Mammalian DGKs represent a large enzyme family that plays an essential role in lipid metabolism and signal transduction. This enzyme class is responsible for catalyzing the conversion of secondary lipid messenger diacylglycerol (DAG) to phosphatidic acid (PA) by phosphorylation. Current research methods are deficient in tools to measure DGK activity in native biological systems. Development of chemical probes to study the unique functions of all ten mammalian DGK isoforms is needed in order to better characterize and understand these multidomain lipid kinases. Here, an ATP acyl phosphate activity-based probe, combined with quantitative mass spectrometry, is utilized to define, for the first time, unique ATP- and small molecule-binding modes for all five mammalian DGK subtypes. These proteomic studies provide support of interactions between the regulatory and catalytic domains of DGKs that can be employed to develop novel, isoform-specific inhibitors. This chemical proteomic strategy was used to study DGK-alpha (DGKα) inhibitor ritanserin, which revealed an unusual binding mode with interactions at the atypical C1 domain remote from the ATP binding region. Fragment-based deconstruction of ritanserin produced a compound that blocked DGKα activity through a conserved binding mode with enhanced selectivity across the kinome. These studies demonstrate the utility of chemical proteomics to profile protein-small molecule interactions at key binding sites of lipid kinases for fragment-based lead discovery. More recently, the described chemical proteomic strategy has been used to assess additional ritanserin- and fragment-based compounds, resulting in the identification of a key pharmacophore responsible for protein-kinase off-target activity.

Development of a chemical proteomic strategy to target diacylglycerol kinases

255th National Meeting (2018)

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