Scalable Thiol Reactivity\nProfiling Identifies Azetidinyl\nOxadiazoles as Cysteine-Targeting Electrophiles

Cysteine reactive groups are a mainstay in the design\nof covalent\ndrugs and probe molecules, yet only a handful of electrophiles are\nroutinely used to target this amino acid. Here, we report the development\nof scalable thiol reactivity (STRP), a method which enables the facile\ninterrogation of large chemical libraries for intrinsic reactivity\nwith cysteine. High throughput screening using STRP identified the\nazetidinyl oxadiazole as a moiety that selectively reacts with cysteine\nthrough a ring opening-based mechanism, capable of covalently engaging\ncysteine residues broadly across the human proteome. We show the utility\nof this reactive group with the discovery of an azetidinyl oxadiazole\ncontaining a small molecule that augments the catalytic activity of\nthe deubiquitinase UCHL1 in vitro and in cells by covalently modifying\na cysteine distal to its enzymatic active site. This study adds a\nnovel cysteine targeting group to the electrophilic lexicon and provides\nrobust methodology to rapidly surveil libraries for reactivity with\ncysteine.