Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering

[Display omitted] •Rapid and selective inhibition of cysteine-dependent enzymes is often challenging.•We showed this can be achieved by bioorthogonal tethering of an electrophile.•Selective inhibition of two E2 enzymes was demonstrated.•The technique developed here represents a valuable biomedical tool. A general approach for the rapid and selective inhibition of enzymes in cells using a common tool compound would be of great value for research and therapeutic development. We previously reported a chemogenetic strategy that addresses this challenge for kinases, relying on bioorthogonal tethering of a pan inhibitor to a target kinase through a genetically encoded non-canonical amino acid. However, pan inhibitors are not available for many enzyme classes. Here, we expand the scope of the chemogenetic strategy to cysteine-dependent enzymes by bioorthogonal tethering of electrophilic warheads. For proof of concept, selective inhibition of two E2 ubiquitin-conjugating enzymes, UBE2L3 and UBE2D1, was demonstrated in biochemical assays. Further development and optimization of this approach should enable its use in cells as well as with other cysteine-dependent enzymes, facilitating the investigation of their cellular function and validation as therapeutic targets.