Activity-Based Chemical Proteomics Accelerates Inhibitor Development for Deubiquitylating Enzymes

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair. [Display omitted] ► Characterization of a broad-range and specific inhibitors for deubiquitylating enzymes (DUBs) ► Activity-based chemical probe screen for specificity of small molecule DUB inhibitors in cells ► Cell-based profiling of inhibitors specific for deubiquitylating enzymes by mass spectrometry ► Evidence for USP7 association with DNA repair pathways