Identifying Reactive Sites on Diacylglycerol Kinases for Covalent Binding in Cells

Diacylglycerol kinases (DGKs) are metabolic kinases involved in regulating cellular levels of diacylglycerol and phosphatidic lipid messengers. The development of selective inhibitors for individual DGKs has been hindered partially by a lack of understanding of ligand binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) to establish a comprehensive map of protein-small molecule interactions for all members of the DGK superfamily in cells. Treatment of recombinant DGK expressing cells with TH211 facilitated chemoproteomic identification of modified tyrosine and lysine sites found in catalytic and regulatory domains. Notably, we discovered that DGK chimera proteins, containing C1 domains exchanged across subtypes, displayed isoform-specific effects on probe recognition and biochemical activity. Collectively, we provide a family-wide ligand binding map of reactive sites found in functional domains of DGKs to guide inhibitor development efforts.