Development of novel PIP4K inhibitors to treat p53-null cancer

项目总结/摘要 该项目的一个长期目标是开发PI 5 P4 K抑制剂作为新的药理学药物，以治疗 p53缺失型癌症。PI 5 P4 K β和PI 5 P4 K β是同源的脂质激酶，在调节细胞凋亡中起重要作用。 细胞代谢和增殖。它们催化PI（5）P的磷酸化形成PI（4，5）P2。虽然这 不是PI（4，5）P2的主要合成途径，它们的活性消除了PI（5）P，一种应激诱导的脂质第二 使者PI 5 P4 K基因敲除的转基因动物对胰岛素过敏， PI 5 P4 K基因敲除可减少人Li-Fraumeni小鼠模型中自发性肿瘤发生 肿瘤抑制基因p53在生殖细胞中发生突变的综合征。在初步研究中， 从高通量筛选中鉴定出二氢蝶啶酮衍生物为PI 5 P4 K β的弱抑制剂。 初步的合成，激酶抑制剂复合物的X-射线晶体学分析的指导下，并利用 PI 5 P4 K κ/κ独特疏水口袋，已经产生了对两者都具有50倍效力的化合物 PI 5 P4 K β和PI 5 P4 K β，以及对蛋白激酶的高度选择性。具体目标1： 继续修饰最有效的抑制剂，基于该化合物与PI 5 P4 K β的共晶结构， 并聚焦于经历构象变化的结合口袋的不同区域。我们计划 用PIKfyve（家族的一个遥远成员）解决抑制剂的晶体结构，以设计 在具体的目标2中，我们研究了p53（+/+）和p53（-/-）细胞如何响应于p53（+/+）和p53（-/-）细胞如何响应于p53（-/-）细胞。 化学探针在培养的肌管中，我们发现脂质激酶抑制剂破坏了细胞能量稳态， 导致AMPK激活，这可以解释在动物研究中观察到的胰岛素敏感性增强。的 脂质激酶抑制可能通过破坏细胞增殖中的能量稳态而导致细胞周期停滞 将检查p53-/-癌细胞。PI 5 P4 K κ B/κ B和p53之间的合成致死相互作用将是 通过化学生物学和遗传学方法进行检查，并将化学探针与 增殖肿瘤细胞内的脂质激酶将通过细胞热位移测定（CETSA）来研究。

PROJECT SUMMARY / ABSTRACT A long-term objective of the project is to develop PI5P4K/ inhibitors as novel pharmacological agents to treat p53-null cancers. PI5P4K and PI5P4K 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 PI5P4K knocked out are hypersensitive to insulin, and combined knockout with PI5P4K 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 PI5P4K. Initial syntheses, guided by X-ray crystallographic analysis of kinase inhibitor complexes, and exploiting a hydrophobic pocket unique to PI5P4K/, have yielded compounds with 50-fold greater potency for both PI5P4K and PI5P4K, 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 PI5P4K, 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 PI5P4K/ 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).