Acetylation of the catalytic lysine inhibits kinase activity in PI3Kδ

Fournier, Julie C. L. and Evans, John P. and Zappacosta, Francesca and Thomas, Daniel A. and Patel, Vipulkumar K. and White, Gemma V. and Campos, Sebastien and Tomkinson, Nicholas C. O. (2021) Acetylation of the catalytic lysine inhibits kinase activity in PI3Kδ. ACS Chemical Biology, 16 (9). pp. 1644-1653. ISSN 1554-8937 (

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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(PI3K delta;) 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 PI3Kδinhibitor. 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 PI3K delta;-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδinhibition 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.


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