PO.ET09.07 · 实验与分子治疗
恢复突变型p53R273H DNA结合与活性的小分子
Small molecule restoring mutant p53R273H DNA binding and activity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
尽管TP53突变是人类癌症中最常见的遗传学改变，但靶向突变型p53以恢复p53野生型（WT）抑癌功能的方法收效有限，目前尚无FDA批准的靶向突变型p53的治疗药物。我们报道了一种膜通透性小分子化合物K，它能选择性增强p53-R273H（癌症中两种最常见p53突变体之一）的抑癌功能。理解p53-R273H抑癌功能丧失的关键在于His273在癌细胞较高的细胞内pH（pHi）下发生去质子化，从而减弱其与DNA带负电磷酸骨架的结合。我们在一项针对350万个小分子的计算筛选中鉴定出化合物K，用于靶向去质子化的His273并赋予正电荷以恢复与DNA的静电结合，类似于带正电的WT p53-Arg273。我们发现，化合物K在癌细胞较高的pH下选择性增强p53-R273H的DNA结合，而对未转化细胞较低pH下p53-R273H的DNA结合，或对较低和较高pH值下的p53-WT、p53-R175H及其他pH调控的转录因子无影响。化合物K还在p53-R273H纯合的人PANC-1胰腺癌细胞中恢复p53-WT功能，而对人HPDE未转化胰腺上皮细胞的影响有限，如RNA-seq所测定，PANC-1中有>4200个差异表达基因（DEG），而HPDE中<200个DEG。在PANC-1细胞中，化合物K增加了功能丧失性、抗肿瘤生成基因的表达，包括GADD45A、KLF6和IFIT2，并降低了功能获得性、促肿瘤生成基因的表达，包括CDC20、PLK1、AURKA以及TGF-beta信号相关基因，如RNA-seq和细胞裂解物免疫印迹所测定。化合物K还选择性降低PANC-1细胞的活力而对HPDE细胞无影响，并降低致瘤性，表现为软琼脂中集落形成减少80%。我们的发现是朝着开发靶向突变型p53以限制癌症进展这一未满足需求迈出的重要一步。此外，除p53-R273H外，Arg>His突变在许多癌症中富集，最显著的是髓母细胞瘤、急性髓系白血病以及胰腺癌和前列腺癌，这使我们靶向Arg>His替换以恢复正电荷的方法具有广泛的癌症治疗前景。
查看英文原文 English abstract
Although mutations in TP53 are the most frequent genetic alteration in human cancers, approaches that target mutant p53 to restore p53-wild type (WT) tumor suppressor functions have had limited success and there are currently no FDA-approved therapeutics targeting mutant p53. We report a membrane-permeant small molecule, Compound K, that selectively increases tumor suppressor functions of p53-R273H, one of the two most common p53 mutants in cancers. Key to understanding the loss of tumor suppressor functions of p53-R273H is deprotonation of His273 at the higher intracellular pH (pHi) of cancer cells, which attenuates binding to the negatively charged phosphate backbone of DNA. We identified Compound K in a computational screen of 3.5 million small molecules for targeting deprotonated His273 and imparting a positive charge to restore electrostatic binding to DNA, like positively charged WT p53-Arg273. We find that Compound K selectively increases DNA binding by p53-R273H at the higher pH of cancer cells with no effect on DNA binding by p53-R273H at the lower pH of untransformed cells, or by p53-WT, p53-R175H, and other pH-regulated transcription factors at lower and higher pH values. Compound K also restores p53-WT functions in human PANC-1 pancreatic cancer cells that are homozygous for p53-R273H, with limited effects in human HPDE untransformed pancreatic epithelial cells, including >4200 DEGs in PANC-1 and <200 DEGs in HPDE, as determined by RNA-seq. In PANC-1 cells Compound K increases expression of loss-of-function, anti-tumorigenic genes, including GADD45A, KLF6, and IFIT2, and decreases expression of gain-of-function pro-tumorigenic genes, including CDC20, PLK1, AURKA, and for TGF-beta signaling, as determined by RNA-seq and immunoblotting cell lysates. Compound K also selectively decreases viability of PANC-1 cells with no effect on HPDE cells, and tumorigenicity, determined by 80% reduced colony formation in soft agar. Our findings are an important step toward the unmet need of developing therapeutics targeting mutant p53 to limit cancer progression. Additionally, beyond p53-R273H, Arg>His mutations are enriched in many cancers, most notably medulloblastoma, acute myeloid leukemia, and pancreatic and prostate cancers, which makes our approach of targeting an Arg>His substitution to restore a positive charge have broad cancer therapeutic promise.
利益披露 Disclosure
A. Kingsland, None..
K. P. Kisor, None..
E. Alexov, None..
D. L. Barber, None.