Modeling Molecular of Interactions Regulating the Activity of the p53 Protein

分子建模核心与实验组合作，为评估和理解他们的结果提供物理分子结构基础。这反过来又为发展新的假设和实验提供了素材。该中心与细胞生物学实验室的Ettore Appellas博士有着长期的合作关系，专注于了解调节p53蛋白的分子相互作用。人类p53是一种同源四聚体，序列特异性转录因子，在细胞凋亡，细胞周期阻滞，细胞衰老和DNA修复中起着至关重要的作用。它在非应激细胞中维持在低水平，但在DNA损伤后通过广泛的翻译后修饰而稳定和激活。启动p53活性的一种机制是通过p300组蛋白乙酰转移酶共激活因子的位点特异性募集，其促进局部染色质解绕。为了理解这种招募，我们最近确定了p53的n端反活化结构域和p300的Taz2结构域之间形成的复合物的结构，并研究了p53的位点特异性磷酸化如何导致更强的结合。此外，我们在p53残基35-59中发现了Taz2的第二个结合位点。第二个位点与Taz2结合的亲和力与第一个位点相似，但结合不受磷酸化的影响。我们也一直在关注Wip1磷酸酶的活性。appeella组的实验表明，Wip1的过表达通过p53的失活促进肿瘤发生。Wip1是一种保守的PP2C磷酸酶，在大多数组织中低水平表达，在DNA损伤后以p53依赖的方式转录诱导。目前发现的Wip1底物有p53、p38MAPK、UNG2、Chk1、Chk2和ATM。在过去的几年里，我们揭示了Wip1在pTXpY和pT/SQ氨基酸序列基序中去磷酸化丝氨酸和苏氨酸残基的能力的分子基础。在此基础上，我们最近设计、合成并测试了几种针对Wip1的多肽和小分子抑制剂。目前的一个是由一个基于吡咯的支架组成的，从它延伸的官能团模仿天然肽底物的极性和疏水残基的三维排列。我们目前正在进行Wip1/inhibitor复合物结构的x射线晶体学测定，以进一步优化其结合选择性和亲和力。我们假设用Wip1抑制剂治疗过表达Wip1的癌症将导致p53激活增加和随后的细胞杀伤。这种抑制剂无论单独使用还是与标准癌症化疗或放疗联合使用，都能提供选择性靶向肿瘤。在类似的情况下，我们最近也开发了一种人类MDM2和MDMX蛋白（HDM2和HDMX）的肽模拟抑制剂。这两种蛋白结合到n端，即p53的反活化结构域，并导致其降解。继我们早期开发MDM2的肽类（聚n -取代甘氨酸）抑制剂之后，我们成功地生产了一种基于多胺骨架的更小、更容易合成的抑制剂。该分子在霍夫曼-罗氏公司开发的著名HDM2抑制剂Nutlin范围内具有结合亲和力。然而，与Nutlin不同的是，我们的先导分子对HDM2和HDMX都有效，这是癌症治疗的两个重要靶点。

The Molecular Modeling Core collaborates with experimental groups by providing a physical molecular, structural basis for evaluating and understanding their results. This in turn provides the grist for developing new hypotheses and experiments. The Core enjoys a long-standing collaboration with Dr. Ettore Appellas section in the Lab of Cell Biology, which has focused on understanding the molecular interactions that regulate the p53 protein. Human p53 is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. It is maintained at low levels in unstressed cells, but is stabilized and activated following DNA damage through extensive post-translational modification. One mechanism of initiating p53 activity is by site-specific recruitment of the p300 histone acetyltransferase coactivators, which promote local chromatin unwinding. To understand this recruitment, we have recently determined the structure of the complex formed between the N-terminal transactivation domain of p53 and the Taz2 domain of p300, and studied how site-specific phosphorylations of p53 lead to stronger binding. Furthermore, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. We have also been focusing of the activity of the Wip1 phosphatase. Experiments from the Appella group suggest that over-expression of Wip1 promotes tumorigenesis through inactivation of p53. Wip1 is a conserved PP2C phosphatase expressed at low levels in most tissues and transcriptionally induced after DNA damage in a p53-dependent manner. Wip1 substrates discovered thus far are p53, p38MAPK, UNG2, Chk1, Chk2 and ATM. In previous years we revealed the molecular bases for the ability of Wip1 to dephosphorylate serine and threonine residues within pTXpY and pT/SQ amino acid sequence motifs. Based on this, we have recently designed, synthesized and tested several peptide, and now, small molecule inhibitors specific for Wip1. The current one is composed of a pyrrole-based scaffold, from which extend functional groups that mimic the three-dimensional arrangement of polar and hydrophobic residues of the native peptide substrates. We are currently pursuing X-ray crystallographic determination of the Wip1/inhibitor complex structure to facilitate further optimization of the binding selectivity and affinity. We hypothesize that treatment of cancers that over-express Wip1 with a Wip1 inhibitor will lead to increased activation of p53 and subsequent cell killing. Such an inhibitor would provide selective targeting of tumors either when given alone or in combination with standard cancer chemo- or radio-therapy. In a similar vein, we have also recently developed a peptide-mimetic inhibitor of both the human MDM2 and MDMX proteins (HDM2 and HDMX). Both these proteins bind to the N-terminal, transactivation domain of p53, and cause its degredation. Following our earlier work developing a peptoid (poly N-substituted glycine) inhibitor of MDM2, we have succeeded in producing a smaller and easier to synthesize inhibitor based on a polyamine backbone. This molecule has a binding affinity in the range of the well-known HDM2 inhibitor Nutlin being developed by Hoffman-La Roche. However, unlike Nutlin, our lead molecule is potent against both HDM2 and HDMX, two important targets for cancer therapy.