Jones, H. B. L.; Draganov, S. D.; Schonbauer Huaman, S.; Wing, P. A.; Nguyen, C.; Liang, Z.; Dorner, J.; Lithgow, J.; Murphy, E.; Beard, A.; Pierangelini, A.; Houghton, J. W.; Robert, M. E.; Flannery, S.; Tumber, A.; Vendrell, I.; Tate, E. W.; Elliott, P. R.; West, G. M.; O'Brien, D. P.; Salah, E.; Turnbull, A. P.; Schofield, C. J.; Kessler, B. M.; Brewitz, L.; Pinto-Fernandez, A.

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.