Ubiquitin C‐Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine‐Based Inhibitor

Abstract

The deubiquitinase (DUB) ubiquitin C‐terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small‐molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine‐based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

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

“…In summary, we report the discovery and characterization of the most potent and selective small-molecule DUB ABP ( 2 ) to date, enabling robust detection of UCHL1 activity in living cells at low nanomolar concentrations. The only previously reported UCHL1 ABP is >150-fold less potent than 2 and has multiple significant off-targets, 14 with no selectivity over UCHL3. UCHL1 labeling by 2 is strictly activity-dependent, occurring only at the catalytic cysteine and providing a new chemical tool to examine UCHL1 activity in various intact cell types.…”

“…We further characterized inhibition kinetics for 1 ( k obs / I = 7400 M –1 s –1 , 95% Cl 5200–9700) and 2 ( k obs / I = 11000 M –1 s –1 , 95% Cl 7700–13 000 M –1 s –1 ); slow recovery of activity following dilution demonstrated these inhibitors are slowly reversible ( Figure S2 ), in line with previous reports on cyanamide warheads. 14 Cross-screening against 20 DUBs demonstrated exquisite selectivity for 1 and 2 for UCHL1 ( Figure 1 b), whereas control compound 3 was inactive against all tested DUBs ( Figure S1 ). Cellular UCHL1 activity was demonstrated in breast cancer cells (Cal51) stably expressing FLAG-UCHL1 using a Ub-vinyl methyl ester probe (HA-Ub-VME) by a homogeneous time-resolved fluorescence (HTRF) assay.…”

Recent Developments in Cell Permeable Deubiquitinating Enzyme Activity-Based Probes

“…Finally, the authors used compound 3 and IMP-1710 (4) to demonstrate the therapeutic potential of UCHL1 inhibition in a model of idiopathic pulmonary fibrosis, without cytotoxicity. Related alkyne-tagged small molecule ABP 7 of cyanopyrrolidine 6 was recently reported by Krabill et al, although this molecule is >150-fold less potent than IMP-1710 (4), and is relatively non-specific (Figure 1F; Krabill et al, 2019).…”

“…Attempts to date to address cell-penetration for DUB ABPs can be divided into four categories: pore-forming toxins (Claessen et al, 2013), electroporation (Mulder et al, 2016), cellpenetrating peptide (CPP) ABPs (Gui et al, 2018) and smallmolecule based cell permeable ABPs (Ward et al, 2016(Ward et al, , 2019Geurink et al, 2019;Krabill et al, 2019;Panyain et al, 2019). These are discussed in further detail below and in Figure 1.…”

Small molecules that target the ubiquitin system

“…Compound 34, for example, was first reported as a UCHL1 inhibitor in a patent application by Mission Therapeutics [108]. But it was found to inhibit both UCHL1 and UCHL3 with IC 50 values of 0.67 ± 1.0 μM and 6.4 ± 1.1 μM, respectively [109]. A more selective and potent UCHL1 inhibitor (35a) [118], as well as a structurally related activity-based probe (35b) [110], were reported to label UCHL1 in [117] living cells at low micromolar concentrations, and block pro-fibrotic responses in IPF cellular models without substantial associated cytotoxicity.…”