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High‐Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor
Author(s) -
Foote Peter K.,
Krist David T.,
Statsyuk Alexander V.
Publication year - 2017
Publication title -
current protocols in chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.503
H-Index - 14
ISSN - 2160-4762
DOI - 10.1002/cpch.24
Subject(s) - ubiquitin , ubiquitin ligase , chemistry , biochemistry , enzyme , drug discovery , small molecule , microbiology and biotechnology , biology , gene
HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off‐target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high‐throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc.

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