Structural basis for specific inhibition of the deubiquitinase UCHL1

Abstract

Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.

Small-molecule probes from bench to bedside: advancing molecular analysis of drug–target interactions toward precision medicine

“…Based on this cyanamide scaffold, Grethe et al have designed and evaluated a series of DUB-specific activity-based probes. 97 This has led to the discovery of potent and non-toxic probes for in situ investigation of UCHL1, which provides structural basis for UCHL1-specific inhibition and drug development.…”

“…95,96 The small size of a terminal alkyne minimizes perturbation of intrinsic properties of small molecule inhibitors after modification, making it suitable for proteomic and mechanistic studies of specific target inhibition, such as GK13S (26), which was used to investigate the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in cells and mice. 97 However, the use of a toxic Cu(I) catalyst in CuAAC restricts live-cell or in vivo applications of terminal alkyne, although this limitation can be partially reduced by using Cu(I)-stabilizing ligands. 98 To obviate this limitation, strain-promoted azide-alkyne cycloaddition (SPAAC) reactions have been developed.…”

Deubiquitylating Enzymes in Cancer and Immunity

“…Oncology [ 603] Oncology [ 603] Oncology [ 603] USP47 Compound 1 Non-selective Oncology [ 584] POH1 8-thioquinoline Capzimin phen Non-selective Highly selective Highly selective Oncology [ 604] Oncology [ 605] Oncology [ 606] UCHL1 LDN-57444 Highly selective Oncology [ 607] GK13S Highly selective Oncology [ 608] 6RK73 Highly selective, irreversible Oncology [ 169] (Continued) [594][595][596] b-AP15 VLX1570 WP1130 Auranofin Non-selective, reversible Non-selective, reversible Non-selective Non-selective Oncology [ 597,598] Oncology [ 599] Oncology [ 582] Oncology [ 600] Trabid NSC112200 Highly selective Inflammation and oncology [ 609,610] CYLD Subquinocin Non-selective Inflammation [ 611] SARS-CoV PLpro GRL0617 Highly selective, reversible Anti-infection [ 276] DDL-701 Highly selective, reversible Anti-infection [ 279]…”

Cell‐Permeable Ubiquitin and Histone Tools for Studying Post‐translational Modifications

“…Thanks for the small molecules which have already been used to study the activity of PTMs related enzymes, here we mainly focus on the synthetic protein tools [6] . The cellular permeability of most chemical synthesized protein tools is very poor due to their hydrophilicity, limiting its utility as pharmaceutical drugs and probes for in‐situ applications.…”

“…Thanks for the small molecules which have already been used to study the activity of PTMs related enzymes, here we mainly focus on the synthetic protein tools. [6] The cellular permeability of most chemical synthesized protein tools is very poor due to their hydrophilicity, limiting its utility as pharmaceutical drugs and probes for in-situ applications. Many cell penetrating techniques have been developed to deliver protein cargoes into cells, such as physical methods, nanocarriers, bio-reversible esterification, cell-penetrating peptides (CPPs), and cell-penetrating poly(disulfide)s. [7] We mainly introduce the cell penetrating techniques that have been used for studying PTMs.…”