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Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis

Nature Chemical Biology volume 9, pages 499–506 (2013)Cite this article

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Abstract

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13F79S mutant conferred high resistance. In vitro, TP inhibited fatty acyl–AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

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Figure 1: Effect of TP2 and TP4 on mycolic acid biosynthesis in M. tuberculosis.
Figure 2: Inhibition of fatty acyl-AMP loading onto purified Pks13 by TP2.
Figure 3: Bactericidal activity of TP2, TP4 and other antituberculosis drugs against M. tuberculosis.

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Acknowledgements

This work was supported in part by US National Institutes of Health (NIH) grant R01 AI080653 to D.A., a United Negro College Fund–Merck Postdoctoral Science Research Fellowship to R.W., a grant from the University of Medicine and Dentistry of New Jersey (UMDNJ) foundation to R.C. and NIH grant R01 AI081736 to M.B.N. W.R.J. acknowledges generous support from the NIH Centers for AIDS Research grant AI-051519 at the Albert Einstein College of Medicine and by NIH grant AI26170. The compounds initially screened in this work were supplied as part of NIH grants AI-95364 and AI-15449. We thank R.C. Goldman (formally Department of Health and Human Services, NIH, and Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), currently RCG Consulting) for his support obtaining Molecular Libraries Probe Centers Network (MLPCN) compounds for the piniBAC screen, R. Reynolds (Southern Research Institute) for his advice on the initial analysis of the MLPCN library and C.E. Barry, III and H.I.M. Boshoff (Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH) for their kind gift of TDM and TMM standards. The MS data were obtained from an Orbitrap instrument funded in part by NIH grant NS046593 for the support of the UMDNJ Neuroproteomics Core Facility.

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Author notes

  1. Romain Veyron-Churlet

    Present address: Present address: Centre d'Infection et Immunité, Institut Pasteur de Lille, Lille, France.,

  2. Regina Wilson and Pradeep Kumar: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Infectious Disease, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA

    Regina Wilson, Pradeep Kumar, Emily Marchiano, Shubhada Shenai, Roberto Colangeli & David Alland

  2. Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA

    Regina Wilson, Pradeep Kumar, Joel S Freundlich, Michael J Szymonifka, Emily Marchiano, Shubhada Shenai, Roberto Colangeli & David Alland

  3. Ruy V. Lourenco Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA

    Regina Wilson, Pradeep Kumar, Joel S Freundlich, Michael J Szymonifka, Emily Marchiano, Shubhada Shenai, Roberto Colangeli & David Alland

  4. Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA

    Vijay Parashar & Matthew B Neiditch

  5. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA

    Catherine Vilchèze & William R Jacobs Jr

  6. Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA

    Catherine Vilchèze & William R Jacobs Jr

  7. Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5235, Université de Montpellier 2, Montpellier, France

    Romain Veyron-Churlet & Laurent Kremer

  8. Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA

    Joel S Freundlich & Michael J Szymonifka

  9. Genomics Institute of the Novartis Research Foundation, San Diego, California, USA

    S Whitney Barnes & John R Walker

  10. Institut National de la Santé et de la Recherche Médicale, Dynamique des Interactions Membranaires Normales, Montpellier, France

    Laurent Kremer

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Contributions

R.W., P.K., C.V., W.R.J., L.K. and D.A. conceived and designed experiments. J.S.F. synthesized compound JSF-1735, and M.J.S. and J.S.F. synthesized FAME standards. V.P. and M.B.N. performed computational docking studies. S.W.B. and J.R.W. performed whole-genome sequencing and analysis. R.W., P.K., C.V., R.V.-C., E.M., S.S., R.C. and L.K. performed whole-cell screening; performed MIC testing; selected resistant mutants; constructed recombinant strains; performed mycolic acid analyses; overexpression studies; and bactericidal, intracellular and synergy assays. R.W., P.K. and D.A. wrote the manuscript. All of the authors discussed the results and commented and contributed to sections of the manuscript.

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Correspondence to David Alland.

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Wilson, R., Kumar, P., Parashar, V. et al. Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat Chem Biol 9, 499–506 (2013). https://doi.org/10.1038/nchembio.1277

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