Acetylation of the catalytic lysine inhibits kinase activity in PI3Kδ

Julie C. L. Fournier, John P. Evans, Francesca Zappacosta, Daniel A. Thomas, Vipulkumar K. Patel, Gemma V. White, Sebastien Campos, Nicholas C. O. Tomkinson

Research output: Contribution to journalArticlepeer-review


Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.
Original languageEnglish
Pages (from-to)1644-1653
Number of pages10
JournalACS Chemical Biology
Issue number9
Early online date16 Aug 2021
Publication statusPublished - 17 Sep 2021


  • kinase inhibitors
  • selective kinase inactivation
  • catalytic lysine


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