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 would benefit from discovery of protein pockets available for inhibitor binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) bearing a DGK fragment ligand for covalent binding to tyrosine and lysine sites on DGKs in cells that map to predicted small molecule binding pockets in AlphaFold structures. We apply this chemoproteomics-AlphaFold approach to evaluate probe binding of DGK chimera proteins engineered to exchange regulatory C1 domains between DGK subtypes (DGKα and DGKζ). Specifically, we discovered loss of TH211 binding to a predicted pocket in the catalytic domain when C1 domains on DGKα were exchanged that correlated with impaired biochemical activity as measured by a DAG phosphorylation assay. Collectively, we provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.
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