Understanding cofactor f420 dependent mechanism(s) in the activation of bicycle nitroimidazoles and in the physiology of mycobacterium tuberculosis

UNDERSTANDING COFACTOR F420 DEPENDENT MECHANISM(S) IN THE ACTIVATION OF BICYCLIC NITROIMIDAZOLES AND IN THE PHYSIOLOGY OF MYCOBACTERIUM TUBERCULOSIS MEERA GURUMURTHY B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Meera Gurumurthy 24 November 2012 ii ACKNOWLEDGEMENTS Manju, for making this thesis work and my four years at NITD so memorable - You have been a tremendous source of inspiration, ideas, challenges, arguments and agreements and are greatly responsible for making science this fascinating to me Novartis Institute for Tropical Diseases (NITD) for funding my PhD and for its vibrant work environment Thomas, for giving me the opportunity to work at NITD Paul Herrling, for always being an email away and for being such a pillar of strength and confidence to the students Collaborators at the Genomics Institute of the Novartis Research Foundation (GNF) and the National Institutes of Health (NIH) for all the scientific input Tathagata, Helena, Clif, Nood, Srini, Raman, Pablo, Sylvie and Madhu for scientific insight and numerous interesting discussions Joseph Cherian and Cynthia Dowd for chemistry support Sabai, Sindhu, Martin Rao, Martin G, Melvin, Jo Ann, Vivian and Jun, for being such wonderful teachers in the lab Wai Yee, Pat, Wenwei, KL, Pramila, Seow Hwee, Jansy and Bee Huat, for laughter, more laughter and some more laughter…. Friends who were flatmates at one point and who are nothing less than family todayAmeek, Kiran, Anshul, Abhishek, Megha, Deepika, Vani, Rishi, Gokul, Pappoo, for just being who you are! Mrs. Bhaskar (fondly, teacher) and Raka (fondly, aunty), for bringing dance into my life the way you did and for so many more things that words cannot justice to… amma, appa and Gayu,what can I say here that I haven’t said in the many phone calls everyday? Paati, This Thesis is dedicated to you, paati – to your love, willpower, determination, open-mindedness, unassuming nature and your characteristic ‘logic’ iii TABLE OF CONTENTS Summary…………………………………………………………………………………ix List of Figures…………………………………………………………………… … xii List of Tables…………………………… .……… .………………………………….xiv List of Abbreviations………………………………………………………………… .xv Publications……………………………………………………………………………xvii Poster Presentations……………………… .…………………………………… xviii Tuberculosis and Thesis: An Introduction 1.1 Tuberculosis: The Global Burden 1.2 TB Drug Development: Challenges Galore . 1.3 TB: Matters of the mycobacterium and its biology . 1.3.1 Mtb Pathogenesis: Infection of alveolar macrophage and survival strategies 1.3.2 Granulomas: The hallmark of TB infection . 1.3.3 Latency and Reactivation . 1.4 TB: Matters of the Clinic . 1.4.1 Preventive Measures against TB 1.4.2 HIV Co-infection 1.4.3 Diagnosis of Active, Latent and Drug-Resistant TB 10 1.4.4 Treatment Regimens and Emergence of Resistance . 10 1.5 TB Drug Development: Global Initiatives . 15 1.6 TB Drug Development: NITD’s Initiative and The genesis of this Thesis . 20 1.6.1 Objectives Overall 23 iv Materials and Methods 2.1 Bacterial strains, media and culture conditions . 24 2.2 Drugs and Chemicals . 25 2.3 F420 purification from M. smegmatis 25 2.4 F420 reduction assay 27 2.5 Cloning, expression and purification of recombinant Ddn proteins 27 2.6 Evaluation of Ddn enzyme activity 28 2.6.1 Absorbance-based Methods 28 2.6.2 Mass Spectrometry (MS)-based Ddn enzyme activity . 30 2.6.3 Fluorometry-based methods . 30 2.6.4 Ddn kinetics studies via NO release assay . 31 2.7 Ddn binding studies to PA-824, F420 and F420H2 . 33 2.8 Modeling of PA-824 at the binding pocket 33 2.9 Sequence Analysis . 34 2.10 Mycobacterial genomic DNA isolation . 37 2.11 Generation of genetic knockout mutants and complemented mycobacterial strains . 37 2.11.1 Construction of knockout (gene replacement) cassette 38 2.11.2 Construction of complementation vector . 38 2.11.3 Electroporation and selection of transformants 38 2.12 Southern blot hybridization 39 2.13 Minimal inhibitory concentration determination (MIC99, MIC90) . 40 2.14 Biochemical characterization of mycobacterial strains for detection of F420 levels . 41 v 2.15 In vivo NO release assay in M. bovis BCG cells 41 2.16 Growth sensitivity assays . 42 2.16.1 Colony Forming Units Assay . 42 2.16.2 Oxidative Stress Sensitivity Zone of Inhibition Assay . 42 2.17 Macrophage infection and assay 43 2.18 Non-replicating persistence . 43 2.18.1 Gradual oxygen depletion (Wayne model) . 43 2.18.2 Rapid oxygen depletion (Anaerobic shiftdown) . 44 2.19 Determination of [NADH] and [NAD+] Concentrations . 44 2.20 Quantification of Intracellular ATP . 45 2.21 RNA isolation and quantitative reverse transcription (qRT) PCR . 45 2.22 Protein extraction and Western Blotting 46 2.23 Mycobacterial membrane vesicle assay for ATP synthesis . 46 Biochemical and structural characteization of Ddn, the activating enzyme of bicyclic nitroimidazoles in Mtb 3.1 The evolution of nitroimidazoles as anti-tuberculars . 48 3.2 PA-824 and OPC-67683 50 3.3 Current Status: Clinical Development . 52 3.4 Mechanism of Activation and Action 54 3.5 Objectives 58 3.6 Summary of findings 58 3.7 Results 61 3.7.1 Optimization of Ddn catalyzed PA-824 reduction . 61 vi 3.7.2 The lipophilic tail of nitroimidazole substrates determines reduction selectivity and efficiency 63 3.7.3 Kinetic mechanism of Ddn catalysis 71 3.7.4 The kinetics of NO generation by Ddn . 73 3.7.5 Ddn: Structural and Mutational studies 75 3.7.6 Ddn binding with PA-824 and F420 by fluorescence quenching . 78 3.7.7 Mutagenesis and characterization of the N-terminus of Ddn . 84 3.8 Discussion 87 Evaluation of the physiological role of cofactor F420 in Mtb pathogenesis 4.1 Cofactors and drug targets . 95 4.2 Cofactor F420: Properties, Distribution and functions . 96 4.3 F420 and Mycobacteria 98 4.4 Structural characterization of F420 dependent enzymes . 101 4.5 Biosynthesis of F420 102 4.5.1 Structures of F420 in various species . 102 4.5.2 F420 biosynthetic pathway . 103 4.5.3 F420 isolation and production 110 4.6 F420’s role in Mycobacteria: Clues from Bioinformatics and Literature 111 4.7 Survival strategies in the phagosome: Mtb’s defence against stress 112 4.8 Objectives 116 4.9 Summary of findings 117 4.10 Results 118 4.10.1 fbiC knockout mutants are compromised for the production of F420 118 vii 4.10.2 F420- mutants show survival defect under in vitro induced nitrosative stress . 123 4.10.3 F420- mutant is hypersensitive to menadione and plumbagin induced oxidative stress 126 4.10.4 Ddn catalyzes F420H2 dependent reduction of quinone to quinol . 129 4.10.5 Ddn, Rv1261 and Rv1558 form a unique class of F420H2 specific quinone reductases. 138 4.10.6 F420- mutants are hypersensitive to bactericidal agents . 140 4.10.7 F420- mutants show survival defect under hypoxic dormancy models 143 4.11 Discussion 146 Conclusions…………………………………………………………………….154 Reference List………………………………………………………………….160 Appendix 7.1 Appendix 1. Synthesis of CGI-17341 isomers 182 7.2 Appendix 2. Synthesis of (R) and (S) phenyl analogues of nitroimidazo-oxazoles . 184 7.3 Appendix 3. Mycobacterium Vessicle assay . 187 viii SUMMARY The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 that are currently in Phase II clinical development represent a promising novel class of therapeutics for Tuberculosis (TB) (Mukherjee and Boshoff, 2011). Both compounds are pro-drugs that are reductively activated within Mycobacterium tuberculosis (Mtb), the causative organism of TB. PA-824 as a potential anti-TB agent has many attractive characteristics - most notably its novel mechanism of action, activity against aerobic replicating and hypoxic non-replicating mycobacterium, activity in vitro against drug-resistant clinical isolates, and activity as both a potent bactericidal and a sterilizing agent in mice. While PA-824 was optimized for its aerobic cellular activity, its anaerobic activity correlated with the amount of nitric oxide (NO) generated when the pro-drug was reduced by an Mtb deazaflavin cofactor (F420) dependent nitroreductase (Ddn) (Singh et al., 2008). The physiological role of both F420 and the activating enzyme Ddn in Mtb is unknown. This thesis delves into two areas that are in line with the Novartis Institute of Tropical Diseases’ initiatives in developing drugs against latent TB and in unravelling novel Mtb ‘targets’ for discovery of therapeutics against drug resistant Mtb. (i) Biochemical and structural characterization of Ddn enzyme, to understand substrate / cofactor specificity of the enzyme and to support a programme that focused on rational optimization of bicyclic nitroimidazoles (ii) Evaluation of the physiological role of cofactor F420 and Ddn in Mtb pathogenesis as a first step in assessing its suitability as a drug discovery target ix In order to further enhance current understanding of the mechanism of action of PA-824, Ddn activity was evaluated with PA-824 and a selected collection of chemically distinct nitroimidazole analogs substrates by investigating reoxidation of F420H2, production of NO and by determining binding constants of the analogs to the protein. A direct chemiluminescence-based NO detection assay to measure the kinetics of NO production by Ddn was developed. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating an enzymatic turnover-independent assessment of affinity. The results from this research work suggest that the tail portion of the nitroimidazole determines the binding orientation of the head group, conferring stereospecificity in orientation of the molecule towards reduction (Cellitti et al., 2012;Gurumurthy et al., 2012). The results presented elucidate structural features important for understanding substrate binding providing insight into the activation of bicyclic 4nitroimidazoles that could facilitate optimization of this class of compounds toward more efficient killing for improved TB treatment. F420 is a low redox potential (-360 mV), soluble 7, 8-didemethyl-8-hydroxy-5-deazariboflavin that is characteristic of methanogenic bacteria where it is involved in anaerobic respiration and energy metabolism. 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Synthesis of CGI-17341 isomers The synthesis of the (R) and (S) isomers of CGI-17341 was carried out stereo-specifically modifying the procedure for racemic synthesis as described previously (Nagarajan et al., 1989). Briefly, 2,4-dinitroimidazole (Sudarsanam VNK et al., 1982) was condensed with either (R) or (S) 1,2-epoxybutane to give the corresponding alcohols. The alcohols were then cyclized using NaH to yield the final products. (S)-1-(2,4-dinitro-1H-imidazol-1-yl)butan-2-ol (CSD-250): The title compound was prepared in the same manner as for the R isomer using (R)-1,2-epoxybutane. The yield after column chromatography (run once) was 1.19 g (5.17 mmol, 82 %). LCMS (ESI) M+H= 230.9. 1H NMR matched the values for CSD-252. (R)-1-(2,4-dinitro-1H-imidazol-1-yl)butan-2-ol (CSD-252): 2,4-dinitroimidazole (1.00g, 6.33 mmol) and (S)-1,2-epoxybutane (0.83 ml, 9.54 mmol) were combined in ethanol (0.6 ml) and heated in a sealed tube at 70 °C overnight. The reaction mixture was cooled to ambient temperature and the solvents were removed in vacuo. The reaction mixture was purified twice by column chromatography (CHCl3:CH3OH, 10: 0.6) to give 0.47 g (2.04 mmol, 32 %)of pure material. 1H NMR (300 MHz, CDCl3) δ: 1.08 (t, 3H, CH3), 1.48-1.78 (m, 2H, CH2), 3.93-4.01 (m, 1H, CH), 4.24 (dd, 1H, J1=13.8, J2=9.3 Hz), 4.81 (dd, 1H, J1=13.8, J2=2.4 Hz), 8.00 (1H, Ar-H). (R)-2-ethyl-6-nitro-2,3-dihydroimidazo[2,1-b]oxazole ((R)-CGI-17341): (S)-1-(2,4-dinitro-1Himidazol-1-yl)butan-2-ol (CSD-250) (0.79 g, 3.43 mmol) was combined with NaH (60 % dispersion in oil, 158 mg, 4.12 mmol) in anhydrous THF (20 ml) under argon at 0˚C. Once the NaH addition was complete, the reaction mixture was brought to ambient temperature and stirred 182 for h. The reaction was quenched with water and the solvent was removed in vacuo. The mixture was extracted between water and EtOAc, and the organic portion was dried over MgSO4. The mixture was filtered and evaporated to give a pale solid (0.40 g). The solid was recrystallized from EtOAc to give 87 mg (0.48 mmol, 14 %) of the desired product. H NMR (300 MHz, CDCl3) δ: 1.10 (t, 3H, CH3), 1.88-2.06 (m, 2H, CH2), 3.93 (dd, 1H, J1=10.2, J2=7.5 Hz), 4.36 (dd, 1H, J1=10.2. J2=8.4 Hz), 5.23-5.32 (m, 1H, CH), 7.53 (1H, Ar-H). (S)-2-ethyl-6-nitro-2,3-dihydroimidazo[2,1-b]oxazole ((S)-CGI-17341): (R)-1-(2,4-dinitro-1Himidazol-1-yl)butan-2-ol (CSD-252) (0.42 g, 1.82 mmol) was combined with NaH (60 % dispersion in oil, 84 mg, 2.2 mmol) in anhydrous THF (10.4 ml) under argon at 0˚C. Once the NaH addition was complete, the reaction mixture was brought to ambient temperature and stirred for h. The reaction was quenched with water and the solvent was removed in vacuo. The mixture was extracted between water and EtOAc, and the organic portion was dried over MgSO4. The mixture was filtered and evaporated to give a pale solid (0.26 g). The solid was recrystallized from EtOAc to give 0.12 g (0.66 mmol, 36 %) of the desired product. H NMR (300 MHz, CDCl3) δ: 1.10 (t, 3H, CH3), 1.88-2.06 (m, 2H, CH2), 3.93 (dd, 1H, J1=10.2, J2=7.5 Hz), 4.36 (dd, 1H, J1=10.2, J2=8.4 Hz), 5.23-5.32 (m, 1H, CH), 7.53 (1H, Ar-H). 183 7.2 Appendix 2. Synthesis of (R) and (S) phenyl analogues of nitroimidazo-oxazoles (R)-2-(2-chloro-4-nitro-1H-imidazol-1-yl)-1-phenylethanol (CSD-297): 2-Chloro-4- nitroimidazole (1.00 g, 6.78 mmol), (S)-(-)-styrene oxide (1.22 g, 10.17 mmol), and K2CO3 (0.13 g, 0.95 mmol) were combined at ambient temperature in ethanol (9 mL). The reaction mixture was heated at 70-75 °C overnight in a sealed tube. TLC showed two spots and no remaining starting material. The mixture was reduced, re-dissolved in CHCl3 and passed through a Celite bed. The eluent was evaporated and purified via column chromatography (Hex: EtOAc, 3:1 to 3:2.25) to give 0.45 g (1.70 mmol, 25 %) of the desired product as a solid. A second column recovered 0.17 g (0.63 mmol) additional desired product, bringing the total yield to 34 %. LCMS (ESI) M+H= 268.1. H NMR (300 MHz, CDCl3) δ: 2.81 (bs, 1H), 4.14 (dd, 1H, J1=14.4 Hz, J2=7.8 Hz), 4.26 (dd, 1H, J1=14.4 Hz, J2=3.3 Hz), 5.07 (1H, dd, J1=7.8 Hz, J2=3.3 Hz), 7.30-7.41 (m, 5H), 7.88 (s, 1H). (S)-2-(2-chloro-4-nitro-1H-imidazol-1-yl)-1-phenylethanol (CSD-296): The desired compound was prepared as for the (R)-isomer using (R)-(+)-styrene. The yield after column chromatography was 0.52 g (1.95 mmol, 29%). LCMS (ESI) M+H=268.1. 1H NMR (300 MHz, CDCl3) δ: 2.40 (bs, 1H), 4.16 (dd, 1H, J1=14.1 Hz, J2=7.2 Hz), 4.27 (dd, 1H, J1=14.1 Hz, J2=3.6 Hz), 5.06 (1H, dd, J1=6.9 Hz, J2=3.0 Hz), 7.30-7.42 (m, 5H), 7.89 (s, 1H). (S)-1-(2-(tert-butyldimethylsilyloxy)-2-phenylethyl)-2-chloro-4-nitro-1H-imidazole (CSD-302): At °C, TBDMS-triflate (0.37 ml, 1.61 mmol) was added to a stirring solution of (S)-2-(2chloro-4-nitro-1H-imidazol-1-yl)-1-phenylethanol (CSD-296) (0.266 g, 0.99 mmol) in CH2Cl2 (10 ml) and 2,6-lutidine (0.217 ml, 1.86 mmol). The reaction mixture was allowed to stir for 3.5 h, when TLC showed no remaining starting material. Aqueous NaHCO3 (10%) was added to quench the reaction. The mixture was extracted between CH2Cl2 and water. The organic 184 portions were combined, washed with 1N HCl, and dried over MgSO4. The mixture was filtered and evaporated to give 0.323 g (0.85 mmol, 86 %) of a crystalline solid that was used in the next reaction without further purification. LCMS (ESI) M+H=382.1 and M+Na=404.0. 1H NMR (300 MHz, CDCl3) δ: -0.20 (s, 3H), -0.13 (s, 3H), 0.85 (s, 9H), 4.06 (dd, 1H, J1=14.4 Hz, J2= 8.1 Hz), 4.17 (dd, 1H, J1=14.1 Hz, J2=3.6 Hz), 4.93 (dd, 1H, J1=8.1 Hz, J2=3.9 Hz), 7.27-7.40 (m, 5H), 7.74 (s, 1H). (S)-6-nitro-2-phenyl-2,3-dihydroimidazo[2,1-b]oxazole ((R)-Phenyl Oxazole): At 0oC and under an argon atmosphere, NaH (60% oil dispersion, 48 mg) was added to (R)-2-(2-chloro-4-nitro-1Himidazol-1-yl)-1-phenylethanol (CSD-297) (0.16 g, 0.60 mmol) in ml dry THF. The reaction mixture became orange and opaque upon addition. After 90 minutes, the reaction was quenched by the addition of MeOH. Roughly one-third of the product was purified via preparative TLC to give 15 mg of the desired compound. HRMS for C11H9N3O3 was calculated 232.0722 and found to be 232.0741. 1H NMR (300 MHz, CDCl3) δ: 4.22 (dd, 1H, J1=10.2, J2=8.1 Hz), 4.67 (dd, 1H, J1=10.5, J2=8.7 Hz), 6.25 (t, 1H, CH), 7.36-7.49 (m, 5H, Ar-H), 7.59 (s, 1H, Ar-H). (R)-6-nitro-2-phenyl-2,3-dihydroimidazo[2,1-b]oxazole ((S) Phenyl Oxazole): At ambient temperature and under a nitrogen atmosphere, TBAF (1M in THF, 1.35 ml, 1.35 mmol) was added to (S)-1-(2-(tert-butyldimethylsilyloxy)-2-phenylethyl)-2-chloro-4-nitro-1H-imidazole (CSD-302) (0.17 g, 0.45 mmol) in 20 ml anhydrous THF. The reaction mixture changed from colorless to yellowish brown. After 1h, the reaction was stopped by the addition of water and was evaporated to dryness. The mixture was purified by preparative TLC (EtOAc: Hex, 2:3) to give 26 mg (25 %) of the target compound. Purification also yielded 39 mg (32.4 %) of the desTBDMS alcohol as an undesired side product. HRMS for C11H9N3O3 calculated 232.0728 and found to be 232.0722. 1H NMR (300 MHz, CDCl3) δ: 4.22 (dd, 1H, J1=10.5, J2=7.8 Hz), 4.68 (dd, 1H, J1=10.5, J2=8.4 Hz), 6.25 (t, 1H, CH), 7.35-7.50 (m, 5H, Ar-H), 7.59 (s, 1H, Ar-H). 185 186 7.3 Appendix 3. Mycobacterium Vessicle assay 150000 no cofactor control NADH NAD 100000 RLUs F420H2 F420H2+ NAD F420 50000 F420+ NAD NADH F420H2 + TMC-207 0 10 20 30 Time (min) 40 50 187 [...]... ethylenediamines (SQ-109) and bicyclic nitroimidazoles (PA-824 and OPC67683) Of these, compounds belonging to only two classes- bicyclic nitroimidazoles and diarylquinolines- offer new mechanisms of action against Mtb Understaning the mechanism of action of bicyclic nitroimidazoles and the physiologic significance of their activating machinery in Mtb form the basis for the research work described in this thesis... contributed to the understanding of the Mtb pathogenesis The following subsections discussing the biology and pathophysiology of Mtb not only present the various challenges that the organism poses in the context of drug development but also highlight some of the key processes that can be targetted by therapeutics that are designed to combat the pathogen Significant advancements in the understanding of Mtb... For latent TB, the standard treatment is six to nine months of INH alone Patients with MDR-TB are treated with the remaining first-line drugs to which Mtb is sensitive, in combination with second-line and other drugs used as TB therapeutics ρ-aminosalicylic acid (PAS), cycloserine, fluoroquinolones (ciprofloxacin, moxifloxacin, ofloxacin), aminoglycosides (streptomycin, amikacin, kanamycin), polypeptides... understand TB infection in humans have lent significant insights into the progression of the disease In vitro analyses of the responses of murine and human macrophages to Mtb infection indicate a robust pro-inflammatory response of the cells Infected macrophages are stimulated to invade the lung epithelium (Ulrichs and Kaufmann, 2006;Flynn and Chan, 2005;Algood et al., 2005) and consequently there is... Second-line Aminoglycosides (Streptomycin,Kanamycin, Amikacin) Capreomycin Fluoroquinolones (Ofloxacin, Levofloxacin, Moxifloxacin) Cycloserine ρ-aminosalicylic acid (PAS) Ethionamide/Prothionamide Thioacetazone Others Clofazimine Amoxicilin + Clavulanate Imipenem Linezolid Clarithromycin Thioridazine * Gene to which resistance is mapped to is indicated in brackets 13 In the last three decades, there... increase in the number of TB cases owing to the HIV epidemic, the emergence of resistance and several other factors discussed in the following sections 8 1.4.1 Preventive Measures against TB The TB vaccine M.bovis Bacille Calmette-Guerin (BCG) - an attenuated strain of M.bovis that underwent several passages - was first tested in humans in 1921 To date, it remains the only licensed TB vaccine and the. .. recruitment of several other components of the immune system that result in the remodelling of the infection site (Ulrichs and Kaufmann, 2006;Flynn and Chan, 2005) The result is the formation of the granuloma; a structure that is responsible for immune containment and therefore results in latent TB infection (LTBI) (Tully et al., 2005;Dheda et al., 2005) The solid granuloma is not only the site of Mtb containment... progression of the disease and most importantly, the adoption of a non-replicating or dormant form by Mtb; all these present a series of complications in the development of therapeutics against the pathogen Nevertheless, in order to truly appreciate the challenge around effective management of TB, one has to understand several other issues closely linked to the management of the disease – some examples being... Before detailing the objectives of the thesis in section 1.6, the following is a brief description of the ten compounds in the pipeline 15 A B Figure 1.2 The Global Pipeline for anti-TB Therapeutics (A) There are ten compounds in clinical development (Phase 1-III) with several other novel novel chemical entities in the lead optimization and preclinical development stages Source: WHO Global Tuberculosis. .. Ribosome Inhibitors: Linezolid, an approved drug of the Oxazolidinone class, has low in- vitro activity against Mtb Oxazolidinones inhibit protein synthesis by binding to the 70S ribosomal initiation complex and have a broad spectrum of activity against anaerobic and gram-positive aerobic bacteria, and mycobacteria (Diekema and Jones, 2001) Phase II studies evaluating the use of low dose linezolid for . UNDERSTANDING COFACTOR F 420 DEPENDENT MECHANISM(S) IN THE ACTIVATION OF BICYCLIC NITROIMIDAZOLES AND IN THE PHYSIOLOGY OF MYCOBACTERIUM TUBERCULOSIS MEERA. developed. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating an enzymatic turnover-independent assessment of affinity. The results. are in line with the Novartis Institute of Tropical Diseases’ initiatives in developing drugs against latent TB and in unravelling novel Mtb ‘targets’ for discovery of therapeutics against