Targeting Deubiquitinases Enabled by Chemical Synthesis of Proteins

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.

Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

“…Earlier studies on the enzyme kinetics of UCH-L1, -L3 and -L5 N240 have established that both UCHs bind to ubiquitin with sub μM K M values161718193536. While UCH-L1 exhibits higher binding affinity for ubiquitin compared to that of UCH-L3, its hydrolysis turnover rate ( k cat ) is about two orders of magnitude slower than that of UCH-L3.…”

Emerging role of DUBs in tumor metastasis and apoptosis: Therapeutic implication

“…Pimozide (an anti-psychotic drug) was identified as inhibitors of USP1, and auranofin (a rheumatoid arthritis drug) is a proteasome-associated DUB inhibitor (Chen et al, 2011; Liu et al, 2014). Benefiting from high-throughput screening studies, LS1 has been identified as a UCHL3 inhibitor and PR-619 has been characterized as a general DUB enzyme inhibitor (Edelmann, Nicholson, & Kessler, 2011; Ohayon, Spasser, Aharoni, & Brik, 2012). Interestingly, the mitochondria-localized DUB USP30 was found to be inhibited by a diterpenoid derivative 15-oxospiramilactone (S3), leading to the increased Mfn1/2 proteins which promote mitochondrial fusion (Yue et al, 2014).…”

The emerging role of deubiquitinating enzymes in genomic integrity, diseases, and therapeutics

“…Pimozide (an anti-psychotic drug) was identified as inhibitors of USP1, and auranofin (a rheumatoid arthritis drug) is a proteasome-associated DUB inhibitor [154, 155]. Benefiting from high-throughput screening studies, LS1 as an UCHL3 inhibitor and PR-619 as a general DUB enzyme inhibitor [156, 157]. Interestingly, the mitochondria-localized DUB USP30 was found to be inhibited by a diterpenoid derivative 15-oxospiramilactone (S3), leading to the increased Mfn1/2 proteins which promote mitochondrial fusion [158].…”