Cell-Based Covalent-Capture Deubiquitinase Assay for Inhibitor Discovery

Ubiquitination
is a post-translational modification that
elicits
a variety of cellular responses. Deubiquitinases (DUBs) remove ubiquitin
moieties from proteins and modulate cellular processes by counteracting
the ubiquitin ligase activities. Ubiquitination and deubiquitination
processes are tightly regulated by different mechanisms and their
dysregulation is associated with many diseases. Discovery of DUB inhibitors
could not only lead to therapeutics but also facilitate the understanding
of ubiquitination/deubiquitination processes and their regulatory
mechanisms. To enable the inhibitor discovery against DUBs, we developed
a cell-based DUB assay that utilizes a cell-permeable ubiquitin probe,
Biotin-cR10-Ub-PA, to covalently label DUBs in their native
cellular environment. Amplified luminescent proximity homogeneous
assay (Alpha, specifically AlphaLISA) is utilized to quantitatively
assess the capture of the target DUB by the Biotin-cR10-Ub-PA probe. We demonstrated that this new cell-based DUB assay
is robust and amenable to high-throughput screening. Human USP15 was
selected as a DUB of interest and screened against a library of protease
inhibitors as a proof of concept. In addition to the widely adopted
pan-DUB inhibitor PR-619, several other DUB inhibitors from the library
were also identified as hits. This new DUB assay can be readily adapted
for inhibitor discovery against many other human DUBs to identify
potent and cell-permeable inhibitors.