DUB inhibitor (Mission Therapeutics/Pfizer)

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.