Structural and molecular investigation of small molecule reactivation of p53

描述（由申请人提供）：肿瘤抑制因子p53是一种重要的细胞周期调节转录因子，是人类癌症中突变最多的基因1，2。这些p53癌症突变中的大多数导致DNA结合结构域中的单个氨基酸取代，导致具有单个氨基酸改变的全长p53蛋白的持续表达。这些p53 DNA结合域错义突变中最常见的R175 H导致蛋白质在生理温度下高度不稳定和无功能1，3，5，20。一种能够恢复R175H或任何这些单氨基酸突变的内源性p53功能的药物可能对我们的癌症治疗产生巨大影响。在开发高通量筛选后，我们发现了一种能够稳定R175 H DNA结合结构域并诱导癌细胞中R175 H依赖性细胞周期停滞的小分子。我们的目标是使用结构和分子技术来了解这种小分子对突变型p53的影响，以及它如何能够恢复其正常功能。我们正在研究的结构变化，在R175 H诱导后，其结合使用X射线晶体学。这将使我们能够理解它稳定和重新激活p53的机制。通过与R175 H结合的小分子的这种相同结构，我们也将能够深入了解允许它结合p53的分子相互作用。有了这些相互作用的知识，我们和其他人可以开始对这种分子支架进行定制修饰，以提高其结合力和功效。目前正在进行研究，以确定这种小分子是否能够恢复正常的DNA结合活性R175 H，或者如果观察到的再活化是通过另一种机制。我们还在探索这种分子的化学类似物对R175H和其他常见错义突变的影响。基于我们目前的数据，我们假设这种小分子能够结合到p53 DNA结合结构域的锌配位区，稳定蛋白质-DNA界面，并允许这种突变体重新获得转录活性。我们预计，这种小分子和它的几个类似物将能够结合和恢复功能的其他构象p53 DNA结合域突变体。

DESCRIPTION (provided by applicant): The tumor suppressor p53 is an important cell cycle regulating transcription factor which is the most mutated gene in human cancers1,2. Most of these p53 cancer mutations cause a single amino acid substitution in the DNA-binding domain, leading to continued expression of full length p53 protein with a single amino acid alteration. The most common of these p53 DNA-binding domain missense mutations, R175H, causes the protein to be highly destabilized and nonfunctional at physiological temperatures1,3,5,20. A pharmaceutical which can restore endogenous p53 function to R175H or any of these single amino acid mutations could have an enormous impact on our treatment of cancer. After developing a high throughput screen, we have discovered a small molecule which is able to stabilize the R175H DNA-binding domain and induce R175H-dependent cell cycle arrest in cancer cells. Our goal is to use structural and molecular techniques to understand the effects of this small molecule on mutant p53 and how it is able to restore its normal function. We are investigating the structural changes in R175H induced upon its binding using X-ray crystallography. This will allow us to understand the mechanism which allows it to stabilize and reactivate p53. Through this same structure of the small molecule bound to R175H we will also be able to gain insight into the molecular interactions which allow it to bind p53. With knowledge of these interactions, we and others can then begin to make tailored modifications to this molecular scaffold in order to improve its binding and efficacy. Studies are being done to determine if this small molecule is able to restore normal DNA-binding activity to R175H, or if the observed reactivation is through another mechanism. We are also exploring the effects of chemical analogs of this molecule on R175H and other common missense mutations. Based on our current data, we hypothesize that this small molecule is able to bind to the zinc-coordinating region of the p53 DNA-binding domain, stabilizing the protein-DNA interface and allowing this mutant to regain transcriptional activity. We anticipate that this small molecule and several of it analogs will be able to bind and restore function to other conformational p53 DNA-binding domain mutants.