Electrophile scanning by chemical prote-omics reveals a potent pan-active DUB probe for investigation of deubiquitinase activity in live cells

ABSTRACT: Deubiquitinases (DUBs) are proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, which can lead to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been im-plicated in many disease states, including cancer, neurodegeneration and inflammation, making them potentially attractive targets for therapeutic intervention. The >100 known DUB enzymes have been classified primarily by their conserved active sites, but we are still building our understanding of their substrate profiles, localization and regulation of DUB activity in di-verse contexts. Ubiquitin-derived covalent activity-based probes (ABPs) are the premier tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which account for local DUB concentration, protein interactions, complexes, and organelle compartmentalization in intact cells or organisms. Here, through comprehensive warhead profiling we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12), a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cell systems. Through chemical prote-omics and targeted assays we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs in live cells across a range of non-toxic concentrations, and in diverse cell lines and disease models, including induction of MYC deregulation in B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the con-text of disease-relevant phenotypes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast, Bone Tissue, Cell Culture, Astrocyte

DISEASE(S): Osteosarcoma,Brain Cancer,Breast Cancer

SUBMITTER: Daniel Conole  

LAB HEAD: Edward William Tate

PROVIDER: PXD035417 | Pride | 2024-01-03

REPOSITORIES: Pride