Chemical proteomic investigation of lipid kinase specificity and druggability

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Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that catalyze phosphorylation of diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA serve as potent lipid messengers to shape cellular responses by altering subcellular localization, activation, and function of essential receptor proteins (ranging from enzymes to transcription factors). DAG and PA also serve as building blocks for phospholipid and triglyceride biosynthesis and integral to membrane architecture and bioenergetics. The significance of our proposed studies is the enormous therapeutic potential of targeting individual DGKs because of their fundamental role in sculpting the lipidome to support metabolic, structural, and signaling demands of healthy and diseased cells. Despite their clinical value and discovery nearly 30 years ago, gaps in knowledge with regards to ligand binding and regulation of DGK active-sites in living systems have confounded basic understanding of how 10 mammalian DGK isoforms, which share a common catalytic domain, are capable of regulating distinct metabolic and signaling functions. We will test our hypothesis that C1 and other non-catalytic domains, which largely differentiate DGK isoforms, function in substrate and inhibitor recognition of DGK active sites. The proposed research program will test whether selective blockade of DGK can restore deficient DAG signaling to overcome immunosuppression of tumor infiltrating lymphocyte activity. Genetic and clinical evidence point to DGKs as promising targets for reversing immunosuppression of T cells although the molecular mechanisms coupling disrupted DGK metabolism to enhanced TCR signaling are not clear. Our mechanistic studies will establish a testable model for fundamental understanding of substrate and inhibitor recognition in DGK active sites to guide development of new chemical strategies to perturb activity of T cell specific DGKs in vivo for immunotherapy applications. Our long-term goals for this proposal are to functionally map novel and druggable small molecule binding sites on DGK and potentially other DGK isoforms in T cells to: 1) gain molecular level insights into DAG fatty acyl chain recognition and specificity, 2) identify molecular features of enzyme active sites to target lipid versus protein kinases, and 3) develop new inhibitors for selective inactivation of DGK isoforms in live cells and animals. We will test 2 independent yet related specific aims directed at: (Aim 1) identification of the DAG binding site, (Aim 1) understanding how individual DGK domains couple extracellular signals to shape T cell responses, (Aim 2) determining how DGK inhibitors amplify T cell activation, (Aim 2) understanding how DGK inhibitors reverse T cell immunosuppression in vivo, and (Aim 2) determining if DGK inhibitors affect membrane translocation. The overall impact of our findings will be to understand how intrinsic features of DGKs cross-talk with extrinsic features of cellular environments to form the basis of a lipid signaling code that can be therapeutically targeted for reversing immunosuppression of T cells.