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Stemming the tide of resistance in TB: development of chemical tools to evaluate mycothiol dependent enzymes in multidrug resistance in mycobacteria Ewelina Rybak Thesis submitted for the degree of Doctor of Philosophy Department of Pharmaceutical and Biological Chemistry School of Pharmacy University College London 2019 I, Ewelina Rybak, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I can confirm that this has been indicated in the thesis. 2 Acknowledgments First, I would like to thank Dr Jess Healy, my research supervisor, for giving me the opportunity to work on this project. I would also like to thank Dr Geoff Wells, my research supervisor, for great support and guidance during my final year. Apart from my UCL supervisors, I would like to express my gratitude to Dr David Andrews for his valuable advice and suggestions over the years. I would also like to acknowledge Dr Steve Stokes for his advice and helping me to find my way around the lab during my placement at AstraZeneca. Lastly, I would like to thank all past and current G25 lab members for creating a great atmosphere in the lab. 3 Abstract Tuberculosis (TB) is an infectious disease that kills more than a million people a year and poses a significant health threat. Because of the global spread of multi- drug resistant TB and high infection rates, there is a significant unmet clinical need for new drugs. M. tuberculosis (Mtb) produces mycothiol (MSH) in place of glutathione as the most abundant low molecular weight thiol. MSH and associated enzymes (e.g. mycothiol-S-transferase, MST) are thought to play pivotal roles in cellular protection against various xenobiotics. Successful synthesis of MST inhibitors may lead to the development of a novel strategy for the treatment of TB. The aim of this project is to validate MST as a novel drug target and understand the role played by MST in mycobacterial physiology via the development of MSH analogues as chemical probes. To achieve this a 2-fold approach was adopted. The first approach includes the development of mycothiol analogues. The synthetic routes towards the synthesis of three different mycothiol analogues containing glucosamine and cysteine moieties are described. These simplified analogues provide a potential scaffold for the synthesis of a library of S- conjugates that would enable us to probe the hydrophobic pocket of MST and gain some information about MST’s structure–activity relationships. The second approach involves the use of kinetic target-guided synthesis (kTGS), a process in which the protein acts as a catalyst or template in the synthesis of its own best inhibitors from the ‘choices’ provided. Towards this goal, two azido and two azidoacetamido derivatives of the simplified mycothiol analogue were successfully synthesised and can act as a handle within the binding site. Moreover, the above-mentioned azides were used to synthesize several triazoles that can be utilised as standards when kTGS is performed. The significance of these findings to future drug discovery efforts in this area is discussed. 4 Impact statement Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis and is among the top 10 causes of death globally. In 2017, 1.6 million people died because of TB with 10 million people estimated to suffer from it worldwide. TB is extremely difficult to eradicate and has to be treated using a cocktail of antimicrobial drugs that must be taken for a minimum of six months. Over the years, the same anti-TB drugs have been used extensively resulting in the rise of multi-drug resistant and extensively drug-resistant strains of TB. Because of the global spread of multi-drug resistant tuberculosis and high infection rates, there is a great need for new drugs to be brought to the clinic. M. tuberculosis produces mycothiol (MSH) as the most abundant low molecular weight thiol. MSH and associated enzymes (e.g. mycothiol-S-transferase, MST) are thought to play a major role in combating oxidative stress and to be involved in the detoxification of electrophilic toxins. This is why it is hypothesised that inhibition of MST will lead to the development of a novel strategy for the treatment of TB. This project resulted in the development of a novel approach towards the synthesis of the cysteine-containing mycothiol scaffolds. Three different mycothiol analogues containing glucosamine and cysteine moieties were successfully obtained. The synthesised scaffolds can be easily derivatized forming a library of S-conjugates that can be utilized to explore the role of MST and other MSH-dependent enzymes. The described methodology can be used to generate more mycothiol-based scaffolds if needed. Moreover, relatively short and easy synthetic strategies were designed and applied to successfully form two azido and two azidoacetamido derivatives of the simplified mycothiol analogue. Because of the introduction of the azido group the analogues can be utilized in kinetic target-guided synthesis (kTGS) to produce triazole containing mycothiol analogues. kTGS is a process in which the protein acts as a catalyst or template in the synthesis of its own best inhibitors from the ‘choices’ provided. The use of kTGS to synthesise MST inhibitors will allow further exploration of this technique in lead discovery. Additionally, a diverse chemical library containing azido mycothiol analogue-based triazoles was successfully synthesised using standard ‘click chemistry’ conditions. Thus, providing an easy way to produce a diverse library of potential MST substrates. The biological activity of all of the synthesised 5 mycothiol analogues will have to be tested. The findings of these tests will allow a better understanding of the role of MSH-dependent enzymes. If a promising candidate is found, it can be further developed into a lead compound and finally novel anti-TB drugs could be identified. 6 Contents ACKNOWLEDGMENTS ..................................................................................... 3 ABSTRACT ........................................................................................................ 4 IMPACT STATEMENT ....................................................................................... 5 LIST OF TABLES ............................................................................................... 9 LIST OF FIGURES ........................................................................................... 10 LIST OF ABBREVIATIONS.............................................................................. 15 PART I INTRODUCTION ................................................................................ 18 1. TUBERCULOSIS .......................................................................................... 19 2. MYCOTHIOL ................................................................................................ 23 2.1. MYCOTHIOL BIOSYNTHESIS ........................................................................ 24 2.2. MYCOTHIOL-DEPENDENT ENZYMES AND THEIR INHIBITORS ............................ 29 2.2.1. Mycothiol disulfide reductase (Mtr) .................................................. 29 2.2.2. Mycothiol S-conjugate amidase (Mca) ............................................. 30 2.2.3. Mycothiol-S-nitrosoreductase/-formaldehyde dehydrogenase (MscR) ................................................................................................................... 33 2.2.4. Mycothiol-S-transferase (MST) ........................................................ 33 2.3. CHEMICAL SYNTHESIS OF MYCOTHIOL ......................................................... 35 2.4. CHEMICAL SYNTHESIS OF MYCOTHIOL ANALOGUES ....................................... 43 3. KINETIC TARGET GUIDED SYNTHESIS .................................................... 52 4. AIMS ............................................................................................................. 54 PART II RESULTS AND DISCUSSION ........................................................... 57 5. SYNTHESIS OF THE CYCLOHEXYL CYSTEINE MYCOTHIOL ANALOGUE .......................................................................................................................... 58 5.1. THE FIRST SYNTHETIC ROUTE ..................................................................... 58 5.1.1. Synthesis of the truncated mycothiol analogue ................................ 69 5.2. THE SECOND SYNTHETIC ROUTE ................................................................. 72 5.3. THE THIRD SYNTHETIC ROUTE .................................................................... 76 5.4. CONCLUSIONS .......................................................................................... 78 6. SYNTHESIS OF OTHER CYSTEINE CONTAINING MYCOTHIOL ANALOGUES ................................................................................................... 79 6.1. THE PHENYL CYSTEINE MYCOTHIOL ANALOGUE ............................................ 79 6.2. THE BENZYL CYSTEINE MYCOTHIOL ANALOGUE............................................. 81 6.3. THE AZIDO CYSTEINE MYCOTHIOL ANALOGUE ............................................... 83 6.4. CONCLUSIONS .......................................................................................... 86 7. SYNTHESIS OF THE NON-CYSTEINE MYCOTHIOL ANALOGUES ......... 88 7.1. THE AZIDO MYCOTHIOL ANALOGUES ............................................................ 88 7.1.1. The cyclohexyl azido mycothiol analogue .......................................

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