Send to:

Choose Destination
See comment in PubMed Commons below
Nat Biotechnol. 2009 Apr;27(4):387-94. doi: 10.1038/nbt.1531. Epub 2009 Mar 29.

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

Author information

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

Erratum in

  • Nat Biotechnol. 2009 May;27(5):485.


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.

[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Nature Publishing Group Icon for PubMed Central
    Loading ...
    Write to the Help Desk