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

Abstract 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.