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Synthetic EthR inhibitors boost antituberculous activity of ethionamide

Nature Medicine volume 15, pages 537–544 (2009)Cite this article

Abstract

The side effects associated with tuberculosis therapy bring with them the risk of noncompliance and subsequent drug resistance. Increasing the therapeutic index of antituberculosis drugs should thus improve treatment effectiveness. Several antituberculosis compounds require in situ metabolic activation to become inhibitory. Various thiocarbamide-containing drugs, including ethionamide, are activated by the mycobacterial monooxygenase EthA, the production of which is controlled by the transcriptional repressor EthR. Here we identify drug-like inhibitors of EthR that boost the bioactivation of ethionamide. Compounds designed and screened for their capacity to inhibit EthR-DNA interaction were co-crystallized with EthR. We exploited the three-dimensional structures of the complexes for the synthesis of improved analogs that boosted the ethionamide potency in culture more than tenfold. In Mycobacterium tuberculosis–infected mice, one of these analogs, BDM31343, enabled a substantially reduced dose of ethionamide to lessen the mycobacterial load as efficiently as the conventional higher-dose treatment. This provides proof of concept that inhibiting EthR improves the therapeutic index of thiocarbamide derivatives, which should prompt reconsideration of their use as first-line drugs.

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Figure 1: Structure of the ligand-binding pocket of EthR.
Figure 2: Sites of BDM14500 interactions with EthR.
Figure 3: Synergistic effect of ethionamide and BDM14500.
Figure 4: Detailed view of the ligand-binding site of EthR co-crystallized with BDM31381.
Figure 5: Comparative measurements of the distance separating the helix-turn-helix DNA-binding motifs (α carbon of Thr60) of EthR upon binding with its ligand.
Figure 6: Ethionamide-boosting effect of BDM31343 on tuberculosis-infected mice.

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References

  1. Corbett, E.L. et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163, 1009–1021 (2003).

    Article  Google Scholar 

  2. Wells, C.D. et al. HIV infection and multidrug-resistant tuberculosis: the perfect storm. J. Infect. Dis. 196 Suppl 1, S86–S107 (2007).

    Article  Google Scholar 

  3. Cox, H. et al. tuberculosis recurrence and mortality after successful treatment: impact of drug resistance. PLoS Med. 3, e384 (2006).

    Article  Google Scholar 

  4. Tomioka, H. Current status of some antituberculosis drugs and the development of new antituberculous agents with special reference to their in vitro and in vivo antimicrobial activities. Curr. Pharm. Des. 12, 4047–4070 (2006).

    Article  CAS  Google Scholar 

  5. Scorpio, A. & Zhang, Y. Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat. Med. 2, 662–667 (1996).

    Article  CAS  Google Scholar 

  6. Zhang, Y., Heym, B., Allen, B., Young, D. & Cole, S. The catalase peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358, 591–593 (1992).

    Article  CAS  Google Scholar 

  7. Manjunatha, U.H. et al. Identification of a nitroimidazo-oxazine–specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 103, 431–436 (2006).

    Article  CAS  Google Scholar 

  8. DeBarber, A.E., Mdluli, K., Bosman, M., Bekker, L.G. & Barry, C.E., III. Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 97, 9677–9682 (2000).

    Article  CAS  Google Scholar 

  9. Baulard, A.R. et al. Activation of the pro-drug ethionamide is regulated in mycobacteria. J. Biol. Chem. 275, 28326–28331 (2000).

    CAS  PubMed  Google Scholar 

  10. Qian, L. & Ortiz de Montellano, P.R. Oxidative activation of thiacetazone by the Mycobacterium tuberculosis flavin monooxygenase EtaA and human FMO1 and FMO3. Chem. Res. Toxicol. 19, 443–449 (2006).

    Article  CAS  Google Scholar 

  11. Dover, L.G. et al. EthA, a common activator of thiocarbamide-containing drugs acting on different mycobacterial targets. Antimicrob. Agents Chemother. 51, 1055–1063 (2007).

    Article  CAS  Google Scholar 

  12. Wilson, T.M., Delisle, G.W. & Collins, D.M. Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis. Mol. Microbiol. 15, 1009–1015 (1995).

    Article  CAS  Google Scholar 

  13. Boshoff, H.I. & Mizrahi, V. Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides. J. Bacteriol. 182, 5479–5485 (2000).

    Article  CAS  Google Scholar 

  14. Engohang-Ndong, J. et al. EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator. Mol. Microbiol. 51, 175–188 (2004).

    Article  CAS  Google Scholar 

  15. Weinstein, H.J., Hallett, W.Y. & Sarauw, A.S. The absorption and toxicity of ethionamide. Am. Rev. Respir. Dis. 86, 576–578 (1962).

    CAS  PubMed  Google Scholar 

  16. Lees, A.W. Ethionamide and isoniazid in previously untreated cases of pulmonary tuberculosis. Dis. Chest 45, 247–250 (1964).

    Article  CAS  Google Scholar 

  17. Holdiness, M.R. Neurological manifestations and toxicities of the antituberculosis drugs. A review. Med. Toxicol. 2, 33–51 (1987).

    Article  CAS  Google Scholar 

  18. Frénois, F., Engohang-Ndong, J., Locht, C., Baulard, A.R. & Villeret, V. Structure of EthR in a ligand bound conformation reveals therapeutic perspectives against tuberculosis. Mol. Cell 16, 301–307 (2004).

    Article  Google Scholar 

  19. Dover, L.G. et al. Crystal structure of the TetR/CamR family repressor Mycobacterium tuberculosis EthR implicated in ethionamide resistance. J. Mol. Biol. 340, 1095–1105 (2004).

    Article  CAS  Google Scholar 

  20. Frénois, F., Baulard, A.R. & Villeret, V. Insights into mechanisms of induction and ligands recognition in the transcriptional repressor EthR from Mycobacterium tuberculosis. Tuberculosis (Edinb.) 86, 110–114 (2006).

    Article  Google Scholar 

  21. Lipinski, C.A., Lombardo, F., Dominy, B.W. & Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001).

    Article  CAS  Google Scholar 

  22. Lipinski, C.A. Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov. Today Technol. 1, 337–341 (2004).

    Article  CAS  Google Scholar 

  23. Veber, D.F. et al. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 45, 2615–2623 (2002).

    Article  CAS  Google Scholar 

  24. Korduláková, J. et al. Isoxyl activation is required for bacteriostatic activity against Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 51, 3824–3829 (2007).

    Article  Google Scholar 

  25. Vieth, M. et al. Characteristic physical properties and structural fragments of marketed oral drugs. J. Med. Chem. 47, 224–232 (2004).

    Article  CAS  Google Scholar 

  26. Kasim, N.A. et al. Molecular properties of WHO essential drugs and provisional biopharmaceutical classification. Mol. Pharm. 1, 85–96 (2004).

    Article  CAS  Google Scholar 

  27. Holdiness, M.R. Clinical pharmacokinetics of the antituberculosis drugs. Clin. Pharmacokinet. 9, 511–544 (1984).

    Article  CAS  Google Scholar 

  28. Zhu, M. et al. Population pharmacokinetics of ethionamide in patients with tuberculosis. Tuberculosis (Edinb.) 82, 91–96 (2002).

    Article  CAS  Google Scholar 

  29. Guo, H., Seet, Q., Denkin, S., Parsons, L. & Zhang, Y. Molecular characterization of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from the USA. J. Med. Microbiol. 55, 1527–1531 (2006).

    Article  CAS  Google Scholar 

  30. Zhang, Y., Vilchèze, C. & Jacobs, W.R. Mechanisms of drug resistance in Mycobacterium tuberculosis. in Tuberculosis and the Tubercle Bacillus (eds. Cole, S.T. et al.) 115–140 (American Society for Microbiology, Washington, DC, 2005).

    Google Scholar 

  31. Morlock, G.P., Metchock, B., Sikes, D., Crawford, J.T. & Cooksey, R.C. ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrob. Agents Chemother. 47, 3799–3805 (2003).

    Article  CAS  Google Scholar 

  32. Schaaf, H.S. et al. Minimal inhibitory concentration of isoniazid in isoniazid-resistant Mycobacterium tuberculosis isolates from children. Eur. J. Clin. Microbiol. Infect. Dis. 26, 203–205 (2007).

    Article  CAS  Google Scholar 

  33. Phetsuksiri, B. et al. Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis. J. Biol. Chem. 278, 53123–53130 (2003).

    Article  CAS  Google Scholar 

  34. Alahari, A. et al. thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria. PLoS One 2, e1343 (2007).

    Article  Google Scholar 

  35. Fajardo, T.T. et al. A clinical trial of ethionamide and prothionamide for treatment of lepromatous leprosy. Am. J. Trop. Med. Hyg. 74, 457–461 (2006).

    Article  CAS  Google Scholar 

  36. Hastings, R.C., Gillis, T.P., Krahenbuhl, J.L. & Franzblau, S.G. Leprosy. Clin. Microbiol. Rev. 1, 330–348 (1988).

    Article  CAS  Google Scholar 

  37. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  38. Winn, M.D., Isupov, M.N. & Murshudov, G.N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122–133 (2001).

    Article  CAS  Google Scholar 

  39. Collaborative-Computational-Project-Number-4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  40. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  41. Snewin, V.A. et al. Assessment of immunity to mycobacterial infection with luciferase reporter constructs. Infect. Immun. 67, 4586–4593 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are indebted to G. Mahieu for his invaluable help with the synergistic experiments on M. tuberculosis, P. Rucktooa for crystal structure acquisitions and manipulations, F. Demirkaya for technical support, M. Flipo for support in chemical synthesis, M. Cynamon for sharing experimental data about ethionamide sensitivity in mice and S. Delaroche for data formatting. We thank H. Gras for critical reading of the manuscript and F. Jean, C. Desruelle and V. Dewailly for grants management. Nuclear magnetic resonance spectra acquisitions were done at the Laboratoire d'Application de Résonance Magnétique Nucléaire, Lille. This work was supported by INSERM, Université de Lille 2 Droit et Santé, Institut Pasteur de Lille, CNRS, the Région Nord-Pas de Calais, Ministère délégué à la Recherche et aux Nouvelles Technologies and the European Union (FEDER OBJ2-4.1-2006/3-n°297/9360), l'Agence Nationale de la Recherche, France (ANR-06-EMPB-033). We are indebted to the European Synchrotron Radiation Facility for beam-time allocation (through the Block Allocation Group BAG MX-485) for this project. X.C. is a recipient of a doctoral fellowship from Institut Pasteur de Lille and the Région Nord-Pas de Calais. B.D. is a recipient of a doctoral fellowship of the Ministère de la Recherche. M.A. and V.V. are researchers from the CNRS.

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  1. Nicolas Willand and Bertrand Dirié: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale (INSERM), U761, Lille, France

    Nicolas Willand, Bertrand Dirié, Matthieu Desroses, Florence Leroux, Rebecca Déprez-Poulain & Benoit Déprez

  2. Faculté de Pharmacie de Lille, Univ Lille Nord de France, Lille, France

    Nicolas Willand, Bertrand Dirié, Matthieu Desroses, Florence Leroux, Rebecca Déprez-Poulain & Benoit Déprez

  3. Institut Pasteur de Lille, France

    Nicolas Willand, Bertrand Dirié, Xavier Carette, Matthieu Desroses, Florence Leroux, Eve Willery, Rebecca Déprez-Poulain, Guy Delcroix, Frédéric Frénois, Marc Aumercier, Camille Locht, Vincent Villeret, Benoit Déprez & Alain R Baulard

  4. Institut Fédératif de Recherche 142, Molecular and Cellular Medicine, Lille, France

    Nicolas Willand, Bertrand Dirié, Xavier Carette, Matthieu Desroses, Florence Leroux, Eve Willery, Rebecca Déprez-Poulain, Frédéric Frénois, Marc Aumercier, Camille Locht, Vincent Villeret, Benoit Déprez & Alain R Baulard

  5. Pôle de Recherche Interdisciplinaire pour le Médicament, Lille, France

    Nicolas Willand, Xavier Carette, Matthieu Desroses, Florence Leroux, Eve Willery, Rebecca Déprez-Poulain, Benoit Déprez & Alain R Baulard

  6. INSERM U629, Lille, France

    Xavier Carette, Eve Willery, Camille Locht & Alain R Baulard

  7. Institut Pasteur, Brussels, Belgium

    Pablo Bifani, Amit Singhal & Vanessa Mathys

  8. Unité Mixte de Recherche 8161, Institut de Biologie de Lille, Centre National de la Recherche Scientifique (CNRS)–Univ Lille Nord de France, Institut Pasteur de Lille, France

    Frédéric Frénois, Marc Aumercier & Vincent Villeret

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Correspondence to Alain R Baulard.

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Willand, N., Dirié, B., Carette, X. et al. Synthetic EthR inhibitors boost antituberculous activity of ethionamide. Nat Med 15, 537–544 (2009). https://doi.org/10.1038/nm.1950

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