Identification of a chemical probe for lipid kinase phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ)

ABSTRACT Phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ), which phosphorylates phosphatidylinositol-5-monophosphate (PI(5)P), is a human lipid kinase with intriguing roles in inflammation, T cell activation, autophagy regulation, immunity, heart failure, and several cancers. To provide a high-quality chemical tool that would enable additional characterization of this protein, we designed and evaluated a potent, selective, and cell-active inhibitor of human PI5P4Kγ. We describe the use of the PI5P4Kγ NanoBRET assay to generate structure–activity relationships (SAR), support chemical probe ( 2 ) design, and identify a structurally related negative control ( 4 ). We have characterized the binding of our chemical probe to PI5P4Kγ using orthogonal assay formats reliant on competition with an ATP-competitive reagent. Based on our results in these assays, we hypothesize that 2 binds in the ATP active site of PI5P4Kγ. Kinome-wide profiling complemented by further off-target profiling confirmed the selectivity of both our chemical probe and negative control. When a breast cancer cell line (MCF-7) was treated with compound 2 , increased mTORC1 signaling was observed, demonstrating that efficacious binding of 2 to PI5P4Kγ in cells results in activation of a negative feedback loop also reported in PI5P4Kγ knockout mice.