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基因p53突变。大多数p53阴性癌症确实表达全长，但突变的p53。在癌症中发现的许多p53突变是p53的200个氨基酸核心结构域中的单个氨基酸变化。这些突变被认为会破坏正确的p53折叠，从而导致p53肿瘤抑制功能的失活。我们的假设是，小分子的存在，可以稳定正确的构象在p53癌症突变体。因此，这些分子可以重新激活癌症中突变形式的p53。在动物模型中的实验已经证明，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预计将显着改善携带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，以鉴定能重新激活人类癌症中常见的p53突变体的化合物。拟议中的药物筛选依赖于独特、廉价和非常有效的基于酵母的测定，使我们能够同时针对70种最常见的p53癌症突变体筛选化合物。我们建议为基于酵母的系统实现基于荧光的读数，以实现HTS。此外，我们提出了二级酵母试验，以确认小分子对p53癌突变体的特定作用，并确定有多少不同的p53突变体被一个特定的小分子拯救。提出了一种生物化学策略，研究所识别的小分子对p53热稳定性的影响，以选择直接改善p53结构完整性的分子。最后，适应三级屏幕提出，将评估救援化合物在哺乳动物细胞。该提案的近期目标是开发框架，以识别恢复p53癌症突变体功能的小分子，并随后使用这些分子作为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，已经证明恢复肿瘤中的p53功能可以逆转肿瘤生长，因此是一种有吸引力的癌症治疗策略。 公共卫生相关性：大约50%的人类癌症携带肿瘤抑制基因p53突变。p53阴性癌症通常对目前的治疗策略更有抗性，并且替代方法对于有效治疗这些癌症是特别必要的。该提案的目的是鉴定恢复p53癌症突变体功能的小分子，并将这些分子开发成恢复肿瘤中p53功能的治疗剂。

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.