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Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes

Nature Biotechnology volume 27, pages 387–394 (2009)Cite this article

An Erratum to this article was published on 01 May 2009

This article has been updated

Abstract

High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein.

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Figure 1: Schematic representation of the fluopol-ABPP assay.
Figure 2: Optimization and validation of the fluopol-ABPP assay for RBBP9.
Figure 3: Identification of RBBP9 primary hits.
Figure 4: Competitive ABPP in proteomes identifies emetine (1) as a selective inhibitor of RBBP9.
Figure 5: Mechanistic characterization of RBBP9 inhibitors.
Figure 6: Fluopol-ABPP identifies a selective inhibitor of GSTO1.

Change history

  • 08 May 2009

    In the version of the article initially published, the IC50 value of cephaeline is given as 2.7 μM in Figure 3d. The correct value is 27 μM. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank the Cravatt laboratory for helpful discussions. We are grateful to S. Schürer and P. Baillargeon for help purchasing emetine analogs, to S. Tully for assistance with the synthesis of 4NPG and to J. Garfunkle for assembly of the Boger chemical library. Full-length cDNA encoding human RBBP9 was a gift of the Cheresh laboratory (UCSD). This work was supported by the National Institutes of Health (CA132630, MH084512), a National Science Foundation Predoctoral Fellowship (D.A.B.), and the Skaggs Institute for Chemical Biology.

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Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA

    Daniel A Bachovchin & Benjamin F Cravatt

  2. Department of Chemical Physiology,

    Daniel A Bachovchin, Steven J Brown, Hugh Rosen & Benjamin F Cravatt

  3. The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, La Jolla, California, USA

    Steven J Brown & Hugh Rosen

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  1. Daniel A Bachovchin
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D.A.B. performed experiments. D.A.B., S.J.B., H.R. and B.F.C. designed experiments and analyzed data. D.A.B. and B.F.C. wrote the paper.

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Correspondence to Benjamin F Cravatt.

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Bachovchin, D., Brown, S., Rosen, H. et al. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat Biotechnol 27, 387–394 (2009). https://doi.org/10.1038/nbt.1531

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