Selective USP32 inhibition reveals endosomal ubiquitin signaling and enables cell-permeable activity-based probes.

Deubiquitinating enzymes (DUBs) regulate ubiquitin signaling and are attractive therapeutic targets, but selective chemical tools remain available for only a limited number of family members. Here, we report Huib32, the first potent and selective covalent inhibitor of ubiquitin-specific protease 32 (USP32). Huib32 rapidly and selectively engages USP32 in vitro and in cells, recapitulating USP32 loss of function by enhancing substrate ubiquitination, disrupting endosomal organization, and defining an expanded network of USP32-regulated membrane trafficking proteins. Building on Huib32, we develop the first cell-permeable activity-based probes for USP32, enabling direct visualization of active USP32 in cells and selective proteomic enrichment of the enzyme. Together, these first-in-class chemical tools establish selective chemical targeting of USP32 as an approach to interrogate endosomal ubiquitin signaling and provide a foundation for therapeutic exploration in cancers and other diseases associated with endocytic trafficking.