Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering

Luke A. Spear, Yang Huang, Jinghao Chen, Alexander R. Nödling, Satpal Virdee (Lead / Corresponding author), Yu-Hsuan Tsai (Lead / Corresponding author)

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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.

Original languageEnglish
Article number167524
Number of pages13
JournalJournal of Molecular Biology
Issue number8
Early online date3 Mar 2022
Publication statusPublished - 30 Apr 2022


  • genetic code expansion
  • non-canonical amino acid
  • chemogenetics
  • E2 ubiquitin-conjugating enzymes
  • bioorthogonal reaction


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