Identification of USP39 Ligands and Their Application in Targeted Protein Degradation

Alternative splicing is a tightly regulated process essential for transcriptomic and proteomic diversity, and its dysregulation has been implicated in various cancers. Ubiquitin-specific protease 39 (USP39) is a core component of the spliceosome that lacks enzymatic activity, rendering it challenging to target with traditional small molecules. Here, we report the identification of small-molecule ligands that bind selectively to USP39 via a 2-aminothiazole scaffold, primarily interacting with its zinc finger domain. Structure–activity relationship studies guided the design of PROTACs (proteolysis-targeting chimeras), leading to the development of USP39_PROTAC_V1, which recruits the VHL E3 ligase. Biophysical and biochemical assays confirmed potent ternary complex formation and nanomolar binding affinities. In cellular models, USP39_PROTACs induced efficient degradation of USP39 at concentrations as low as 1 nM, with minimal off-target effects as shown by proteome-wide profiling. The degradation was abrogated by proteasome and neddylation inhibitors and was dependent on VHL-mediated recruitment. Importantly, PROTAC-induced USP39 depletion reproduced known splicing defects at 5’ splice sites, validating both the mechanism of action and the therapeutic relevance. Our study provides the demonstration of targeted USP39 degradation and highlights its potential as a tractable target in splicing-related pathologies.