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ACS Chem Biol. 2017 Sep 15;12(9):2399-2407. doi: 10.1021/acschembio.7b00543. Epub 2017 Aug 24.

Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.

Author information

1
Department of Chemistry and Biochemistry, University of Delaware , 214A Drake Hall, Newark, Delaware 19716, United States.
2
National Center for Advancing Translational Sciences, NIH , Bethesda, Maryland 20892, United States.

Abstract

The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes.

PMID:
28836754
PMCID:
PMC5947317
DOI:
10.1021/acschembio.7b00543
[Indexed for MEDLINE]
Free PMC Article

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