INVESTIGATIONS OF THE NATURAL PRODUCT ANTIBIOTIC THIOSTREPTON FROM STREPTOMYCES AZUREUS AND ASSOCIATED MECHANISMS OF RESISTANCE by Cullen Lucan Myers A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2013 © Cullen Lucan Myers 2013 AUTHOR’S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii ABSTRACT The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co- factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket iii and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP- AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non- covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase. iv ACKNOWLEDGEMENTS My sincerest thanks are expressed to my supervisor Professor John Honek, for his steady tutelage, guidance and support throughout my doctoral studies. I am deeply grateful for his fostering the growth of my self-confidence as a researcher, by encouraging open discussions about ideas and his unfailing way of steering me in a right direction without belittlement and always ensuring that I was learning in the process. I also wish to thank the members of my committee, Drs. Dieckman, Guillemette and Wettig for their constructive advice which helped to advance my project. My gratitude extends to the many members of the Honek Laboratory over the years that provided their friendship and support, foremost among whom is Dr.Pei Hang, who began the work on this project. I am immensely grateful for her spending the time to train me (in spite of being in the process of preparing her own doctoral thesis). I would also especially like to thank Drs. Zhengding Su, Uthiawan Suttisansanee and Betsey Daub for sharing scientific expertise and advice that was tremendously helpful at many different stages of my studies. Thanks are also owed Joshua Yuen, Jesse Harris and Jacky Yeung, who assisted in the synthesis of thiostrepton analogues and in studies of thiostrepton resistance proteins. I am deeply grateful to the Department of Chemistry at the University of Waterloo for wonderful technical support that was vital to the success and understanding of many aspects of this project. In particular, I would like to thank Dr. Richard Smith for mass spectrometry training, invaluable advice and suggestions; Jan Venne for NMR training and assistance with two-dimensional NMR experiments; and Valarie Goodfellow and the Dmitrienko group for allowing me access to their HPLC equipment. I am additionally grateful to all the administrative and support staff of the Department of Chemistry. As well, I greatly appreciate the gracious assistance of Marcie Chaudet and the Rose group (Department of Biology) with X-ray crystallography. v Finally, I would like to express my deepest and heartfelt gratitude to my family for their unwavering support, love and understanding, which has been a great source of motivation and inspiration throughout my studies. Thank you, all of you, for your encouragement during my times of despair. For Mama & Janine vi TABLE OF CONTENTS LIST OF TABLES ............................................................................ xiii LIST OF FIGURES ........................................................................... xv LIST OF ABBREVIATIONS .............................................................. xix CHAPTER 1: INTRODUCTION ........................................................ 1 1.1. Antibiotic action and mechanisms of resistance ................................................... 2 1.1.1. Target sites and modes of action for common antibiotics ............................ 4 1.1.1.1. Cell wall synthesis and cell membrane disruption ............................. 4 1.1.1.1.1. Inhibitors of peptidogylcan synthesis ....................................... 4 1.1.1.1.2. Bacterial cell membrane .......................................................... 5 1.1.1.2. Ribosome targeting antibiotics ........................................................... 5 1.1.1.3. Antibiotics targeting DNA synthesis and replication ......................... 10 1.1.2. Mechanisms of antibiotic resistance ............................................................. 11 1.1.2.1. Drug efflux ......................................................................................... 11 1.1.2.2. Antibiotic modification ...................................................................... 12 1.1.2.3. Target site modification...................................................................... 13 1.1.2.4. Molecular neutralization ..................................................................... 13 1.1.3. Emergence and transfer of antibiotic resistance ........................................... 14 1.1.4. Antibiotic development ................................................................................ 17 1.2. Thiostrepton and the thiopeptide family of antibiotics ......................................... 18 1.2.1. Structure and physical properties ................................................................. 18 1.2.2. Total synthesis and biosynthesis of thiostrepton .......................................... 21 1.2.3. Biological properties of thiostrepton ............................................................ 26 1.2.3.1. Antibacterial activity .......................................................................... 26 1.2.3.2. Additional biological properties of thiostrepton ................................. 30 1.3. Thiostrepton resistance ......................................................................................... 31 1.3.1. Resistance in thiostrepton producing organisms .......................................... 31 1.3.1.1. RNA methylation and antibiotic resistance ........................................ 34 1.3.1.2. AdoMet dependent methyltransferases .............................................. 36 1.3.1.3. Mechanism of transmethylation ......................................................... 37 1.3.1.4. AdoMet-dependent methyltransferases as tools ................................. 39 1.3.2. Resistance in organisms that do not produce thiostrepton ............................ 43 1.3.2.1. Thiostrepton resistant mutants ............................................................ 43 1.3.2.2. Thiostrepton binding proteins ............................................................ 45 vii 1.4. Research objectives ............................................................................................... 47 CHAPTER 2: SYNTHESIS AND BIOLOGICAL ACTIVITY OF THIOSTREPTON ANALOGUES 2.1. Preface .................................................................................................................