N‐Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1

Abstract

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross‐reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ‐1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N’‐alkyl,N‐cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with potent target engagement in cells. Notably, transitioning from 5‐membered pyrrolidines to 6‐membered heterocycles eliminated PARK7 binding and introduced context‐dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound‐induced conformational restriction of the cross‐over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead.

Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell

“…The reason for the disagreement between the fluorescent labeling and SLC-ABPP experiments could be due to the changes in the chemical composition of JYQ-173 by installment of dye as the similar effect observed in the previous studies for UCHL1-selective probes. 17 , 18 , 43 Previously, Sieber and co-workers reported an alkyne-equipped probe to monitor PARK7 in different cell lines after a postlysis click reaction with rhodamine- or biotin-azide. 14 The presented probes here provide great advantages because of the highly efficient one-step labeling of cellular PARK7 without a need for an intermediate click chemistry reaction, thus omitting all disadvantages associated with two-step labeling.…”