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A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

Nature Chemical Biology volume 13, pages 943–950 (2017)Cite this article

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Abstract

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

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Figure 1: BRD4592 activity against Mtb.
Figure 2: BRD4592 inhibition of TrpAB in vitro.
Figure 3: BRD4592 stabilizes closed, active states of the β-subunit.
Figure 4: Crystal structures of Mtb TrpAB.
Figure 5: TrpAB is required for survival of Mtb in various conditions.

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Acknowledgements

We thank J. Gomez, M. Serrano-Wu, and B. Hubbard for helpful discussion. We would also like to thank S. Minami for assistance with in vivo studies. This work was supported by the Broad Institute Tuberculosis donor group, the Pershing Square Foundation, the Bill and Melinda Gates Foundation (grant OPP1032518 to S.L.S.), the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (HHSN272200700058C and HHSN272201200026C to A.J.) and the US Department of Energy, Office of Biological and Environmental Research (contract DE-AC02-06CH11357, A.J.). For this work, the University of Chicago is a subgrantee of Structural Genomics Consortium under a Bill and Melinda Gates Foundation grant for the Structure-Guided Drug Discovery Coalition (A.J. and K.M.).

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Author notes

  1. Partha P Nag and Karolina Michalska: These authors contributed equally to this work.

Authors and Affiliations

  1. The Broad Institute, Cambridge, Massachusetts, USA

    Samantha Wellington, Partha P Nag, Stephen E Johnston, Virendar K Kaushik, Patrick McCarren, Besnik Bajrami, Stewart L Fisher, Stuart L Schreiber & Deborah T Hung

  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Samantha Wellington & Deborah T Hung

  3. Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Samantha Wellington, Anne E Clatworthy, Senya Combs & Deborah T Hung

  4. Center for Structural Genomics of Infectious Diseases, University of Chicago, Chicago, Illinois, USA

    Karolina Michalska, Robert P Jedrzejczak, Natalia I Maltseva & Andrzej Joachimiak

  5. Biosciences Division, Structural Biology Center, Argonne National Laboratory, Argonne, Illinois, USA

    Karolina Michalska, Robert P Jedrzejczak & Andrzej Joachimiak

  6. Chan School of Public Health, Boston, Massachusetts, USA

    Noman Siddiqi

  7. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    Andrzej Joachimiak

  8. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Stuart L Schreiber

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Contributions

S.W. performed experiments with Mtb as well as kinetic and biophysical experiments with TrpAB. P.P.N. was responsible for chemistry. K.M., N.I.M., R.P.J., and A.J. were responsible for protein expression and purification, X-ray crystallography, and structure refinement and analysis. S.E.J. and B.B. developed and performed LC-MS analysis of kinetic assays. V.K.K. and S.W. performed calorimetry. N.S., A.E.C., S.C., and S.W. performed in vivo experiments. P.M. contributed to structural analysis and modeling. S.L.F., A.J., S.L.S., and D.T.H. advised on experiments and interpretation and together with S.W., K.M., and P.P.N. wrote the paper.

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Correspondence to Deborah T Hung.

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Wellington, S., Nag, P., Michalska, K. et al. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase. Nat Chem Biol 13, 943–950 (2017). https://doi.org/10.1038/nchembio.2420

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