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