Search

Deubiquitinating enzymes (DUBs) are a largely understudied and untapped resource in the toolkit of protein degradation functionalities. They comprise a large repertoire of enzymes that remove ubiquitin from substrates in a variety of cellular and pathophysiological contexts, and have enormous potential for research and clinical use. It is only within the last 5 years that potent, selective, and well-characterized small-molecule inhibitors of DUBs have been described. These compounds are now being used to study the biological roles of DUBs. Here, we describe downstream applications of small-molecule inhibitors for studying DUBs and provide a framework for future studies. We highlight recent examples of using these inhibitors to confirm and explore the role of these enzymes in both normal and pathological contexts. These studies represent the first steps in the burgeoning field of pharmacological and chemoproteomic studies of DUBs, which will be critical for the continued advancement of DUB field. [Display omitted] Magin et al. review the development of small-molecule inhibitors of deubiquitinating enzymes (DUBs), and their application in studying DUB function and therapeutic potential. Examples of well-validated inhibitors are given and future perspectives for this growing field are discussed. [ABSTRACT FROM AUTHOR]

Summary Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5–10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors. [ABSTRACT FROM AUTHOR]

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]

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents. • Commonly used DUB inhibitors show a wide range of selectivity and cellular activity • Cellular proteomics of USP7, USP47, USP25, USP28, USP30, UCHL1, and USP1 inhibitors • Use of multiple USP7 inhibitors to confirm known and new targets of USP7 • Practical insights for profiling DUB inhibitors earlier in the development pipeline Bushman et al. profile commonly used inhibitors of deubiquitinating enzymes to assess their biochemical selectivity and cellular activity. USP7 is used to demonstrate proof-of-concept for using potent and selective inhibitors of deubiquitinating enzymes to identify their substrates by mass spectrometry. [ABSTRACT FROM AUTHOR]