Development of a covalent inhibitor of gut bacterial bile salt hydrolases

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Ridlon, J. M., Kang, D.-J. & Hylemon, P. B. Bile salt biotransformations by human intestinal bacteria. J. Lipid Res. 47, 241–259 (2006).

    CAS  PubMed  Google Scholar 

  2. 2.

    Fiorucci, S. & Distrutti, E. Bile acid-activated receptors, intestinal microbiota, and the treatment of metabolic disorders. Trends Mol. Med. 21, 702–714 (2015).

    CAS  PubMed  Google Scholar 

  3. 3.

    Setchell, K. D., Lawson, A. M., Tanida, N. & Sjövall, J. General methods for the analysis of metabolic profiles of bile acids and related compounds in feces. J. Lipid Res. 24, 1085–1100 (1983).

    CAS  PubMed  Google Scholar 

  4. 4.

    Hamilton, J. P. et al. Human cecal bile acids: concentration and spectrum. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G256–G263 (2007).

    CAS  PubMed  Google Scholar 

  5. 5.

    Modica, S., Gadaleta, R. M. & Moschetta, A. Deciphering the nuclear bile acid receptor FXR paradigm. Nucl. Recept Signal 8, e005 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Vavassori, P., Mencarelli, A., Renga, B., Distrutti, E. & Fiorucci, S. The bile acid receptor FXR is a modulator of intestinal innate immunity. J. Immunol. 183, 6251–6261 (2009).

    CAS  PubMed  Google Scholar 

  7. 7.

    Pols, T. W. H. et al. Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor. PLoS One 12, e0176715 (2017).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Begley, M., Hill, C. & Gahan, C. G. M. Bile salt hydrolase activity in probiotics. Appl. Environ. Microbiol. 72, 1729–1738 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Chiang, J. Y. Recent advances in understanding bile acid homeostasis. F1000Res 6, 2029 (2017).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Song, Z. et al. Taxonomic profiling and populational patterns of bacterial bile salt hydrolase (BSH) genes based on worldwide human gut microbiome. Microbiome 7, 9 (2019).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Strelow, J. M. A perspective on the kinetics of covalent and irreversible inhibition. SLAS Disco. 22, 3–20 (2017).

    CAS  Google Scholar 

  12. 12.

    Roberts, A. B. et al. Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential. Nat. Med. 24, 1407–1417 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Rossocha, M. et al. Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product. Biochem. 44, 5739–5748 (2005).

    CAS  Google Scholar 

  14. 14.

    Huijghebaert, S. M. & Hofmann, A. F. Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase or human fecal cultures. J. Lipid Res. 27, 742–752 (1986).

    CAS  PubMed  Google Scholar 

  15. 15.

    Kawamoto, K., Horibe, I. & Uchida, K. Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus. J. Biochem. 106, 1049–1053 (1989).

    CAS  PubMed  Google Scholar 

  16. 16.

    Yao, L. et al. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife 7, 675 (2018).

    Google Scholar 

  17. 17.

    Liu, Q. et al. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 20, 146–159 (2013).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Wilson, A. J., Kerns, J. K., Callahan, J. F. & Moody, C. J. Keap calm, and carry on covalently. J. Medicinal Chem. 56, 7463–7476 (2013).

    CAS  Google Scholar 

  19. 19.

    Serafimova, I. M. et al. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat. Chem. Biol. 8, 471–476 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Henise, J. C. & Taunton, J. Irreversible Nek2 kinase inhibitors with cellular activity. J. Med. Chem. 54, 4133–4146 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Xie, T. et al. Pharmacological targeting of the pseudokinase Her3. Nat. Chem. Biol. 10, 1006–1012 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Quintás-Cardama, A., Kantarjian, H., Cortes, J. & Verstovsek, S. Janus kinase inhibitors for the treatment of myeloproliferative neoplasias and beyond. Nat. Rev. Drug Discov. 10, 127–140 (2011).

    PubMed  Google Scholar 

  23. 23.

    Cohen, M. S., Zhang, C., Shokat, K. M. & Taunton, J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 308, 1318–1321 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Angliker, H., Wikstrom, P., Rauber, P. & Shaw, E. The synthesis of lysylfluoromethanes and their properties as inhibitors of trypsin, plasmin and cathepsin B. Biochem. J. 241, 871–875 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Miller, R. M. & Taunton, J. Targeting protein kinases with selective and semipromiscuous covalent inhibitors. Meth. Enzymol. 548, 93–116 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Coleman, J. P. & Hudson, L. L. Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens. Appl. Environ. Microbiol. 61, 2514–2520 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Tanaka, H., Hashiba, H., Kok, J. & Mierau, I. Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization. Appl. Environ. Microbiol. 66, 2502–2512 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Wang, Z. et al. Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters. Appl. Environ. Microbiol. 78, 8795–8802 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Stellwag, E. J. & Hylemon, P. B. Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis. Biochimica et. Biophysica Acta–Enzymol. 452, 165–176 (1976).

    CAS  PubMed  Google Scholar 

  30. 30.

    Smith, K., Zeng, X. & Lin, J. Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system. PLoS One 9, e85344 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Sayin, S. I. et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell Metab. 17, 225–235 (2013).

    CAS  PubMed  Google Scholar 

  32. 32.

    Li, F. et al. Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity. Nat. Commun. 4, 2384 (2013).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Hofmann, A. F. The function of bile salts in fat absorption. The solvent properties of dilute micellar solutions of conjugated bile acids. Biochem. J. 89, 57–68 (1963).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Ferruzza, S., Rossi, C., Scarino, M. L. & Sambuy, Y. A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium. Toxicol. Vitr. 26, 1252–1255 (2012).

    CAS  Google Scholar 

  35. 35.

    Lanning, B. R. et al. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nat. Chem. Biol. 10, 760–767 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Parasar, B. et al. Chemoproteomic profiling of gut microbiota-associated bile salt hydrolase activity. ACS Cent. Sci. 5, 867–873 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Alnouti, Y. Bile Acid sulfation: a pathway of bile acid elimination and detoxification. Toxicol. Sci. 108, 225–246 (2009).

    CAS  PubMed  Google Scholar 

  38. 38.

    Padmanabhan, P., Grosse, J., Asad, A. B. M. A., Radda, G. K. & Golay, X. Gastrointestinal transit measurements in mice with 99mTc-DTPA-labeled activated charcoal using NanoSPECT-CT. EJNMMI Res. 3, 60–68 (2013).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Singh, J., Petter, R. C., Baillie, T. A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10, 307–317 (2011).

    CAS  PubMed  Google Scholar 

  40. 40.

    Turk, B. Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov. 5, 785–799 (2006).

    CAS  PubMed  Google Scholar 

  41. 41.

    Johnson, D. S., Weerapana, E. & Cravatt, B. F. Strategies for discovering and derisking covalent, irreversible enzyme inhibitors. Future Med. Chem. 2, 949–964 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Joyce, S. A. et al. Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut. Proc. Natl Acad. Sci. USA 111, 7421–7426 (2014).

    CAS  PubMed  Google Scholar 

  43. 43.

    Ma, C. et al. Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells. Science 360, eaan5931 (2018).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Wallace, B. D. et al. Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 330, 831–835 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Xie, C. et al. An intestinal farnesoid X receptor-ceramide signaling axis modulates hepatic gluconeogenesis in mice. Diabetes 66, 613–626 (2017).

    CAS  PubMed  Google Scholar 

  46. 46.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D. 68, 352–367 (2012).

    CAS  PubMed  Google Scholar 

  48. 48.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  49. 49.

    Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Ficarro, S. B., Alexander, W. M. & Marto, J. A. mzStudio: a dynamic digital canvas for user-driven interrogation of mass spectrometry data. Proteomes 5, 20 (2017).

    PubMed Central  Google Scholar 

  51. 51.

    Verhoeckx, K. et al. Caco-2 Cell Line. Impact Food Bioact. Health 175, 103–111 (2015).

    Google Scholar 

  52. 52.

    Weerapana, E., Speers, A. E. & Cravatt, B. F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)–a general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2, 1414–1425 (2007).

    CAS  PubMed  Google Scholar 

  53. 53.

    Ficarro, S. B. et al. Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells. Anal. Chem. 81, 3440–3447 (2009).

    CAS  PubMed  Google Scholar 

  54. 54.

    Alexander, W. M., Ficarro, S. B., Adelmant, G. & Marto, J. A. multiplierz v2.0: a Python-based ecosystem for shared access and analysis of native mass spectrometry data. Proteomics 17, 1700091 (2017).

    Google Scholar 

  55. 55.

    Zybailov, B. et al. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J. Proteome Res. 5, 2339–2347 (2006).

    CAS  PubMed  Google Scholar 

  56. 56.

    Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Schloss, P. D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Weber, N. et al. Nephele: a cloud platform for simplified, standardized and reproducible microbiome data analysis. Bioinformatics 34, 1411–1413 (2018).

    CAS  PubMed  Google Scholar 

  60. 60.

    Wrzosek, L. et al. Transplantation of human microbiota into conventional mice durably reshapes the gut microbiota. Sci. Rep. 8, 6854–6859 (2018).

    PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Ethics declarations

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–10, Figs. 1–18 and Synthetic Procedures

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing