Development of novel PIP4K inhibitors to treat p53-null cancer

项目摘要/摘要 该项目的一个长期目标是开发PI5P4K/抑制剂作为治疗 P53基因缺失的癌症。PI5P4K和PI5P4K是在调节中起重要作用的同源脂蛋白激酶 细胞的新陈代谢和增殖。它们催化PI(5)P的磷酸化生成PI(4，5)P2。虽然这件事 不是PI(4，5)P2的主要合成途径，它们的活性消除了PI(5)P，应激诱导的第二种脂质 信使。剔除PI5P4K的转基因动物对胰岛素过敏，并联合 PI5P4K基因敲除可降低人LI-Flaumeni小鼠模型的自发成瘤率 肿瘤抑制基因P53在生殖系中发生突变的综合征。在初步研究中， 高通量筛选结果表明，二氢蝶呤类化合物是PI5P4K的弱抑制剂。 在X射线结晶学分析的指导下，初步合成了激酶抑制剂复合体，并利用了一种 PI5P4K/独有的疏水口袋，产生的化合物对两者的效力都提高了50倍 PI5P4K和PI5P4K，对蛋白激酶具有高度的选择性。在具体目标1中，我们将 继续修饰最有效的缓蚀剂，基于与PI5P4K化合物的共晶结构， 并聚焦于结合口袋中经历构象变化的不同区域。我们计划 用该家族的远亲PIKfyve解算缓蚀剂的晶体结构，以便设计 没有交叉抑制作用的类似物。在特定目标2中，我们研究了P53(/)和P53(-/-)细胞如何对 化学探头。在培养的肌管中，我们发现脂激酶抑制剂破坏了细胞的能量平衡， 引起AMPK激活，这可能解释了在动物研究中观察到的胰岛素敏感性增强。这个 脂蛋白激酶抑制通过破坏细胞增殖过程中的能量平衡而导致细胞周期停滞的可能性 将检查P53-/-癌细胞。PI5P4K/和P53之间的合成致死作用将是 通过化学、生物学和遗传学方法进行检查，并与化学探针直接接触 用细胞热漂移分析法(CETSA)研究增殖期肿瘤细胞内的脂蛋白激酶。

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).