Deconstructing Lipid Kinase Inhibitors by Chemical Proteomics.

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.

Identification of ritanserin analogs that display DGK isoform specificity.

The diacylglycerol kinase (DGK) family of lipid enzymes catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Both DAG and PA are lipid signaling molecules that are of notable importance in regulating cell processes such as proliferation, apoptosis, and migration. There are ten mammalian DGK enzymes that appear to have distinct biological functions. DGKα has emerged as a promising therapeutic target in numerous cancers including glioblastoma (GBM) and melanoma as treatment with small molecule DGKα inhibitors results in reduced tumor sizes and prolonged survival. Importantly, DGKα has also been identified as an immune checkpoint due to its promotion of T cell anergy, and its inhibition has been shown to improve T cell activation. There are few small molecule DGKα inhibitors currently available, and the application of existing compounds to clinical settings is hindered by species-dependent variability in potency, as well as concerns regarding isotype specificity particularly amongst other type I DGKs. In order to resolve these issues, we have screened a library of compounds structurally analogous to the DGKα inhibitor, ritanserin, in an effort to identify more potent and specific alternatives. We identified two compounds that more potently and selectively inhibit DGKα, one of which (JNJ-3790339) demonstrates similar cytotoxicity in GBM and melanoma cells as ritanserin. Consistent with its inhibitor profile towards DGKα, JNJ-3790339 also demonstrated improved activation of T cells compared with ritanserin. Together our data support efforts to identify DGK isoform-selective inhibitors as a mechanism to produce pharmacologically relevant cancer therapies.