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Volume 169, Issue 1

Assay development and inhibition of the -DprE2 essential reductase from Open Access

This article is part of the Diversity in Microbiology collection

Abstract

DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β--arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through -DprE2-target overexpression studies. The -DprE1-DprE2 complex was co-purified and a new DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The -DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where -DprE2 was found to enhance -DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving and .

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Keyword(s): decaprenylphosphoryl-D-arabinose , enzyme kinetics , Mt-DprE1 , Mt-DprE2 , mycobacterial cell wall , Mycobacterium tuberculosis and substrate inhibition
Funding
This study was supported by the:
  • Medical Research Foundation (Award MR/R001154/1)
    • Principle Award Recipient: GurdyalS. Besra
  • Medical Research Foundation (Award MR/S000542/1)
    • Principle Award Recipient: GurdyalS. Besra
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-01-23
2024-05-05
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References

  1. Chakaya J, Khan M, Ntoumi F, Aklillu E, Fatima R et al. Global tuberculosis report 2020 - Reflections on the Global TB burden, treatment and prevention efforts. Int J Infect Dis 2021; 113 Suppl 1:S7–S12 [View Article]
     [Google Scholar]
  2. Mirzayev F, Viney K, Linh NN, Gonzalez-Angulo L, Gegia M et al. World Health Organization recommendations on the treatment of drug-resistant tuberculosis, 2020 update. Eur Respir J 2021; 57:2003300 [View Article]
     [Google Scholar]
  3. Abrahams KA, Besra GS. Mycobacterial drug discovery. RSC Med Chem 2020; 11:1354–1365 [View Article]
     [Google Scholar]
  4. Ballell L, Bates RH, Young RJ, Alvarez-Gomez D, Alvarez-Ruiz E et al. Fueling open-source drug discovery: 177 small-molecule leads against tuberculosis. ChemMedChem 2013; 8:313–321 [View Article]
     [Google Scholar]
  5. Rebollo-Lopez MJ, Lelièvre J, Alvarez-Gomez D, Castro-Pichel J, Martínez-Jiménez F et al. Release of 50 new, drug-like compounds and their computational target predictions for open source anti-tubercular drug discovery. PLoS ONE 2015; 10:e0142293 [View Article]
     [Google Scholar]
  6. Macalino SJY, Billones JB, Organo VG, Carrillo MCO. In silico strategies in tuberculosis drug discovery. Molecules 2020; 25:665 [View Article]
     [Google Scholar]
  7. Pauli I, dos Santos RN, Rostirolla DC, Martinelli LK, Ducati RG et al. Discovery of new inhibitors of Mycobacterium tuberculosis InhA enzyme using virtual screening and a 3D-pharmacophore-based approach. J Chem Inf Model 2013; 53:2390–2401 [View Article]
     [Google Scholar]
  8. Wilsey C, Gurka J, Toth D, Franco J. A large scale virtual screen of DprE1. Comput Biol Chem 2013; 47:121–125 [View Article]
     [Google Scholar]
  9. Wolucka BA. Biosynthesis of D-arabinose in mycobacteria - a novel bacterial pathway with implications for antimycobacterial therapy. FEBS J 2008; 275:2691–2711 [View Article]
     [Google Scholar]
  10. Alderwick LJ, Harrison J, Lloyd GS, Birch HL. The mycobacterial cell wall--peptidoglycan and arabinogalactan. Cold Spring Harb Perspect Med 2015; 5:a021113 [View Article]
     [Google Scholar]
  11. Mikusová K, Huang H, Yagi T, Holsters M, Vereecke D et al. Decaprenylphosphoryl arabinofuranose, the donor of the D-arabinofuranosyl residues of mycobacterial arabinan, is formed via a two-step epimerization of decaprenylphosphoryl ribose. J Bacteriol 2005; 187:8020–8025 [View Article]
     [Google Scholar]
  12. Alderwick LJ, Radmacher E, Seidel M, Gande R, Hitchen PG et al. Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core. J Biol Chem 2005; 280:32362–32371 [View Article] [PubMed]
     [Google Scholar]
  13. Kolly GS, Boldrin F, Sala C, Dhar N, Hartkoorn RC et al. Assessing the essentiality of the decaprenyl-phospho-d-arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants. Mol Microbiol 2014; 92:194–211 [View Article]
     [Google Scholar]
  14. Crellin PK, Brammananth R, Coppel RL. Decaprenylphosphoryl-β-D-ribose 2’-epimerase, the target of benzothiazinones and dinitrobenzamides, is an essential enzyme in Mycobacterium smegmatis. PLOS ONE 2011; 6:e16869 [View Article]
     [Google Scholar]
  15. Carroll P, Faray-Kele MC, Parish T. Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach. Appl Environ Microbiol 2011; 77:5040–5043 [View Article]
     [Google Scholar]
  16. Makarov V, Manina G, Mikusova K, Möllmann U, Ryabova O et al. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 2009; 324:801–804 [View Article]
     [Google Scholar]
  17. Pasca MR, Degiacomi G, Ribeiro AL de JL, Zara F, De Mori P et al. Clinical isolates of Mycobacterium tuberculosis in four European hospitals are uniformly susceptible to benzothiazinones. Antimicrob Agents Chemother 2010; 54:1616–1618 [View Article]
     [Google Scholar]
  18. World Health Organisation Global Tuberculosis Report 2019 [Internet]. Geneva: World Health Organisation Press; 2019 https://www.who.int/tb/publications/global_report/en/
  19. Makarov V, Lechartier B, Zhang M, Neres J, van der Sar AM et al. Towards a new combination therapy for tuberculosis with next generation benzothiazinones. EMBO Mol Med 2014; 6:372–383 [View Article]
     [Google Scholar]
  20. Batt SM, Jabeen T, Bhowruth V, Quill L, Lund PA et al. Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors. Proc Natl Acad Sci 2012; 109:11354–11359 [View Article]
     [Google Scholar]
  21. Batt SM, Cacho Izquierdo M, Castro Pichel J, Stubbs CJ, Vela-Glez Del Peral L et al. Whole cell target engagement identifies novel inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β- d -ribose oxidase. ACS Infect Dis 2015; 1:615–626 [View Article]
     [Google Scholar]
  22. Gao Y, Xie J, Tang R, Yang K, Zhang Y et al. Identification of a pyrimidinetrione derivative as the potent DprE1 inhibitor by structure-based virtual ligand screening. Bioorg Chem 2019; 85:168–178 [View Article]
     [Google Scholar]
  23. Piton J, Vocat A, Lupien A, Foo CS, Riabova O et al. Structure-based drug design and characterization of sulfonyl-piperazine benzothiazinone inhibitors of DprE1 from Mycobacterium tuberculosis. Antimicrob Agents Chemother 2018; 62:e00681-18 [View Article]
     [Google Scholar]
  24. Chikhale R, Menghani S, Babu R, Bansode R, Bhargavi G et al. Development of selective DprE1 inhibitors: Design, synthesis, crystal structure and antitubercular activity of benzothiazolylpyrimidine-5-carboxamides. Eur J Med Chem 2015; 96:30–46 [View Article]
     [Google Scholar]
  25. Wang F, Sambandan D, Halder R, Wang J, Batt SM et al. Identification of a small molecule with activity against drug-resistant and persistent tuberculosis. Proc Natl Acad Sci 2013; 110:E2510–7 [View Article]
     [Google Scholar]
  26. Chatterji M, Shandil R, Manjunatha MR, Solapure S, Ramachandran V et al. 1,4-azaindole, a potential drug candidate for treatment of tuberculosis. Antimicrob Agents Chemother 2014; 58:5325–5331 [View Article]
     [Google Scholar]
  27. Taneja NK, Tyagi JS. Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis. J Antimicrob Chemother 2007; 60:288–293 [View Article]
     [Google Scholar]
  28. Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K et al. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics. Appl Environ Microbiol 2013; 79:6134–6139 [View Article]
     [Google Scholar]
  29. Trefzer C, Škovierová H, Buroni S, Bobovská A, Nenci S et al. Benzothiazinones are suicide inhibitors of mycobacterial decaprenylphosphoryl-β-D-ribofuranose 2’-oxidase DprE1. J Am Chem Soc 2012; 134:912–915 [View Article]
     [Google Scholar]
  30. Neres J, Pojer F, Molteni E, Chiarelli LR, Dhar N et al. Structural basis for benzothiazinone-mediated killing of Mycobacterium tuberculosis. Sci Transl Med 2012; 4:150ra121 [View Article]
     [Google Scholar]
  31. Richter A, Rudolph I, Möllmann U, Voigt K, Chung C-W et al. Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1. Sci Rep 2018; 8:13473 [View Article]
     [Google Scholar]
  32. Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 2000; 405:962–966 [View Article]
     [Google Scholar]
  33. Haver HL, Chua A, Ghode P, Lakshminarayana SB, Singhal A et al. Mutations in genes for the F420 biosynthetic pathway and a nitroreductase enzyme are the primary resistance determinants in spontaneous in vitro-selected PA-824-resistant mutants of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2015; 59:5316–5323 [View Article]
     [Google Scholar]
  34. Fujiwara M, Kawasaki M, Hariguchi N, Liu Y, Matsumoto M. Mechanisms of resistance to delamanid, a drug for Mycobacterium tuberculosis. Tuberculosis 2018; 108:186–194 [View Article]
     [Google Scholar]
  35. Manjunatha UH, Boshoff H, Dowd CS, Zhang L, Albert TJ et al. Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci 2006; 103:431–436 [View Article]
     [Google Scholar]
  36. Matsumoto M, Hashizume H, Tomishige T, Kawasaki M, Tsubouchi H et al. OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice. PLoS Med 2006; 3:e466 [View Article]
     [Google Scholar]
  37. Choi KP, Kendrick N, Daniels L. Demonstration that fbiC is required by Mycobacterium bovis BCG for coenzyme F(420) and FO biosynthesis. J Bacteriol 2002; 184:2420–2428 [View Article]
     [Google Scholar]
  38. Vilchèze C, Jacobs WRJ. The isoniazid paradigm of killing, resistance, and persistence in Mycobacterium tuberculosis. J Mol Biol 2019; 431:3450–3461 [View Article]
     [Google Scholar]
  39. Peterson FJ, Mason RP, Hovsepian J, Holtzman JL. Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes. J Biol Chem 1979; 254:4009–4014
     [Google Scholar]
  40. McOsker CC, Fitzpatrick PM. Nitrofurantoin: mechanism of action and implications for resistance development in common uropathogens. J Antimicrob Chemother 1994; 33 Suppl A:23–30 [View Article]
     [Google Scholar]
  41. Spain JC. BIODEGRADATION OF NITROAROMATIC COMPOUNDS. Annu Rev Microbiol 1995; 49:523–555 [View Article]
     [Google Scholar]
  42. Race PR, Lovering AL, Green RM, Ossor A, White SA et al. Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. J Biol Chem 2005; 280:13256–13264 [View Article]
     [Google Scholar]
  43. Singh R, Manjunatha U, Boshoff HIM, Ha YH, Niyomrattanakit P et al. PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science 2008; 322:1392–1395 [View Article]
     [Google Scholar]
  44. Kamata K, Mitsuya M, Nishimura T, Eiki J-I, Nagata Y. Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. Structure 2004; 12:429–438 [View Article]
     [Google Scholar]
  45. Kurosu M, Mahapatra S, Narayanasamy P, Crick DC. Chemoenzymatic synthesis of Park’s nucleotide: toward the development of high-throughput screening for MraY inhibitors. Tetrahedron Letters 2007; 48:799–803 [View Article]
     [Google Scholar]
  46. Besra GS, Morehouse CB, Rittner CM, Waechter CJ, Brennan PJ. Biosynthesis of mycobacterial lipoarabinomannan. J Biol Chem 1997; 272:18460–18466 [View Article]
     [Google Scholar]
  47. Reed MC, Lieb A, Nijhout HF. The biological significance of substrate inhibition: a mechanism with diverse functions. Bioessays 2010; 32:422–429 [View Article]
     [Google Scholar]
  48. Best JA, Nijhout HF, Reed MC. Homeostatic mechanisms in dopamine synthesis and release: a mathematical model. Theor Biol Med Model 2009; 6:21 [View Article]
     [Google Scholar]
  49. Vuong TV, Vesterinen A-H, Foumani M, Juvonen M, Seppälä J et al. Xylo- and cello-oligosaccharide oxidation by gluco-oligosaccharide oxidase from Sarocladium strictum and variants with reduced substrate inhibition. Biotechnol Biofuels 2013; 6:148 [View Article]
     [Google Scholar]
  50. Lachmann P, Huang Y, Reimann J, Flock U, Adelroth P. Substrate control of internal electron transfer in bacterial nitric-oxide reductase. J Biol Chem 2010; 285:25531–25537 [View Article]
     [Google Scholar]
  51. Leeds JA, Sachdeva M, Mullin S, Barnes SW, Ruzin A. In vitro selection, via serial passage, of Clostridium difficile mutants with reduced susceptibility to fidaxomicin or vancomycin. J Antimicrob Chemother 2014; 69:41–44 [View Article]
     [Google Scholar]
  52. Gupta A, Bhakta S. An integrated surrogate model for screening of drugs against Mycobacterium tuberculosis. J Antimicrob Chemother 2012; 67:1380–1391 [View Article]
     [Google Scholar]
  53. Kuzmic P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem 1996; 237:260–273 [View Article]
     [Google Scholar]
  54. Scherman H, Kaur D, Pham H, Skovierová H, Jackson M et al. Identification of a polyprenylphosphomannosyl synthase involved in the synthesis of mycobacterial mannosides. J Bacteriol 2009; 191:6769–6772 [View Article]
     [Google Scholar]
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Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis
Microbiology 169, 001288 (2023); https://doi.org/10.1099/mic.0.001288
/content/journal/micro/10.1099/mic.0.001288
/content/journal/micro/10.1099/mic.0.001288
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