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Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence

Nature Chemical Biology volume 13, pages 218–225 (2017)Cite this article

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Abstract

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme–artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

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Figure 1: Discovery of inhibitors of the DosRST pathway.
Figure 2: Transcriptional profiling shows artemisinin, HC102A and HC103A inhibit the core genes of the DosRST regulon during hypoxia.
Figure 3: Artemisinin, HC102A and HC103A inhibit TAG synthesis, survival and isoniazid (INH) tolerance.
Figure 4: Artemisinin directly inhibits DosS and DosT by targeting sensor-kinase heme.
Figure 5: Amino acid substitutions in DosS or DosT promote resistance to artemisinin.
Figure 6: HC103A inhibits DosS and DosT autophosphorylation.

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Acknowledgements

High-throughput screening libraries and their preparation was supported by the New England Regional Center of Excellence (U54 AI057159) and the Institute of Chemistry and Cell Biology (ICCB) at Harvard Medical School. M. Farrugia and B. Hausinger provided support with the UV–visible spectroscopy experiments. The MSU RTSF provided technical support for the RNA–seq library preparation and sequencing. The Vahlteich Medicinal Chemistry Core is grateful for ongoing support from the Ella and Hans Vahlteich Fund and Beverly Vahlteich Delaney. We thank members of the Abramovitch lab for critical reading of the manuscript. This project was supported by start-up funding from Michigan State University and AgBioResearch, a grant from the NIH-NIAID (R21AI105867), and Grand Challenges Explorations awards (OPP1059227 and OPP1119065) from the Bill & Melinda Gates Foundation.

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Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA

    Huiqing Zheng, Christopher J Colvin, Benjamin K Johnson & Robert B Abramovitch

  2. Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, USA

    Paul D Kirchhoff, Michael Wilson & Scott D Larsen

  3. Sweet Briar College, Sweet Briar, Virginia, USA

    Katriana Jorgensen-Muga

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  1. Huiqing Zheng
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  2. Christopher J Colvin
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Contributions

H.Z., C.J.C., B.K.J. and R.B.A. conducted high throughput screen and follow-up experiments. M.W. and S.D.L. synthesized chemical compounds; K.J.-M. contributed reagents. P.D.K. performed structural modeling; H.Z. and R.B.A. wrote the manuscript.

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Correspondence to Robert B Abramovitch.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–8 (PDF 7417 kb)

Supplementary Dataset 1

Differential gene expression data of WT Mtb treated with inhibitors and the DMSO treated DosR mutant. (XLSX 165 kb)

Supplementary Dataset 2

Differential gene expression data of the DosR mutant treated with the inhibitors. (XLSX 81 kb)

Supplementary Dataset 3

Complete gene expression tables for transcriptional profiling experiments. (XLSX 3320 kb)

Supplementary Note

Synthetic procedures. (PDF 103 kb)

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Zheng, H., Colvin, C., Johnson, B. et al. Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence. Nat Chem Biol 13, 218–225 (2017). https://doi.org/10.1038/nchembio.2259

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