Targeting the Spliceosomal Protein USP39 Through Allosteric Ligands and PROTAC-Induced Degradation.

The precise regulation of gene expression is fundamental to cellular homeostasis and diversity. Dysregulation of splicing has been implicated in a range of diseases, including cancer and neurodegeneration. Ubiquitin-specific protease 39 (USP39), an essential spliceosome component lacking enzymatic activity, has remained an elusive target for pharmacological intervention. Here, we report the discovery of small-molecule ligands that selectively engage with USP39 through a thiazole scaffold, primarily interacting with its zinc finger domain. Guided by AlphaFold-based structure-activity relationship studies, we designed and optimized proteolysis-targeting chimeras (PROTACs), culminating in the development of USP39_PROTAC_V1, which harnesses the von Hippel-Lindau (VHL) E3 ubiquitin ligase for targeted degradation. Biophysical and biochemical assays demonstrated potent ternary complex formation and nanomolar-range binding affinities. In cellular models, USP39_PROTACs achieved efficient degradation of USP39 at concentrations as low as 1 nM, with minimal off-target effects as confirmed by proteome-wide profiling. Mechanistic studies revealed that degradation was dependent on VHL recruitment and was abrogated by proteasome or neddylation inhibition. Notably, USP39 depletion recapitulated 5'-splice-site-specific splicing patterns previously described, thereby validating both the mechanism of action and the therapeutic relevance of this approach-particularly for modulating splicing-associated disease pathways such as cancer and retinitis pigmentosa.