Identifying stabilizers of p53 using pocket complementarity

 描述（由申请人提供）：使用口袋互补性鉴定p53的稳定剂。 肿瘤抑制蛋白p53在超过一半的人类癌症中突变或缺失。最常见的这些功能丧失突变定位于p53“核心结构域”，但不涉及直接负责功能的表面残基。相反，这些点突变体降低了这种边缘稳定的蛋白质的热力学稳定性，使得细胞活性降低，因为不足量的p53被正确折叠。该提议的目标是鉴定有效结合并稳定正确折叠的p53的化合物。我们预期通过这种机制的稳定化将恢复这类最常见的p53点突变体的活性，并进一步恢复这些不稳定突变体的活性-无论确切地是哪种突变导致蛋白质稳定性的潜在丧失。 我们已经从一个小的试点筛选中确定了几种稳定化合物，我们发现这些化合物可以恢复含有p53不稳定突变体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范围，并通过药物化学优化所产生的命中化合物，我们将识别出作用更强的化合物。我们建议通过追求以下具体目标来实现这一目标：1）使用尖端的计算方法来识别与p53结合的化合物。2)使用直接稳定性试验在体外测试预测命中。3)使用结构导向药物化学优化验证命中。 传统的方法来确定化合物，稳定p53结合到新的表面位点可能需要基于结构的虚拟筛选，加上生化筛选的预测命中。这些方法中的每一种在应用于这个问题时都会遇到特定的障碍：拟议研究中的主要创新在于我们使用Karanicolas和Fisher实验室新开发的工具来解决这些特定挑战。 使用这些工具，我们希望确定一组新的p53“再激活剂”，这反过来又可能代表开发一类新的广谱癌症治疗剂的起点。我们进一步期望，通过这些p53的研究，我们的筛选平台的改进将额外增强其用于鉴定其他选择蛋白的再激活剂的效用，这些蛋白在人类癌症中经常通过不稳定突变而失活。

 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.