Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity

Abstract

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.

Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe

“…In contrast, small-molecule, cell-permeable DUB ABPs complement the strengths and weaknesses of Ub-ABPs. Whilst DUB-selective small molecule probes are more challenging to develop and exhibit narrower DUB profiles, they offer powerful tools for profiling and imaging DUB activity in cells 9,10 or whole organisms, 11 accounting for the impact of physiological localization, biomolecular interactions and tissue context. They offer the further advantage of identifying off-targets for a given DUB inhibitor scaffold which would not be identified with the highly DUB-specific Ub-ABPs, therefore potentially better predicting unintended effects of future therapeutic agents.…”

“…Isatin O-acyl oxime LDN-57444 is most widely used as a putative tool to study the role of UCHL1 in both cellular and animal models, 17,18 although recent reports have cast considerable doubt on the on-target activity of this compound. 10,11 Several series of UCHL1 inhibitors have been reported in the patent literature, including cyanamide-containing compounds which likely react with the DUB active site cysteine residue forming an isothiourea adduct (Fig. 1a).…”

“…For example, we recently reported a potent and selective cyanopyrrolidinecontaining covalent inhibitor and an ABP for UCHL1 (IMP-1710), which selectively bind to Cys90 in the UCHL1 active site with minimal off-targets in intact cells. 10 Whilst other reported cyanopyrrolidine UCHL1 ABPs may show good selectivity toward UCHL1 among DUBs they also exhibit suboptimal proteome selectivity, highlighting the importance of both onand off-target profiling for ABPs. 11,21 Herein, we report novel thiazole cyanopyrrolidine inhibitors targeting UCHL1, related to a previously reported UCHL1 ABP.…”

Bioorthogonal Reactions in Activity-Based Protein Profiling

“…Other examples include cyanimides 1 [16] and 2 [17], derived from inhibitors of ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a DUB associated with various human diseases, including neurodegeneration and cancer. Interestingly, these small-molecule ABPs are cell permeable, allowing the study of DUBs in a cellular environment [16,17] and even in an in vivo zebrafish embryo animal model [17]. (3) A tag that enables detection of the covalent probe-protein complex [18].…”

“…For example, 4-chloro-isocoumarin ( Figure 1 D), a common heterocyclic scaffold used for covalent inhibition of serine proteases [ 10 ], has been equipped with biotin, alkyne, or fluorophores to target various soluble serine proteases [ 11 , 12 ], the intramembrane protease family of rhomboids [ 13 , 14 ], as well as acyl protein thioesterase, a serine hydrolase that cleaves fatty acids from S-acylated cysteine residues [ 15 ]. Other examples include cyanimides 1 [ 16 ] and 2 [ 17 ], derived from inhibitors of ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a DUB associated with various human diseases, including neurodegeneration and cancer. Interestingly, these small-molecule ABPs are cell permeable, allowing the study of DUBs in a cellular environment [ 16 , 17 ] and even in an in vivo zebrafish embryo animal model [ 17 ].…”