Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe

Abstract

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme (DUB), is a potential drug target in various cancers, and liver and lung fibrosis. However, <i>bona fide</i> functions and substrates of UCHL1 remain poorly understood. Herein, we report the characterization of UCHL1 covalent inhibitor <b>MT16-001</b> based on a thiazole cyanopyrrolidine scaffold. In combination with chemical proteomics, a closely related activity-based probe (<b>MT16-205</b>) was used to generate a comprehensive quantitative profile for on- and off-targets at endogenous cellular abundance. Both compounds are selective for UCHL1 over other DUBs in intact cells but also engage a range of other targets with good selectivity over the wider proteome, including aldehyde dehydrogenases, redox-sensitive Parkinson's disease related protein PARK7, and glutamine amidotransferase. Taken together, these results underline the importance of robust profiling of activity-based probes as chemical tools and highlight the cyanopyrrolidine warhead as a versatile platform for liganding diverse classes of protein with reactive cysteine residues which can be used for further inhibitor screening, and as a starting point for inhibitor development.