A versatile fluorescence polarization-based deubiquitination assay using an isopeptide bond substrate mimetic (IsoMim)

Zhang, Jiatong; Allen, Jed; Ward, Stephanie J.; Dekker, Lodewijk V.; Dreveny, Ingrid

Abstract

Deubiquitinases (DUBs) play a critical role in the regulation of various cellular processes, such as protein homeostasis and signaling, rendering them attractive drug targets. However, the generation of reagents for measuring DUB activity typically involves several steps and is not straightforward. Here, we report the development and characterization of a novel fluorescent polarization assay using an isopeptide bond substrate mimetic (IsoMim) that can be made recombinantly in high yields. The IsoMim assay was able to discern the differential activity of ubiquitin-specific protease family members (USP4, USP15, USP11, and USP2), the ubiquitin C-terminal hydrolase UCHL3, and the Machado-Joseph Domain deubiquitinase JOSD2. A competition assay format of the assay was developed that discerned differences between the close paralogues USP15, USP4, and USP11 in interacting with mono-ubiquitin, the isopeptide mimetic ubiquitin-GGG, and the C-terminal truncation variant ubiquitin (1–74). Moreover, dose–response curves and associated pIC50 values using the broad-spectrum inhibitor PR-619 confirmed differential inhibition in the low μM range for four tested DUBs. The successful discrimination of DUB activity and inhibition and the easily scalable generation of the substrate make the IsoMim assay method applicable for high-throughput screening (HTS). This was ascertained in a “pseudo HTS screen” for USP4 inhibitors in which PR-619 was successfully identified as a “pseudo hit.” The developed assay provides a valuable tool for probing DUB activity and the identification and characterization of DUB inhibitors and has the potential to accelerate drug discovery efforts in this area.

Citation

Zhang, J., Allen, J., Ward, S. J., Dekker, L. V., & Dreveny, I. (2025). A versatile fluorescence polarization-based deubiquitination assay using an isopeptide bond substrate mimetic (IsoMim). Journal of Biological Chemistry, 301(7), Article 110342. https://doi.org/10.1016/j.jbc.2025.110342

• Journal Article Type: Article
• Acceptance Date: Jun 3, 2025
• Online Publication Date: Jun 4, 2025
• Publication Date: 2025-07
• Deposit Date: Jul 18, 2025
• Publicly Available Date: Jul 22, 2025
• Journal: Journal of Biological Chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 301
• Issue: 7
• Article Number: 110342
• DOI: https://doi.org/10.1016/j.jbc.2025.110342
• Public URL: https://nottingham-repository.worktribe.com/output/50163206
• Publisher URL: https://www.jbc.org/article/S0021-9258(25)02192-1/fulltext