Discovery of a Potent Deubiquitinase (DUB) Small‐Molecule Activity‐Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells

• Published in: Angewandte Chemie International Edition 2023-11, Vol.62 (47), p.e202311190-n/a
• Main Authors: Conole, Daniel, Cao, Fangyuan, am Ende, Christopher W., Xue, Liang, Kantesaria, Sheila, Kang, Dahye, Jin, Jun, Owen, Dafydd, Lohr, Linda, Schenone, Monica, Majmudar, Jaimeen D., Tate, Edward W.
• Format: Article
• Language: English
• Subjects: B-cell lymphoma; Chemical Probe; Deregulation; Deubiquitinase; Electrophilic Warhead; Lymphoma; Myc protein; MYC-Deregulated Cancer; Neurodegeneration; Permeability; Phenotypes; Proteasomes; Proteomics; Substrates; Ubiquitin; Ubiquitin Activity-Based Probe
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202311190

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto‐inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin‐derived covalent activity‐based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP‐2373 (12) as a small molecule pan‐DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP‐2373 quantitatively engages more than 35 DUBs across a range of non‐toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP‐2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease‐relevant phenotypes. IMP‐2373 is the first activity‐based probe (ABP) that captures the activity of a large portion of the “DUBome” across multiple deubiquitinase (DUB) sub‐classes with limited off‐target effects in living cells, providing a complementary tool to ubiquitin ABPs, which function only in lysates. IMP‐2373 enables researchers to profile DUB inhibitors target engagement and selectivity in cells, and to understand DUB activity in disease‐relevant systems.