2. Academic Validation
  3. Targeting the Spliceosomal Protein USP39 Through Allosteric Ligands and PROTAC-Induced Degradation

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

  • Angew Chem Int Ed Engl. 2025 Dec 21:e16809. doi: 10.1002/anie.202516809.
Daniel Schäfer 1 2 3,  Cristian Prieto-Garcia 4,  Jianhui Wang 1 2,  Marcel Heinz 5,  Vigor Matkovic 1 4,  Pavel Kielkowski 6,  Sebastian Hasselbeck 1 2,  Varun Jayeshkumar Shah 4,  Stefan Knapp 1 2 3,  Gerhard Hummer 5 7,  Ivan Dikic 1 4,  Xinlai Cheng 1 2 3 8
Affiliations

Affiliations

  • 1 Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University Frankfurt am Main, Max-von-Laue-Str. 15, D-60438, Frankfurt am Main, Germany.
  • 2 Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University Frankfurt am Main, Max-von-Laue-Str. 9, D-60438, Frankfurt am Main, Germany.
  • 3 Frankfurt Cancer Institute, Paul-Ehrlich-Str. 42-44, D-60596, Frankfurt am Main, Germany.
  • 4 Institute of Biochemistry II Frankfurt, University Hospital Building 75, Faculty of Medicine Frankfurt am Main, Theodor-Stern-Kai 7, D-60596, Frankfurt am Main, Germany.
  • 5 Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438, Frankfurt am Main, Germany.
  • 6 Department of Chemistry, Ludwig Maximilian University München, Würmtalstrasse 201, 81375, Munich, Germany.
  • 7 Institute of Biophysics, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 1, D-60438, Frankfurt am Main, Germany.
  • 8 Mildred-Scheel-Nachwuchszentrum (MSNZ), University Cancer Center (UCT) Frankfurt, University Hospital Frankfurt am Main, Theodor-Stern-Kai 7, D-60596, Frankfurt am Main, Germany.
PMID: 41423877 DOI: 10.1002/anie.202516809
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.

Keywords

Biological chemistry and chemical biology; Drug discovery; PROTAC; Spliceosome; USP39.

Figures
Products