Identification of Small Molecules for Reactivation of p53 Cancer Mutants

描述（由申请人提供）：一半的人类癌症携带肿瘤抑制因子p53突变。大多数p53阴性的癌症确实表达全长但突变的p53。在癌症中发现的许多p53突变是p53的200个氨基酸核心区域的单个氨基酸变化。这些突变被认为破坏了p53正确折叠的稳定性，从而导致p53肿瘤抑制功能失活。我们的假设是存在能够稳定p53癌突变体正确构象的小分子。因此，这些分子可以重新激活癌症中p53的突变形式。动物模型实验表明，即使在晚期肿瘤中，恢复p53功能也会导致肿瘤消退。因此，可以恢复p53癌症突变体功能的小分子有望显著改善携带p53突变的患者的前景。我们建议开发一个与高通量小分子筛选（HTS）兼容的筛选平台，以识别重新激活p53突变体的化合物，这些突变体在人类癌症中经常发现。拟议的药物筛选依赖于独特、廉价和非常有效的基于酵母的检测，使我们能够同时筛选针对70种最常见的p53癌症突变体的化合物。我们建议在酵母为基础的系统中实现基于荧光的读数，以实现HTS。此外，我们提出二次酵母试验，以确认小分子对p53癌症突变体的特定作用，并确定一个特定的小分子拯救了多少不同的p53突变体。研究人员提出了一种生物化学策略，通过观察所鉴定的小分子对p53热稳定性的影响，来选择直接改善p53结构完整性的分子。最后，提出了一种适应的三级筛选，将评估哺乳动物细胞中的拯救化合物。该提案的直接目标是建立一个框架，以识别恢复p53癌症突变体功能的小分子，并将这些分子用作先导化合物来合成衍生物，以增加其治疗活性。在动物模型中，恢复肿瘤中的p53功能已被证明可以逆转肿瘤生长，因此是一种有吸引力的癌症治疗策略。

DESCRIPTION (provided by applicant): Half of all human cancers carry mutations in the tumor suppressor p53. The majorities of p53 negative cancers do express full length, but mutated p53. Many of the p53 mutations found in cancers are single amino acid changes in the 200 amino acid core domain of p53. These mutations are thought to destabilize the correct p53 fold and thus lead to inactivation of the p53 tumor suppressor function. Our hypothesis is that small molecules exist that can stabilize the correct conformation in p53 cancer mutants. These molecules could therefore reactivate mutated forms of p53 in cancers. Experiments in animal models have demonstrated that restoring p53 function even in advanced tumors leads to tumor regression. Small molecules that can restore function to p53 cancer mutants are therefore predicted to significantly improve the outlook for patients carrying a p53 mutation. We propose to develop a screening platform compatible with high throughput small molecule screens (HTS) to identify compounds that reactivate p53 mutants that are frequently found in human cancers. The proposed drug screen relies on unique, cheap and very efficient yeast-based assays that allow us to screen compounds simultaneously against the 70 most commonly found p53 cancer mutants. We propose to implement fluorescence-based readouts for our yeast-based system to enable HTS. In addition we propose secondary yeast assays to confirm a specific effect of small molecules on p53 cancer mutants and to determine how many different p53 mutants are rescued by one specific small molecule. A biochemical strategy that looks at effects of the identified small molecules on thermostability of p53 is proposed to select molecules that directly improve structural integrity of p53. Lastly, adaptation of a tertiary screen is proposed that will evaluate rescue compounds in mammalian cells. The immediate goal of the proposal is to develop the framework to identify small molecules that restore function to p53 cancer mutants and use these molecules later as lead compounds to synthesize derivatives to increase their therapeutic activity. Restoring p53 function in tumors has been demonstrated to reverse tumor growth in animal models and is therefore an attractive strategy for cancer treatment. PUBLIC HEALTH RELEVANCE: About 50% of all human cancers carry mutations in the tumor suppressor p53. p53 negative cancers generally more resistant to current treatment strategies and alternative approaches are particularly necessary for effective treatment of these cancers. The objective of this proposal is to identify small molecules that restore function to p53 cancer mutants and to develop these molecules into therapeutics to restore p53 function in tumors.