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.