Modeling Molecular of Interactions Regulating the Activity of the p53 Protein

分子建模核心通过为评估和理解他们的结果提供物理的分子、结构基础来与实验小组合作。这反过来又为发展新的假设和实验提供了基础。Core与细胞生物学实验室的Ettore Appellas博士长期合作，该实验室专注于了解调节P53蛋白的分子相互作用。人P53是一种同源四聚体、序列特异性转录因子，在细胞凋亡、细胞周期停滞、细胞衰老和DNA修复中发挥重要作用。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稳定和激活。启动P53活性的一种机制是通过部位特异性地招募p300组蛋白乙酰转移酶共活化子，促进局部染色质的解离。为了理解这种招募，我们最近确定了P53的N端反式激活结构域和p300的Taz2结构域之间形成的复合体的结构，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中确定了Taz2的第二个结合位点。第二个位点与Taz2的结合具有与第一个位点相似的亲和力，但结合不受磷酸化的影响。今年我们的重点是确定与Taz2结合的P53的第二个反式激活结构域的结构，该结构几乎已经完成。与第一个反式激活结构域的结构进行比较，将进一步阐明Taz2是如何调节P53活性的。我们也一直在关注Wip1磷酸酶的活性。来自Appella小组的实验表明，Wip1的过度表达通过使P53失活来促进肿瘤的发生。Wip1是一种保守的PP2C磷酸酶，在大多数组织中低水平表达，并在DNA损伤后以P53依赖的方式转录诱导。到目前为止发现的Wip1底物是p53、p38MAPK、UNG2、Chk1、Chk2和ATM。在前些年，我们揭示了Wip1对pTXpY和PT/SQ氨基酸序列基序中的丝氨酸和苏氨酸残基进行去磷酸化的分子基础。基于此，我们最近设计、合成和测试了几种针对Wip1的多肽，现在是针对Wip1的小分子抑制剂。后者是由基于吡咯的支架组成的，从支架上延伸出的官能团模拟了天然多肽底物的极性和疏水残基的三维排列。今年，我们专注于优化我们的环肽抑制剂，使其活性增加了30倍。我们目前正在进行Wip1/抑制剂复合体结构的X射线结晶学测定，以便于进一步优化结合选择性和亲和力。我们假设，用Wip1抑制剂治疗过度表达Wip1的癌症将导致P53的激活和随后的细胞杀伤。这种抑制剂将提供选择性的肿瘤靶向，无论是单独给药，还是与标准的癌症化疗或放射治疗联合使用。在类似的脉络中，我们最近也开发了一种对人类MDM2和MDMX蛋白(Hdm2和HDMX)都有抑制作用的多肽模拟物。这两种蛋白都与P53的N端反式激活结构域结合，并导致其降级。继我们早期开发MDM2的多肽(聚N-取代甘氨酸)抑制剂的工作之后，我们成功地生产了一种基于多胺骨架的更小且更容易合成的抑制剂。这种分子的结合亲和力在霍夫曼-拉罗氏公司正在开发的众所周知的HdM2抑制剂Nutlin的范围内。然而，与Nutlin不同的是，我们的铅分子对Hdm2和HDMX都有效，这是癌症治疗的两个重要靶点。今年，我们将所学到的经验融入到一种新的、相对容易合成的、具有增强活性的环二肽抑制剂中。

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. This year we have focused on determining the structure of this second transactivation domain of p53 bound to Taz2, which is nearly completed. Comparison with the structure of the first transactivation domain will further elucidate how the activity of p53 is regulated by Taz2. 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 latter 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. This year we have focused on optimizing our cyclic peptide inhibitor, which has resulted in a 30-fold increase in activity. 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. This year we incorporated the lessons learned into a new, relatively easy to synthesize, cyclic-di-peptide inhibitor with enhance activity.