Targeting deubiquitinases enabled by chemical synthesis of proteins

Shimrit Ohayon, Liat Spasser, Amir Aharoni, Ashraf Brik

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

Ubiquitination/ubiquitylation is involved in a wide range of cellular processes in eukaryotes, such as protein degradation and DNA repair. Ubiquitination is a reversible post-translational modification, with the removal of the ubiquitin (Ub) protein being catalyzed by a family of enzymes known as deubiquitinases (DUBs). Approximately 100 DUBs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. DUBs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets. Several directions have been pursued in the search for lead anti-DUB compounds. However, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay. In this study, we report on the design and preparation of a FRET-based assay for DUBs based on the application of our recent chemical method for the synthesis of Ub bioconjugates. In the assay, the ubiquitinated peptide was specifically labeled with a pair of FRET labels and used to screen a library comprising 1000 compounds against UCH-L3. Such analysis identified a novel and potent inhibitor able to inhibit this DUB in time-dependent manner with k inact = 0.065 min -1 and K i = 0.8 μM. Our assay, which was also found suitable for the UCH-L1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of DUBs in health and disease.

Original languageEnglish
Pages (from-to)3281-3289
Number of pages9
JournalJournal of the American Chemical Society
Volume134
Issue number6
DOIs
StatePublished - 15 Feb 2012

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