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Inhibition of a Mycothiol Biosynthetic Enzyme and a Detoxification Enzyme As Anti Tubercular Drug Targets

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Inhibition of a Mycothiol Biosynthetic Enzyme and a Detoxification Enzyme As Anti Tubercular Drug Targets The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University Mycothiol Disulfide Reductase as a Drug Target A thesis Presented in fulfilment of the requirement of the degree of DOCTOR OF PHILOSOPHY Department of Clinical Laboratory Sciences UNIVERSITY OF CAPE TOWN In the Division of Chemical Pathology APRIL 2010 Vuyo Bhongolethu Mavumengwana ii DECLARATION The work contained in this thesis is original, except where indicated and acknowledged. No portion of this work has been submitted for another degree at this or any other university. The University of Cape Town may reproduce the contents in whole or in part for the purposes of research. V.B. Mavumengwana April 2010 iii ACKNOWLEDGEMENTS I wish to express my gratitude to my supervisors Prof D.J Steenkamp and Prof. D.W. Gammon for their supervision, guidance and teaching throughout this study. I would also like to thank Dr Levecque, Mr Munyololo and Dr Kinfe for their support and assistance during my stay in the chemistry department. I’m indebted to my colleagues in the Division of Chemical Pathology, Dr Marakalala for testing my synthetic compounds against MshB and Mca and Dr Williams for cloning mtr in pSD26. I’m grateful to Gabriel and Nick for their friendship and willingness to help whenever I needed them. Ms Di James (Department of Molecular and Cell Biology, University of Cape Town) and Dr Wiid (University of Stellenbosch, South Africa) are gratefully acknowledged for sequencing experiments and anti-tubercular testing of compounds respectively. I am deeply indebted to Dr Tricia Owens (Division of Chemical Pathology, UCT) for her support and encouragement throughout my stay in the department, and to Dr André Trollip (BiotecSA) for caring and creating a work environment that facilitated the completion of my studies. You stood by me and gave me strength when I was ready to quit. I really have no words to describe my appreciation. Support from the National Research Foundation, and the University of Cape Town is gratefully acknowledged. iv ABSTRACT Tuberculosis, a common and deadly infectious disease is caused by the pathogen Mycobacterium tuberculosis . The recent emergence of multi-drug and extreme multi- drug resistant strains poses an even greater obstacle to controlling the disease, particularly in areas where efficient and effective health systems have not been properly addressed. Current treatments using first line drugs typically require extended use and strict compliance by patients, which is often not adhered to. As a result, there is an urgent need for new and highly effective drugs to combat this disease. Mycobacterium tuberculosis relies on mycothiol, a low molecular mass thiol used to circumvent oxidative stress generated by activated macrophages and to deal with antibiotics such as rifamycin. Enzymes involved in the biosynthesis of mycothiol have been elucidated over the past ten years and some have been designated to be essential in the growth of Mtb , with mycothiol disulfide reductase (Mtr) being one. Mtr is involved in the reduction of oxidized mycothiol (MSSM) to mycothiol (MSH) upon exposure of the microbe to oxidative stress. The rational behind this study involves the use of naphthoquinones grafted onto the mycothiol template as subversive substrates against Mtr. Initially, we had planned to clone and express Mtr in Corynebacterium glutamicum (C. glutamicum ) to try to obtain enhanced expression levels of Mtr, and although the expression of the enzyme was not successful we were able to confirm that C. glutamicum does indeed depend on MSH as has been previously suggested but not actually proven. MSH was isolated from C. glutamicum with the aid of a new derivatising agent 2-bromo- acetonaphthone (BAN) and characterized using a combination of NMR and mass spectrometry. Consequently, we were also able to establish that MSSnaph (obtained during the isolation process of MSH) is an alternative and better substrate for Mtr with a -1 5 km,app of 23 µM, a kcat and kcat /k m of 15.56 s and 8.17 x 10 respectively, as compared to the natural substrate (MSSM). v Synthesis of subversive substrates proceeded through the initial formation of 2-azido-2- deoxy-3,4,6-tri-O-acetyl-D-glucopyranosyl acetate (7). Phenyl-3,4,6-tri-O-acetyl-2-azido- 1-thio-α-D-glucopyranoside (12 ) was generated from this and, after reduction of the azide, linked to the carboxy terminus of substituted naphthoquinones using standard N-3- (dimethylaminopropyl)-N-ethyl-carbodiimide (EDC) and Hydroxybenzotriazole (HOBt) coupling procedures. Deacetylation using the Zemplén procedure gave the target naphthoquinone derivatives 35b (34%), 36b (46%), 37b (60%), and 38b (52%). R R1 1 R1 O O O R R R1 1 1 R R1 R1 1 OAc N NH2 N3 3 SPh SPh 7 12 R2 R1 O O R2 R1 R2 NH R1 O OH NH SPh O OH 35: n = 0 O SPh 36: n = 1 O n 37: n = 2 n 38: n = 3 O O a R1 = OAc deacetylation b R2 = OH The compounds exhibited specific activities ranging between 0.29 µmol/min/mg and 0.42 µmol/min/mg at 40 µM, and inhibition levels ranging between 4 and 14% at 40 µM when tested against Mtr. The Phenyl-2-deoxy-2-[3'-(8''-hydroxy-3''-methyl-1'',4''-dioxo-1'',4''- dihydronaphthalen-2''-yl)propanamido]-1-thio-α-D-glucopyranoside (35b) was the only compound that exhibited bacteriocidal effects when tested against Mtb using the radiometric respiratory technique based on the Bactec system. A range of thioglycosides and O-glycosides was also evaluated (at 500 µM) as possible inhibitors of Mtr and found to be of poor inhibitory nature exhibiting percentage inhibitions that ranged between 1.86-12.75% and 3.39-20.2% respectively. We also prepared INH-NAD(P) adduct mixtures based on published procedure and found for the vi first time that Mtr is also inhibited by the activated isoniazid, with percentage inhibitions ranging from 16.4% to 66%. Attempts to prepare Ethionamide-NAD(P) adduct mixtures using the same procedure were however, not successful. The finding that INH-NAD(P) adducts also inhibit Mtr offers a new direction for the design of inhibitors targeted at Mtr. vii Abbreviations AcCys Acetyl-cystein AcCySmB Bimane derivative of N-acetylcysteine Accq-fluor 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate ATP Adenosine triphosphate BAN 2-bromo-2’-acetonaphtone BF3.OEt2 Boron triflouride etherate Boc 2O Di-tert -butyldicarbonate CD 3OD Deuterated methanol CH 3CN Acetonitrile Cl 3CCN Trichloroacetonitrile COSY Correlation spectroscopy DCM Dichloromethane DEPT-NMR Distortionless enhancement by polarization transfer DMAP 4-Dimethyl aminopyridine DMSO Dimethyl sulfoxide DNA D eoxyribonucleic acid EDC N-3-(dimethylaminopropyl)-N-ethyl-carbodii mide EDTA Ethylenediaminetetraacetic acid EPO Medium used for growing electroporation competent cells (described in Appl Microbiol Biotechnol, 1999, 52: 541-545) EtOAc Ethyl acetate FAD Flavin adenine dinucleotide GlcNAc N-acetylglucosamine GlcNAc-Ins 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside GSH Glutathione HBr Hydrogen bromide HOBt Hydroxybenzotriazole HPLC High performance liquid chromatography viii INH-NAD(P) Isonicotinoylated nicotinamide adenine dinuclotides IPTG Isopropyl-beta-D-thiogalactopyranoside Kcat kcat Turn over number kcat /k m Overall catalytic efficiency LB Lurea broth LBG Lurea broth containing filter sterilized glucose mBBr monobromobimane Mca Mycothiol S-conjugate amidase MDR Multidrug resistance MeOH Methanol MSH Mycothiol MshA 1L-myo-inositol-1- phosphate 1--D-N-acetylglucosaminyltransferase MshB 1-O-(2-acetamido-2-deoxy--D-glucopyranosyl)-D-myo -inositol deacetylase MshC ATP-dependent L-cysteine: 1-O-(2-amino-2-deoxy-D-glucopyranosyl)-D- myo -inositol ligase MshD Mycothiol synthase MSmB Monobromobimane derivative of mycothiol MSSM Mycothiol disulfide Mtr Mycothiol disulfide reductase NADPH Reduced nicotinamide adenine dinucleotide phosphate NADP + Oxidized nicotinamide adenine dinucleotide phosphate NADH Reduced nicotinamide adenine dinucleotide NAD + Oxidised nicotinamide adenine dinucleotide NaN 3 Sodium azide NMR Nuclear magnetic resonance NQ Naphthoquinone PCR Polymerase chain reaction PMSF Phenylmethylsulphonyl fluoride RNI Reactive nitrogen intermediate ix ROI Reactive oxygen intermediate SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis Tf 2O Trifluoromethanesulfonic (triflic) anhydride xv TfN 3 Triflyl azide THF Tetrahydrofuran TLCK N-p-tosyl-L-lysine-chloromethyl ketone TMS Trimethyl silyl TPCK N-p-tosyl-L-phenylalanine-chloromethyl ketone TR Trypanothione reductase TS 2 Trypanothione disulfide XDR Extensively drug resistant tuberculosis α-DGI 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside xiv TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENTS iii ABSTRACT iv ABBREVIATIONS vii LIST OF FIGURES AND TABLES x CONTENTS xiv CHAPTER 1 INTRODUCTION 1 1.1 Origins of Mycobacterium tuberculosis 1 1.2 Mtb pathogenesis and macrophage interaction 2 1.3 Thiols in intracellular redox reactions 5 1.3.1 Glutathione (GSH) 5 1.3.2 Mycothiol (MSH) 7 1.3.3 MSH Biosynthesis 8 1.3.4 Mycothiol dependent detoxification 12 1.3.41 NAD/MSH-dependent formaldehyde dehydrogenase 12 1.3.4.2 Mycothiol S-Conjugate Amidase 13 1.4 Mycothiol disulfide reductase 16 1.5 Mtb essential genes in MSH biosynthetic pathway 20 1.6 Mycothiol analogs 24
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