A long-term objective of the project is to develop PI5P4Kα/β inhibitors as novel pharmacological agents to treat p53-null cancers. PI5P4Kβ and PI5P4Kβ are homologous lipid kinases that play important roles in regulating cell metabolism and proliferation. They catalyze the phosphorylation of PI(5)P to form PI(4,5)P2. Although this is not a major synthetic route for PI(4,5)P2, their activities eliminate PI(5)P, a stress-induced lipid second messenger. Transgenic animals with PI5P4Kβ knocked out are hypersensitive to insulin, and combined knockout with PI5P4Kα reduce spontaneous tumorigenesis in a mouse model of human Li-Fraumeni syndrome where tumor suppressor p53 is mutated in the germline. In preliminary studies several dihydropteridinone derivatives were identified from high throughput screening as weak inhibitors for PI5P4Kα. Initial syntheses, guided by X-ray crystallographic analysis of kinase inhibitor complexes, and exploiting a hydrophobic pocket unique to PI5P4Kα/β, have yielded compounds with 50-fold greater potency for both PI5P4Kα and PI5P4Kβ, and a high degree of selectivity against protein kinases. In specific aim 1, we will continue to modify the most potent inhibitor, based on a co-crystal structure of the compound with PI5P4Kβ, and focusing on a different region of the binding pocket that undergoes conformational change. We plan to solve the crystal structure of the inhibitor with PIKfyve, a distant member of the family, in order to design analogs that do not cross-inhibit it. In specific aim 2, we study how p53(+/+) and p53(-/-) cells respond to the chemical probe. In cultured myotubes, we found that lipid kinase inhibitor disrupted cell energy homeostasis, causing AMPK activation, which may explain enhanced insulin sensitivity observed in animal studies. The possibility that lipid kinase inhibition causes cell cycle arrest by disrupting energy homeostasis in proliferating p53-/- cancer cells will be examined. The synthetic lethal interaction between PI5P4Kα/β and p53 will be examined by both chemical biological and genetic approaches, and direct engagement of chemical probe with the lipid kinase within proliferating tumor cells will be studied by cellular thermal shift assay (CETSA).