Electrophile scanning by chemical proteomics reveals a potent pan-active DUB probe for investigation of deubiquitinase activity in live cells

ABSTRACT Deubiquitinases (DUBs) are proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, which can lead to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many disease states, including cancer, neurodegeneration and inflammation, making them potentially attractive targets for therapeutic intervention. The >100 known DUB enzymes have been classified primarily by their conserved active sites, but we are still building our understanding of their substrate profiles, localization and regulation of DUB activity in diverse contexts. Ubiquitin-derived covalent activity-based probes (ABPs) are the premier tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which account for local DUB concentration, protein interactions, complexes, and organelle compartmentalization in intact cells or organisms. Here, through comprehensive warhead profiling we identify cyanopyrrolidine (CNPy) probe IMP-2373 ( 12 ), a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cell systems. Through chemical proteomics and targeted assays we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs in live cells across a range of non-toxic concentrations, and in diverse cell lines and disease models, and we demonstrate its application to quantification of changes in intracellular DUB activity during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes. SYNOPSIS TOC Graphical Abstract