Article abstract
Nature Biotechnology 27, 387 - 394 (2009)
Published online: 29 March 2009 | Corrected online: 8 May 2009 | doi:10.1038/nbt.1531
There is an Erratum (May 2009) associated with this Article.
Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes
Daniel A Bachovchin1,2, Steven J Brown2,3, Hugh Rosen2,3 & Benjamin F Cravatt1,2
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.
- The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.
- Department of Chemical Physiology.
- The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, La Jolla, California, USA.
Correspondence to: Benjamin F Cravatt1,2 e-mail: cravatt@scripps.edu
M in Figure 3d. The correct value is 27 M. The error has been corrected in the HTML and PDF versions of the article.MORE ARTICLES LIKE THIS
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