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

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.

• Authors: Daniel A Bachovchin, Steven J Brown, Hugh Rosen, Benjamin F Cravatt
• Journal: Nature biotechnology (Nat Biotechnol) Vol. 27 Issue 4 Pg. 387-94 (Apr 2009) ISSN: 1546-1696 [Electronic] United States
• PMID: 19329999 (Publication Type: 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.)

Chemical References

• Enzyme Inhibitors
• Fluorescent Dyes

Topics

• Enzyme Inhibitors (analysis, chemistry)
• Fluorescent Dyes
• Spectrometry, Fluorescence (methods)