We also discovered important clues to domain binding sites
We also discovered important clues to domain Ketoconazole of DGKs and how to exploit these regions for development of DGKα-selective inhibitors. The identification of a probe-modified site at the C1 domain provided the first evidence of a ligand binding site remote from the ATP binding region of DGKs. Although we cannot rule out the possibility of alternative mechanisms, e.g., probe binding due to domain (Nordin et al., 2015) or protein interactions (Okerberg et al., 2014), we do provide evidence that the C1 domain serves as a ligand binding site for ritanserin distinct from the ATP binding region of DGKα (Figures 5A and 6A). The overlapping (DAGKa) and distinct (C1) binding sites of ritanserin compared with ATP helps explain previous kinetic findings of a mixed competitive mechanism of inhibition whereby ritanserin prefers to bind a DGKα-ATP complex (Boroda et al., 2017). We investigated how the binding mode of ritanserin affects selectivity against other DGK isoforms as well as >50 native kinases detected in cell proteomes. While ritanserin showed good selectivity within the DGK superfamily, we discovered substantial cross-reactivity against protein kinases, including the non-receptor tyrosine kinase FER that was inactivated to a similar magnitude as DGKα (SR = 7.9; Figures 7A and 7B). An unexpected finding was the discovery that a ritanserin fragment (RF001) functioned as a DGKα inhibitor that retained binding at C1 and DAGKa sites (Figure 7D), and largely removed FER and other kinase off-target activity (Figures 7A and 7E). Conservation of fragment binding mode is characteristic of ligand binding hotspots (Hall et al., 2015, Kozakov et al., 2015) of proteins suitable for fragment-based lead and drug discovery (Erlanson et al., 2016). In this regard, future studies are needed to investigate whether RF001 can serve as a core fragment for synthetic elaboration of high-affinity ligands with selectivity for DGKα.
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.
Introduction Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid [, , , ]. Ten mammalian DGK isozymes have been identified and classified into five groups based on their primary structures [, , , ]. 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  isozymes. Moreover, alternatively spliced variants of DGKδ (δ1 and δ2)  and η (η1 – η4) [10,12,13] have been reported.