Identification and validation of selective deubiquitinase inhibitors.

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes. [Display omitted] • High-throughput screen of ∼50k compound library against 8 deubiquitinases • Identification and validation of selective inhibitors for individual DUBs • Study demonstrates importance of validation experiments as part of hit triage • Generation of robust dataset of compound activities against selected DUBs Deubiquitinases (DUBs) have emerged as therapeutically interesting targets due to their involvement in protein turnover in cells. Varca et al. describe a multi-DUB high-throughput screen and orthogonal validation campaign to identify new tool compounds for elucidating DUB function and provide a roadmap for future DUB inhibitor screens. [ABSTRACT FROM AUTHOR]