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Investigation of Antibiotic Targets in the Decaprenyl

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Investigation of Antibiotic Targets in the Decaprenyl INVESTIGATION OF ANTIBIOTIC TARGETS IN THE DECAPRENYL-PHOSPHORYLARABINOSE BIOSYNTHESIS IN M. TUBERCULOSIS by SZILVIA TÓTH a thesis submitted to University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences University of Birmingham July 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Declaration The work presented in this thesis is original except where citing relevant references. Studies were conducted from 2014 to 2017 in the School of Biosciences, University of Birmingham, B15 2TT, UK and also in research and development facility of GlaxoSmithKline, laboratory for Diseases of the Developing World, Tres Cantos, Spain. No part of this work has been submitted for a degree or a diploma to this or any other university. ii Abstract An estimated 1.67 million people died of tuberculosis (TB) in 2016 and it is a threat to human life on a global-scale. To shorten current treatments and battle drug resistant strains it is important to discover and develop new drugs against the causative agent, Mycobacterium tuberculosis. Phenotypic screens have delivered potent hit and lead molecules in the past but the need to target new pathways in M. tuberculosis metabolism to circumvent pre-existing drug resistance mechanisms. The decaprenyl-phosphorylarabinose (DPA) biosynthetic pathway produces irreplaceable building blocks of the unique mycobacterial cell wall structure. Decaprenyl-phosphorylribose (DPR) Epimerase sub-units 1 and 2 (DprE1 and DprE2) are essential enzymes of this pathway. Whilst DprE1 has received much attention, however DprE2 has been overlooked as a potential drug target. In this thesis the role of a small disordered region of DprE1 in ligand binding and physiological function has been investigated. This region appears to interact with the substrate and based on enzyme activity assays, physiological importance of specific residues was found to be in line with the level of conservation between species. In addition, a high-throughput screen against a DprE2 overexpressing M. bovis BCG strain has been utilised to discover new hit compounds against this target, coupled with the development of an enzymatic assay to evaluate specific DprE2 inhibition. The screen resulted nitrofuran hit compounds that probably act as prodrugs, which was indicated by spontaneous resistant mutants resembling the mutations necessary to activate new TB pro- drug pretomanid. iii Acknowledgement I would like to take this opportunity to thank all the people who helped me in completing this work and overcome obstacles along the way. Firstly, I am most grateful to my supervisors, Prof. Gurdyal Besra and Dr. Klaus Fütterer, who guided me in these past four years and are role models to look up to in science research. I am thankful for their global influence on my professional development as well as for every small constructive criticism that made this thesis better. I am thankful for my colleagues that helped my laboratory work in two different locations. The members of the Besra, Alderwick and Bhatt laboratories in University of Birmingham have always been supportive and a pleasure to work with. In particular, I would like to thank the support of Sarah M. Batt, Natacha Veerapen, Patrick Moynihan, Katherine Abrahams, Usha Veeraraghavan and Cristian Varela postdoctoral members of the lab who were knowledgeable and patient to answer my questions at all times. My fellow PhD students at the time, Monika Jankute, Panchali Kanvatirth, Asma Javid have also been always kind and a pleasure to work with. Regarding my Spanish research placement, I am honestly grateful to the people of GSK Tres Cantos, our coordinator Joel Lelievre, my mentors Esther Perez-Herran and Beatriz Rodriguez-Miquel and the TB and Malaria research teams. Also, to Open Lab fellows Shipra Grover, Kaj Kreutzfeldt, Cynthia Tallant and Hugo Fraga who were once more very helpful in laboratory work and troubleshooting. iv Finally, I am happy that as one of four Marie Sklodowska-Curie fellows of the project CooperaTB I could work along Giacomo Chiodarelli, Bogdan Duma and Ramon Soto Garcia. This journey would have not been the same without your company. v Table of contents Table of contents Declaration.................................................................... ii Abstract ......................................................................... iii Acknowledgement ........................................................ iv Table of contents .......................................................... vi List of figures ............................................................... x List of tables ................................................................. xv List of abbreviations ..................................................... xvii 1 Introduction .................................................................................................................. 1 1.1 Tuberculosis disease ......................................................................................................................... 1 1.1.1 Current global status ................................................................................................................... 1 1.1.2 Historical background of TB ......................................................................................................... 3 1.1.3 Pathobiology of tuberculosis with implications to drug development ....................................... 8 1.1.3.1 Latent TB infection .............................................................................................................. 9 1.1.3.2 Unique environmental niches ........................................................................................... 10 1.2 Characteristics of the mycobacterial cell wall ................................................................................ 14 1.2.1 Lipoarabinomannan (LAM), lipomannan (LM) and phosphatidyl-myo-inositol mannosides (PIMs) 16 1.2.2 Peptidoglycan (PG) .................................................................................................................... 17 1.2.3 Arabinogalactan (AG)................................................................................................................. 18 1.2.4 The outer membrane of M. tuberculosis ................................................................................... 19 1.2.5 Capsule material ........................................................................................................................ 21 1.3 Present state of tuberculosis treatment......................................................................................... 21 1.3.1 Current set of anti-tuberculosis drugs ....................................................................................... 21 vi Table of contents 1.3.2 Adjunct therapies in TB treatment ............................................................................................ 22 1.3.3 Typical drug targets of M. tuberculosis ..................................................................................... 23 1.4 Drug targets Mt-DprE1 and Mt-DprE2 ........................................................................................... 25 1.4.1 Mt-DprE1, validated drug target ............................................................................................... 25 1.4.1.1 Physiological function of DprE1/2..................................................................................... 25 1.4.1.2 Structure and inhibition of Mt-DprE1 ............................................................................... 27 1.4.1.3 Biochemical assay of Mt-DprE1 ........................................................................................ 31 1.4.2 Mt-DprE2, potential drug target ................................................................................................ 31 1.5 Phenotypic screens in TB early stage drug discovery ..................................................................... 33 1.5.1 Early stage drug discovery ......................................................................................................... 33 1.5.2 Whole cell screening design in TB drug development ............................................................... 35 1.5.2.1 Basic assay requirements .................................................................................................. 35 1.5.2.2 Indicators of cell viability .................................................................................................. 37 1.5.3 Biological hit compound characterisation ................................................................................. 38 1.5.3.1 Toxicity screening of hit compounds ................................................................................ 38 1.5.3.2 Target validation ............................................................................................................... 39 1.6 Aims ..............................................................................................................................................
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