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Development of a covalent inhibitor of gut bacterial bile salt hydrolases

Nature Chemical Biology volume 16, pages 318–326 (2020)Cite this article

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Abstract

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

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Fig. 1: Rational design of gut bacterial BSH inhibitors.
Fig. 2: Identification of 7 as a potent, nontoxic, pan-BSH inhibitor.
Fig. 3: Compound 7 covalently modifies B. theta BSH at the active site cysteine residue.
Fig. 4: Compound 7 exhibits BSH engagement and minimal off-target effects.
Fig. 5: Compound 7 inhibits BSH activity in vivo and can be gut-restricted.

Data availability

The 16S rDNA datasets analyzed in the manuscript are available through the NCBI under accession number PRJNA574158. The coordinates for both the apo and covalently inhibited forms of the BSH are deposited in the PDB and have PDB ID codes 6UFY and 6UH4, respectively. Raw mass spectrometry data were deposited at MassIVE (massive.ucsd.edu). Native mass spectrometry data files are available for download from the MassIVE archive at the University of California, San Diego (ftp://massive.ucsd.edu/MSV000084491/). All other data generated or analyzed during this study are included in this article and its Supplementary Information files.

Code availability

No custom code or mathematical algorithms were used in this study.

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Acknowledgements

This research was supported National Institutes of Health (NIH) grant nos. R35 GM128618 (to A.S.D.), R35 CA220340 (to S.C.B.), R01 CA222218 (to J.A.M.), an Innovation Award from the Center for Microbiome Informatics and Therapeutics at MIT (to A.S.D.), a grant from Harvard Digestive Diseases Center (supported by NIH grant no. 5P30DK034854-32 to A.S.D.), a Karin Grunebaum Cancer Research Foundation Faculty Research Fellowship (to A.S.D.), a John and Virginia Kaneb Fellowship (to A.S.D.), a Quadrangle Fund for the Advancement and Seeding of Translational Research at Harvard Medical School (Q-FASTR) grant (to A.S.D.) and an HMS Dean’s Innovation Grant in the Basic and Social Sciences (to A.S.D.). L.Y. and S.N.C. acknowledge a Wellington Postdoctoral Fellowship and an American Heart Association Postdoctoral Fellowship, respectively. M.D.M. acknowledges an NSF Graduate Research Fellowship (no. DGE1745303). D.R. is supported by the Early postdoc mobility fellowship from the Swiss National Science Foundation. We thank N. Gray, D. Scott, J. M. Hatcher, J. Wang, J. Clardy, M. Henke and members of the Clardy group for helpful discussions. We thank the ICCB-Longwood Screening Facility for use of their fluorescent plate reader.

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Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Arijit A. Adhikari, Tom C. M. Seegar, Megan D. McCurry, Lina Yao, Snehal N. Chaudhari, Sula Ndousse-Fetter, Stephen C. Blacklow & A. Sloan Devlin

  2. Department of Cancer Biology, Department of Oncologic Pathology, Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA

    Scott B. Ficarro & Jarrod A. Marto

  3. Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Scott B. Ficarro & Jarrod A. Marto

  4. Division of Endocrinology, Metabolism, and Diabetes, Beth Israel Deaconness Medical Center, Boston, MA, USA

    Deepti Ramachandran & Alexander S. Banks

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  1. Arijit A. Adhikari
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Contributions

A.A.A. and A.S.D. conceived the project and designed the experiments. A.A.A. performed most of the experiments. T.C.M.S. and S.C.B. performed the crystallization studies. S.B.F. and J.A.M. performed the mass spectrometry studies. D.R. and A.S.B. performed the in vivo experiments and provided fresh mouse feces. M.D.M. purified and performed experiments with B. longum BSH and performed kinetic studies with B. theta BSH. L.Y. performed the in vitro FXR assays and provided help with experiments. S.N.C. performed the cell culture assays. S.N.F. assisted with bacterial culture experiments. A.A.A. and A.S.D. wrote the manuscript. All authors edited and contributed to the critical review of the manuscript.

Corresponding author

Correspondence to A. Sloan Devlin.

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Competing interests

A.S.D. is an ad hoc consultant for Kintai Therapeutics and HP Hood. S.C.B. serves on the SAB for Erasca, Inc., is a consultant on unrelated projects for Ayala Pharmaceutical and IFM Therapeutics and receives funding from Novartis for an unrelated project. J.A.M. serves on the SAB of 908 Devices (Boston, MA). The other authors declare that no competing interests exist.

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Adhikari, A.A., Seegar, T.C.M., Ficarro, S.B. et al. Development of a covalent inhibitor of gut bacterial bile salt hydrolases. Nat Chem Biol 16, 318–326 (2020). https://doi.org/10.1038/s41589-020-0467-3

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