA-3-ALA. Gradient is 5% to 20 % solvent B over 30 min.

263

2500 962.121 Intens. [a.u.]

2000

1500

1000 1000.004 984.133

500

500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Figure A 8. MALDI TOF-MS of pure compound EA-3-ALA. (Calculated mass = 962.46 [M], observed mass = 962.223 [M+H]+).

264

Figure A 9. RP-HPLC Chromatogram of compound EA-4-LAC. Gradient is 5% to 20% solvent B over 30 min.

265

3500 Intens. [a.u.] 963.115

3000

2500

2000

1500

1000 985.086 1001.078

500

500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Figure A 10. MALDI TOF-MS of pure compound EA-4-LAC. (Calculated mass = 963.26 [M], observed mass = 963.115 [M+H]+).

266 Table A.1. Peptide sequences identified through Phage Display with target molecule EA- 1-CEP, EA-2-ALA, and EA-3-ALA.

Peptide name Peptide Sequence MW (cald.) MW (obs.) EA-L12-01 SGVYKVAYDWQH 1709.82 1708.858 EA-L12-02 DRWVARDPASIF 1689.84 1689.077 EA-L12-03 HRDPHSALTRSW 1719.84 1718.928 EA-L12-04 VGSNLRLLHQWK 1707.94 1708.67 EA-L12-05 GQSEHHMRVASF 1642.76 1642.801 EA-L12-06 TYTANDlHLADL 1603.7 1602.903 EA-L12-07 ITGLGSGSSTST 1324.36 1323.87 EA-L12-08 RKVKRRPRVSNL 1766.07 1765.034 EA-L12-09 VEAKCCFSMHKT 1553.84 1553.87 EA-L12-10 SSSVPPVSALHG 1307.41 1307.46 EA-L12-11 VSNFTKPHKPKA 1524 1524.73 EA-L12-12 SLDGAGAALRTS 1288 1289.01 EA-L12-13 VHWDFRQWWQPS 1841.98 1842.34 EA-L12-14 GTGLVTLPRLTV 1396.64 1396.98 EA-L7-15 LPVRLDW 1068.23 1068.56 EA-L7-16 TTLLTVS 904.02 904.63 EA-L7-17 VTSPYAF 954.04 954.36 EA-L7-18 KCCYTLP 997.2 997.66 EA-L7-19 VLTRCCG 921.11 921.78 EA-L7-20 NIVSRES 974.03 974.32 EA-L7-21 YPWFIRA 1122.28 1123.03 EA-L7-22 TGFLVNV 919.04 920.03 EA-L7-23 TTQVLEA 931 931.88 EA-L7-24 ALSYSRG 922.99 923.16 EA-L7-25 ALQPQKH 991.11 991.62 EA-L7-26 HLFTTGV 944.05 944.46 EA-L7-27 VSRDTPQ 972.01 972.84 EA-L7-28 HGGVRLY 971.07 971.08 EA-L7-29 AHGRSRG 997.04 997.04 EA-L7-30 KVKKRPD 1127.3 1127.3 EA-L7-31 RTYPREK 1206.47 1206.47 EA-L7-32 MYVAPSR 1080.22 1080.22 EA-L7-33 PRLPRTR 1152.33 1152.33 EA-L7-34 HYIDFRW 1293.41 1293.41 EA-L7-35 GASESYL 982.99 982.99 EA-L7-36 MMVLRNQ 1148.37 1148.37 EA-L7-37 RHDIRKT 1182.3 1182.3 EA-L7-38 AVRGYEW 1131.21 1131.21

267 Table A.2. Peptide sequences identified through Phage Display with target molecule EA- 1-CEP, EA-2-ALA, and EA-3-ALA.

Peptide name Peptide Sequence MW (cald.) MW (obs.) EA-L7-39 WSWGEQK 1177.24 1177.24 EA-L7-40 MIRGTTV 1034.2 1034.2 EA-L7-41 RPTAHMA 1040.17 1040.17 EA-L7-42 QLAVAPS 942.03 942.03 EA-L7-43 GHRVRFP 1125.26 1125.26 EA-L7-44 GNVGSVR 945 945 EA-L7-45 ADRFQAL 1077.16 1077.16 EA-L7-46 SLDKRKK 1131.29 1131.29 EA-L7-47 GKDYMGY 1090.17 1090.17 EA-C7C-48 KLTTQMM 1299.62 1299.89 EA-C7C-49 LKLGEKW 1320.59 1321.09 EA-C7C-50 GYSSFNR 1277.39 1278.01 EA-C7C-51 RGATPMS 1166.36 1166.45 EA-C7C-52 TNANHYF 1313.43 1313.93 EA-C7C-53 SISSLTH 1191.34 1191.58 EA-C7C-54 MARYMSA 1276.54 1277.12 EA-C7C-55 EDLTTLS 1225 1225.98 EA-C7C-56 KTLQPWT 1320.55 1320.56 EA-C7C-57 VNLQKDM 1294.53 1295.03 EA-C7C-58 EGQRWMQ 1381.57 1382.15 EA-C7C-59 ILLPDKL 1258.56 1258.67 EA-C7C-60 LWSTGAT 1269.4 1269.4 EA-C7C-61 NTGSPYE 1299.36 1299.99 EA-C7C-62 VPILEGT 1262.45 1262.78 EA-C7C-63 DNIMTPV 1323.53 1323.87 EA-C7C-64 SSPFPEF 1344.48 1344.78 EA-C7C-65 MAPDSRV 1307.5 1307.88 EA-C7C-66 NWGDRIL 1405.58 1405.65

268

Table A.3. Peptide sequences identified throguh Phage Display with target molecule EA- 4-LAC

Peptide name Peptide Sequence MW (cald.) MW (obs.) EA-L12-01 SGVYKVAYDWQH 1709.82 1708.858 EA-L12-08 RKVKRRPRVSNL 1766.07 1765.034 EA-L12-67 GLHTSATNLYLH 1496.63 1496.89 EA-L12-68 FIPFDPMSMRWE 1725.99 1726.03 EA-L12-69 HKHWSTPEFLSS 1625.74 1626.62 EA-L12-70 QSHYDSHLAMLV 1570.73 1570.89 EA-L7-21 YPWFIRA 1122.28 1123.03 EA-L7-71 LPVRLDW 1068.23 1068.337 EA-L7-72 KFYAHLD 1063.17 1064.02 EA-L7-73 VPIYHLT 1012.16 1012.17 EA-L7-34 HYIDFRW 1293.41 1293.67 EA-C7C-74 KHLLGEN 1257 1258.03 EA-C7C-75 GLKALKE 1205.46 1206.78 EA-C7C-76 LVSQHTD 1246.38 1246.84 EA-C7C-77 QMQLRSA 1280.51 1280.73 EA-C7C-78 NQTAARV 1206.56 1207.03 EA-C7C-52 TNANHYF 1313.43 1313.95 EA-C7C-54 MARYMSA 1276.54 1277.12 EA-C7C-51 RGATPMS 1166.36 1166.78 EA-C7C-50 GYSSFNR 1277.39 1278.05 EA-C7C-49 LKLGEKW 1320.59 1321.09 EA-C7C-61 NTGSPYE 1299.36 1299.78

269

Table A.4. Peptide Sequences identified through Bcyclic Peptide Libraries, Library A and Library B from biopanning against EA-1-CEP, EA-2-ALA, EA-4-LAC

Peptide name Peptide Sequence MW (cald.) MW (obs.) MWTBMB mod (cald.) MW TBMB mod (obs.) EA-BC1-78 ACVKSCGQSV 1284.55 1285.15 1398.55 1397.936 EA-BC1-79 ACQYNDCDMLH 1615.85 1615.92 1729.85 1728.91.3 EA-BC1-80 ACLSQFCVID 1401.69 1402.91 1515.69 1515.01 EA-BC2-81 ACPQSSCQGLRL 1565.86 1566.62 1679.86 1678.916 EA-BC2-82 ACTRVCSSSQLY 1620.89 1620.34 1734.89 1733.78 EA-BC1-83 ACQFHIGCYSN 1545.78 1545.99 1659.78 1659.01 EA-BC1-84 ACREVTCHHLQ 1599.88 1600.66 1713.88 1713.63 EA-BC1-85 ACQTDVCQRTI 1540.8 1541.49 1654.8 1653.66 EA-BC2-86 ACYESCRVQSAL 1632.9 1633.69 1746.9 1746.73 EA-BC2-87 ACETRGCYQRFR 1793.08 1794.81 1907.08 1907.02 EA-BC2-88 ACDQGDCHQKIN 1634.83 1635.55 1748.83 1748.13 EA-BC1-89 ACGQVCNQKV 1352.62 1353.13 1466.62 1466.34 EA-BC1-90 ACYAQRCGVTG 1431.68 1432.83 1545.68 1545.56 EA-BC2-91 ACFKQNCSQSRS 1661.9 1661.72 1775.9 1775.02 EA-BC2-92 ACIPIKRCNDQL 1677.04 1678.44 1791.04 1791.24 EA-BC2-93 ACIEQPACPNIF 1608.92 1609.82 1722.92 1722.56 EA-BC1-94 ACSTLDRCYQL 1575.85 1576.15 1689.85 1688.78

270