A small molecule inhibitor prevents gut bacterial genotoxin production

Matthew R. Volpe; José A. Velilla; Martin Daniel-Ivad; Jenny J. Yao; Alessia Stornetta; Peter W. Villalta; Hsin Che Huang; Daniel A. Bachovchin; Silvia Balbo; Rachelle Gaudet; Emily P. Balskus

doi:10.1038/s41589-022-01147-8

A small molecule inhibitor prevents gut bacterial genotoxin production

Matthew R. Volpe, José A. Velilla, Martin Daniel-Ivad, Jenny J. Yao, Alessia Stornetta, Peter W. Villalta, Hsin Che Huang, Daniel A. Bachovchin, Silvia Balbo, Rachelle Gaudet, Emily P. Balskus

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12 Scopus citations

Abstract

The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin’s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. [Figure not available: see fulltext.]

Original language	English (US)
Pages (from-to)	159-167
Number of pages	9
Journal	Nature Chemical Biology
Volume	19
Issue number	2
DOIs	https://doi.org/10.1038/s41589-022-01147-8
State	Published - Feb 2023

Bibliographical note

Funding Information:
This work was funded in part by National Cancer Institute (NCI) grant R01CA208834 (E.P.B.) and National Institute of General Medical Sciences (NIGMS) grant R01GM120996 (R.G.). E.P.B. is a Howard Hughes Medical Institute (HHMI) Investigator. M.R.V. acknowledges support from NCI Fellowship F31CA247069 and J.A.V. acknowledges support from an HHMI Gilliam fellowship. P.W.V. acknowledges support from the NCI grant R50CA211256. M.D.-I. acknowledges support from Canadian Institutes of Health Research postdoctoral fellowship 202012MFE-458776. Diffraction data reported in this study were collected at the GM/CA beamline in the Advanced Photon Source. GM/CA has been funded by the NCI (ACB-12002) and the NIGMS (AGM-12006, P30GM138396). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. LC–MS DNA adduct analysis was performed in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center at the University of Minnesota, supported in part by NCI grant P30CA077598. The authors wish to thank C. Jobin for providing the NC101 strains used in this study, B. Liau and N. Lue for help with western blotting experiments, W. Garrett for sharing samples of mouse fecal pellets, and M. Shair for providing shared workspace and the use of imaging equipment for western blotting.

Publisher Copyright:
© 2022, The Author(s).

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Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

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title = "A small molecule inhibitor prevents gut bacterial genotoxin production",

abstract = "The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin{\textquoteright}s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. [Figure not available: see fulltext.]",

author = "Volpe, {Matthew R.} and Velilla, {Jos{\'e} A.} and Martin Daniel-Ivad and Yao, {Jenny J.} and Alessia Stornetta and Villalta, {Peter W.} and Huang, {Hsin Che} and Bachovchin, {Daniel A.} and Silvia Balbo and Rachelle Gaudet and Balskus, {Emily P.}",

note = "Funding Information: This work was funded in part by National Cancer Institute (NCI) grant R01CA208834 (E.P.B.) and National Institute of General Medical Sciences (NIGMS) grant R01GM120996 (R.G.). E.P.B. is a Howard Hughes Medical Institute (HHMI) Investigator. M.R.V. acknowledges support from NCI Fellowship F31CA247069 and J.A.V. acknowledges support from an HHMI Gilliam fellowship. P.W.V. acknowledges support from the NCI grant R50CA211256. M.D.-I. acknowledges support from Canadian Institutes of Health Research postdoctoral fellowship 202012MFE-458776. Diffraction data reported in this study were collected at the GM/CA beamline in the Advanced Photon Source. GM/CA has been funded by the NCI (ACB-12002) and the NIGMS (AGM-12006, P30GM138396). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. LC–MS DNA adduct analysis was performed in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center at the University of Minnesota, supported in part by NCI grant P30CA077598. The authors wish to thank C. Jobin for providing the NC101 strains used in this study, B. Liau and N. Lue for help with western blotting experiments, W. Garrett for sharing samples of mouse fecal pellets, and M. Shair for providing shared workspace and the use of imaging equipment for western blotting. Publisher Copyright: {\textcopyright} 2022, The Author(s).",

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AU - Velilla, José A.

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AU - Stornetta, Alessia

AU - Villalta, Peter W.

AU - Huang, Hsin Che

AU - Bachovchin, Daniel A.

AU - Balbo, Silvia

AU - Gaudet, Rachelle

AU - Balskus, Emily P.

N1 - Funding Information: This work was funded in part by National Cancer Institute (NCI) grant R01CA208834 (E.P.B.) and National Institute of General Medical Sciences (NIGMS) grant R01GM120996 (R.G.). E.P.B. is a Howard Hughes Medical Institute (HHMI) Investigator. M.R.V. acknowledges support from NCI Fellowship F31CA247069 and J.A.V. acknowledges support from an HHMI Gilliam fellowship. P.W.V. acknowledges support from the NCI grant R50CA211256. M.D.-I. acknowledges support from Canadian Institutes of Health Research postdoctoral fellowship 202012MFE-458776. Diffraction data reported in this study were collected at the GM/CA beamline in the Advanced Photon Source. GM/CA has been funded by the NCI (ACB-12002) and the NIGMS (AGM-12006, P30GM138396). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. LC–MS DNA adduct analysis was performed in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center at the University of Minnesota, supported in part by NCI grant P30CA077598. The authors wish to thank C. Jobin for providing the NC101 strains used in this study, B. Liau and N. Lue for help with western blotting experiments, W. Garrett for sharing samples of mouse fecal pellets, and M. Shair for providing shared workspace and the use of imaging equipment for western blotting. Publisher Copyright: © 2022, The Author(s).

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N2 - The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin’s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. [Figure not available: see fulltext.]

AB - The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin’s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. [Figure not available: see fulltext.]

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A small molecule inhibitor prevents gut bacterial genotoxin production

Abstract

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