A chemoproteomic platform for selective deubiquitinase inhibitor discovery

Chemoproteomics is a powerful method capable of detecting interactions between smallmolecules and the proteome; however, its use as a high-throughput screening method for chemical libraries has so far been limited. To address this need, we have further developed a chemoproteomics workflow to screen cysteine-reactive covalent fragments in cell lysates against the deubiquitinating (DUB) enzymes using activity-based protein profiling. By using targeted ubiquitin probes, we have addressed sensitivity and affinity limitations, enabling target identification and covalent fragment library profiling in a 96-well plate format. The use of data-independent acquisition (DIA) methods for mass spectrometry (MS) analysis combined with automated Evosep liquid chromatography (LC) reduced instrument runtimes to 21 min per sample and simplified the workflow. In this proof-of-concept study, we profile 138 covalent fragments against 57 DUB proteins and validate four hit fragments against OTUD7B and UCHL3 through site identification experiments and orthogonal biochemical activity assays.