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Acetyltransferase/Uridylyltransferase Glmu Mechanistic characterisation and inhibitor identification of Mycobacterium tuberculosis bifunctional N- acetyltransferase/uridylyltransferase GlmU Peter D. Craggs University College London and The Francis Crick Institute PhD Supervisor : Dr Luiz Pedro Sório de Carvalho A thesis submitted for the degree of Doctor of Philosophy University College London May 2019 Declaration I Peter David Craggs confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The mycobacterial cell envelope is a defining feature of the bacteria, primarily due to its highly-ordered, relatively impermeable nature that is likely one of the key attributes that has contributed to the success of this pathogenic over the last thousand years. Peptidoglycan is a unique and essential structural element that provides much of the strength and rigidity of the mycobacterial cell envelope. Most of the enzymes involved in the biosynthetic pathway of peptidoglycan have been shown to be essential for Mycobacterium tuberculosis growth . Mycobacterium tuberculosis GlmU is an essential bifunctional N-acetyltransferase, uridylyltransferase enzyme involved in the formation of uridine-diphosphate N- acetylglucosamine, which is the universal donor of N-acetylglucosamine for both peptidoglycan and lipopolysaccharide biosynthesis. This enzyme catalyses acetylation of glucosamine 1-phosphate, followed by uridylylation of N- acetylglucosamine 1-phosphate. Detailed characterisation of the kinetic mechanism ascertained that acetyl transfer progresses by the formation of a ternary complex, with acetyl coenzyme A binding preceding glucosamine 1-phosphate and coenzyme A the last product to dissociate. A novel ternary complex crystal structure, with glucose 1-phosphate and acetyl- coenzyme A, identified a candidate general base involved in the deprotonation of glucosamine 1-phosphate, as well as other important active site residues for substrate binding and catalysis. pH-rate studies and site-directed mutagenesis led to assignment of Histidine 374 as the catalytic general base. Solvent kinetic isotope effect experiments and pH-rate studies identified that acetyl transfer is partially rate-limiting. Small molecule screening led to the identification of novel inhibitors of GlmU catalysed acetyl transfer reaction. Inhibitors identified from the GSK TB set, were confirmed as GlmU interacting compounds and shown to inhibit the growth of M. tuberculosis . These results show that knowledge of the kinetic and catalytic mechanism enabled optimisation of a thorough screening approach that identified novel inhibitors that demonstrated that GlmU acetyltransferase activity is essential for M. tuberculosis growth. 3 Impact Statement The work presented in this thesis initially focusses on characterising the kinetic and chemical mechanism of the left-handed beta helix (LβH) domain containing acetyltransferase enzyme, GlmU. Further studies focussed on identifying novel small molecule inhibitors of the GlmU acetyltransferase activity and the subsequent confirmation that these compounds can inhibit the growth of Mycobacterium tuberculosis . In addition to catalysing acetyl transfer from acetyl-coenzyme A (Ac- CoA) to glucosamine 1-phosphate (GlcN-1P), GlmU is also a pyrophosphorylase, which catalyses uridylyl transfer from uridine-triphosphate (UTP) to N- acetylglucosamine 1-phosphate (GlcNAc-1P), a product of the acetyltransferase activity. The formation of uridine-diphosphate N-acetylglucosamine (UDP-GlcNAc) is essential in most bacteria as it is the universal donor of N-acetylglucosamine (GlcNAc) for both peptidoglycan and lipopolysaccharide biosynthesis. These biosynthetic pathways are critical for the formation of the cell envelope of Mycobacterium tuberculosis (M. tuberculosis ), the causative agent of tuberculosis (TB). Genetic target validation experiments, such as Transposon Hybridisation (TraSH) studies, identified several of the enzymes involved in both the peptidoglycan and lipopolysaccharide biosynthetic pathways as essential for M. tuberculosis growth, including GlmU. The global impact of TB is still hugely significant, in 2017, the disease was the ninth leading cause of death worldwide, with an estimated 1.3 million fatalities. The emergence and spread of antibiotic resistant TB is an increasing global health threat. For instance, in 2017, there were 558,000 cases of multidrug-resistant TB (MDR-TB), with 8.9% of these incidences estimated to be extensively drug-resistant TB (XDR- TB). To exacerbate this issue, only two new TB drugs have been developed in the last 40 years and global spending on TB drug discovery and development is still well below the estimated required levels. The magnitude of this global health threat underscores the urgent need to discover new antimycobacterial agents that have novel mechanisms of action (MOA). To this end, it is critical to understand how genetically validated, essential M. tuberculosis enzymes, such as GlmU, which could be attractive drug targets, function. 4 There were many facets of the GlmU acetyltransferase mechanism unknown, or kinetically untested, at the outset of this project, which are important to define and understand prior to progressing with drug discovery studies. The aim of this study is to thoroughly characterise the kinetic and chemical mechanisms of the acetyltransferase activity of GlmU and to utilise this information to develop a robust, sensitive small molecule screening strategy that could be utilised to screen both synthetic compound libraries, as well as initiating knowledge-based inhibitor design. Additionally, this project has highlighted the combination of drug discovery strategies, utilising the results of M. tuberculosis phenotypic HTS studies, as effective compound collections for the identification of inhibitors by a target-based approach. The final aims of this project are to both demonstrate the chemical tractability of the acetyltransferase activity of GlmU, while ultimately, using novel inhibitors to prove the essentiality of this enzyme for M. tuberculosis growth. Demonstration of the chemical tractability, or druggability, of GlmU acetyltransferase activity has provided evidence that it is possible to identify and confirm drug-like inhibitors. Identification of dual inhibitors of both GlmU catalysed acetyl transfer and M. tuberculosis growth has confirmed the essentiality of the enzyme. This approach of thorough enzyme characterisation, in combination with hit identification utilising phenotypically validated compounds, could be systematically applied to other genetically validated, essential M. tuberculosis enzymes. 5 Acknowledgement I would like to start by acknowledging my PhD supervisor Luiz Pedro Carvalho for allowing me the opportunity to embark on this project. Luiz has been a supportive and enthusiastic supervisor and has taken the time to help interpret and discuss data, in addition to guiding the next steps. Luiz has taught me many core enzymology techniques and has encouraged me to think small, by concentrating on the details of each experiment and reinforcing the importance of thoroughly interrogating data. Luiz has also supported my attendance of the Gordon Research Conference on Enzymology in the USA for three consecutive years, which has allowed me to learn from discipline experts from around the world. Argyrides (Archie) Argyrou was instrumental in the genesis of this project, providing the initial contact with Luiz, as well as extensively contributing to the initial proposal. Archie is a hugely knowledgeable enzymologist and his guidance and advice has been invaluable throughout this project. This project would not have been possible without the initial support from David Powell, who, as my department head at GSK was responsible for providing the financial assistance required. Dave also showed trust and belief in my abilities and has remained a supportive influence throughout the project. Melanie Leveridge, who succeeded Dave as department head, has continued to provide invaluable support, which has enabled the completion of this project. I’d like to acknowledge and thank Stephane Mouilleron for carrying out all GlmU X- ray crystallography experiments and taking the time to discuss the data and contribute to a publication. I’d like to thank Cesira de Chiara for carrying out the GlcN-1P pH titration 1H NMR Spectroscopy experiments and allowing me to be involved in both the practical and analytical aspects of this study. UDP-GlcN was provided by Rob Field and Martin Rejzek, which enabled the inclusion of this reagent in a panel of analogues for substrate specificity experiments. This reagent is not commercially available, and I’d like to take this opportunity to thank both Rob and Martin for their help. The GlcN-1P inhibitor analogues were kindly contributed by Ben Whitehurst and Rob Young, who took the time to design and make these novel compounds. The 6 synthesis and purification of the GlcN-1P inhibitor analogues were one of the subjects of Ben’s PhD project. Collaboratively working with both Ben and Rob was a productive and rewarding experience. I’d also like to thank Rob for his continued chemistry guidance and tuition throughout this project. Ryan Bingham, my line manager at GSK, throughout most of this project, was largely responsible for developing me as a scientist prior to the initiation of this PhD. Ryan has helped balance my GSK and PhD workload, while continually
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