Identifying stabilizers of p53 using pocket complementarity

 描述(申请人提供)：使用口袋互补鉴定P53的稳定剂。肿瘤抑制蛋白P53在超过一半的人类癌症中发生突变或缺失。这些功能缺失突变中最常见的是定位于P53“核心区”，但不涉及与功能直接相关的表面残基。相反，这些点突变降低了这种边缘稳定的蛋白质的热力学稳定性，从而降低了细胞活性，因为不足的p53被正确折叠。这项建议的目标是识别能够有效结合和稳定正确折叠的p53的化合物。我们预计，通过这一机制的稳定将恢复这类最频繁发生的p53点突变的活性，并进一步恢复这些不稳定突变的活性-无论确切地说，哪个突变导致了潜在的蛋白质稳定性的丧失。我们已经从一个小的中试筛选中鉴定了几种稳定的化合物，我们发现这些化合物可以恢复含有不稳定的p53突变的细胞系的转录活性。我们的中心假设是，通过扩大我们的计算研究的范围，并通过药物化学优化得到的热门化合物，我们将识别出作用更有效的化合物。我们建议通过追求以下具体目标来实现这一目标：1)使用尖端计算方法来识别与P53结合的化合物。2)用直接稳定性试验进行体外预测HITS。3)利用结构导向的药物化学优化有效的HITS。通过结合新的表面位点来鉴定稳定p53的化合物的传统方法可能需要基于结构的虚拟筛选，再加上对预测的命中的生化筛选。这些方法中的每一种在应用于这一问题时都预计会遇到特定的障碍：拟议研究中的主要创新在于我们使用卡拉尼科拉斯和费舍尔实验室新开发的工具来应对每一个具体的挑战。使用这些工具，我们希望确定一组新的P53“再激活子”，这反过来可能代表着开发一类新的广谱癌症疗法的起点。我们还预计，通过这些对p53的研究，我们筛选平台的改进将进一步增强其在识别其他精选蛋白的再激活因子方面的效用，这些蛋白在人类癌症中经常因不稳定突变而失活。

 DESCRIPTION (provided by applicant): Identifying stabilizers of p53 using pocket complementarity. The tumor suppressor protein p53 is mutated or deleted in more than half of human cancers. The most frequently occurring of these loss-of-function mutations are localized to the p53 "core domain," but do not involve surface residues directly responsible for function. Rather, these point mutants reduce the thermodynamic stability of this marginally stable protein, such that cellular activity is diminished because an insufficient amount of p53 is correctly folded The goal of this proposal is to identify compounds that potently bind and stabilize correctly folded p53. We expect that stabilization through this mechanism will restore activity to this most frequently occurring class of p53 point mutants, and further will restore activity to these destabilized mutants - regardless of precisely which mutation is responsible for the underlying loss of protein stability. Already we have identified several stabilizing compounds from a small pilot screen, and we find that these compounds can restore transcriptional activity in cell lines harboring destabilized mutants of p53. Our central hypothesis is that by extending the scope of our computational studies and optimizing the resulting hit compounds through medicinal chemistry, we will identify compounds that act even more potently. We propose to meet this objective through pursuit of the following specific aims: 1) Use cutting-edge computational methods to identify compounds that bind to p53. 2) Test predicted hits in vitro using direct stability assays. 3) Optimize validated hits using structure-guided medicinal chemistry. Conventional approaches to identify compounds that stabilize p53 by binding to new surface sites might entail structure-based virtual screening, coupled with biochemical screening of the predicted hits. Each of these approaches would be expected to encounter particular hurdles when applied to this problem: the primary innovations in the proposed research lie in our use of newly-developed tools from the Karanicolas and Fisher labs to address each of these specific challenges. Using these tools we expect to identify a set of novel p53 "re-activators", which in turn may represent a starting point for developing a new class of broad-spectrum cancer therapeutics. We further expect that refinement of our screening platform through these studies of p53 will additionally enhance its utility for identifying re-activators of other select proteinsthat are frequently deactivated in human cancers by destabilizing mutations.