Deconstructing Lipid Kinase Inhibitors by Chemical Proteomics

作者：McCloud Rebecca L.; Franks Caroline E.; Campbell Sean T.; Purow Benjamin W.; Harris Thurl E.; Hsu Ku-Lung^*

来源：Biochemistry, 2018, 57(2): 231-236.

DOI：10.1021/acs.biochem.7b00962

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-alpha (DGK alpha) inhibitors responsible for selective binding to the DGK alpha active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGK alpha 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 DGK alpha inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGK alpha 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 DGK alpha 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.

• 出版日期2018-1-16