Development of novel PIP4K inhibitors to treat p53-null cancer

项目概要/摘要 该项目的长期目标是开发 PI5P4Kα/β 抑制剂作为新型药物来治疗 p53 缺失的癌症。 PI5P4K 和 PI5P4K 是同源脂质激酶，在调节中发挥重要作用 细胞代谢和增殖。它们催化 PI(5)P 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） 使者。敲除 PI5P4K 的转基因动物对胰岛素过敏，并且结合 PI5P4Kα 敲除可减少人类 Li-Fraumeni 小鼠模型中的自发肿瘤发生 抑癌基因 p53 在种系中发生突变的综合征。在初步研究中几个 通过高通量筛选，鉴定出二氢蝶啶酮衍生物是 PI5P4Kα 的弱抑制剂。 初始合成，以激酶抑制剂复合物的 X 射线晶体学分析为指导，并利用 PI5P4K/ 独有的疏水口袋，产生的化合物对两者的效力均高出 50 倍 PI5P4K 和 PI5P4K，并对蛋白激酶具有高度选择性。在具体目标1中，我们将 基于化合物与 PI5P4K 的共晶结构，继续修改最有效的抑制剂， 并关注经历构象变化的结合口袋的不同区域。我们计划 使用该家族的远亲成员 PIKfyve 解析抑制剂的晶体结构，以便设计 不交叉抑制它的类似物。在具体目标 2 中，我们研究 p53( / ) 和 p53(-/-) 细胞如何响应 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，这可以解释动物研究中观察到的胰岛素敏感性增强的原因。的 脂质激酶抑制可能通过破坏增殖过程中的能量稳态而导致细胞周期停滞 将检查 p53-/- 癌细胞。 PI5P4K/ 和 p53 之间的合成致死相互作用为 通过化学生物学和遗传学方法进行检查，并通过化学探针直接接触 将通过细胞热位移测定（CETSA）研究增殖肿瘤细胞内的脂质激酶。

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).