AT9283 br Significance Our studies describe the first functi

Significance Our studies describe the first functional proteomic map of ligand binding regions that mediate substrate (ATP) and inhibitor binding in the poorly annotated active site of the mammalian diacylglycerol kinase (DGK) superfamily. Given the dearth of lipid kinase inhibitors available in the clinic, and the emerging role of DGKs as anticancer and immunotherapy targets, we believe our findings offer exciting new prospects for development of new chemical probes to study and target lipid kinases. We define, for the first time, the location of the ATP binding site of representative isoforms from all five principal DGK subtypes (types 1 to 5). Inspection of DGK ATP binding sites identified conserved features that are distinct from protein kinases, providing the first experimental evidence in support of a DGK-specific ATP binding motif that was postulated over 20 years ago. We discovered clues to domain regions of DGKs important for inhibitor development by identifying probe-modified sites in C1 and accessory (DAGKa) domains that serve as primary binding sites for the DGKα inhibitor ritanserin. An unexpected finding was the discovery that a fragment of ritanserin (RF001) functioned as a DGKα inhibitor that retained binding at C1 and DAGKa domains, and largely removed protein kinase off-target activity. While few examples have been reported, conservation of fragment binding mode is characteristic of ligand binding hotspots of proteins suitable for fragment-based lead discovery. Thus, we believe the C1 and DAGKa sites are key binding regions of DGKs to enable development of high-affinity, isoform-selective inhibitors of this lipid kinase superfamily.
STAR★Methods
Author Contributions
Introduction Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic AT9283 [[1], [2], [3], [4]]. Ten mammalian DGK isozymes have been identified and classified into five groups based on their primary structures [[1], [2], [3], [4]]. DGK isozymes have been demonstrated to be involved in a wide variety of physiological events and diseases [5,6]. The type II DGK group comprises the δ [7,8], η [9,10] and κ [11] isozymes. Moreover, alternatively spliced variants of DGKδ (δ1 and δ2) [8] and η (η1 – η4) [10,12,13] have been reported. DGKδ is highly expressed in skeletal muscle [7], which is a primary target of insulin for glucose disposal [14]. Notably, Chibalin et al. reported that DGKδ contributes to hyperglycemia-induced peripheral insulin resistance and that decreased protein levels of DGKδ in skeletal muscle attenuated glucose uptake, which is critical for the pathogenesis of type 2 diabetes [15]. In addition, Miele et al. demonstrated that acute exposure (within 5 min) to high glucose levels augmented DGKδ activity in L6 myotubes, which was followed by a decrease in protein kinase C (PKC) α activity and an increase of insulin receptor activity [16]. We observed that DGKδ translocated from the cytoplasm to the plasma membrane in C2C12 myoblasts within 5 min of short term exposure to a high glucose concentration [17]. Moreover, we recently found that DGKδ preferentially utilizes palmitic acid (the 16-carbon saturated fatty acid (16:0))-containing DG species such as 14:0/16:0-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG that are likely supplied from the phosphatidylcholine-specific phospholipase C-dependent (D609-sensitive) pathway in C2C12 cells in response to extracellular high glucose concentration [18,19]. Therefore, an understanding of the regulation of the DGKδ protein level is essential for revealing the pathogenic and exacerbation mechanisms of type 2 diabetes and for its treatment and prevention.