Activity-based proteomics discovery of deubiquitinating enzyme inhibitors with immunomodulatory activity

Deubiquitinases (DUBs) play key roles in human pathologies, including cancer, infectious, autoimmune, and neurodegenerative diseases. The progress of potent and selective active site-targeting DUB inhibitors into clinical trials has demonstrated the therapeutic opportunities of such molecules; however, the shared catalytic geometry among DUB family members poses a challenge for achieving inhibitor selectivity. We describe a high-throughput, DUB-focused, activity-based proteomics workflow to identify new types of cysteine-reactive DUB inhibitors. We demonstrate the potential of αβ,α'β'-diepoxyketones (DEKs) and ubiquitin-derived covalently reacting alkynes as the first selective, cell-active compounds targeting USP47, OTUD7B (Cezanne), and USP5. These DUBs are reported to have critical roles in inflammasome formation, hypoxia, and viral replication, respectively. The identified compounds phenocopy these findings, demonstrating the tractability of these DUBs as immunotherapeutic targets.