Discovery of potent antagonists of the interaction between human double minute 2 and tumor suppressor p53
Discovery of potent antagonists of the interaction between human double minute 2 and tumor suppressor p53
复制标题
DOI：
10.1021/jm990966p
复制
发表时间：
2000-08-24
影响因子：
7.3
通讯作者：
Furet, P
中科院分区：
文献类型：
--
作者：
García-Echeverría, C;Chène, P;Furet, P
介绍。 p53 肿瘤抑制基因是一种多功能蛋白，通过响应 DNA 损伤和/或应激刺激诱导生长停滞或凋亡来调节细胞增殖。 1 尽管这种蛋白在肿瘤转化过程中突变的频率和敏感性很高，但相当一部分癌细胞表达野生型 p53，但在这些细胞中 p53 似乎是失活的。在消除 p53 肿瘤抑制功能的已知机制中 2，我们对人双微体 2 (hdm2) 癌蛋白对 p53 的调节感兴趣。 3 hdm2蛋白与p534的反式激活结构域结合，下调其激活转录的能力；反过来，p53 在自动调节负反馈环路中激活 hdm2 基因的表达。 2-4 在肿瘤中观察到 hdm2 对 p53 的抑制，其中基因扩增和其他改变可导致 hdm2 升高（例如，三分之一的软组织肉瘤）。 3c, 5 最近，hdm2 可以调节 p53 反应的另一种机制已被报道。 hdm2 以 p53 为目标，通过泛素途径进行降解。 6 如果该途径在含有低水平 p53 的肿瘤细胞中发挥作用，则 p53/hdm2 蛋白质-蛋白质相互作用的破坏应导致 p53 积累并激活 p53 响应报告基因。因此，破坏 p53/hdm2 蛋白质-蛋白质相互作用对于癌症治疗来说是一种有吸引力的方法，因为它提供了调节治疗剂的 p53 反应阈值的可能性。作为我们识别 p53/hdm2 蛋白质-蛋白质相互作用拮抗剂的药物发现计划的一部分，我们尝试确定 hdm2 结合袋的氨基酸特异性，以便建立这种蛋白质-蛋白质相互作用的药效团模型。这项工作导致了高效肽拮抗剂的鉴定。结果和讨论。最初使用一系列单克隆抗体研究 p53/hdm2 复合物，以鉴定 p53 与 hdm2 相互作用的区域，反之亦然。使用源自 p53 N 末端部分的合成肽文库对 p53 上的 hdm2 结合位点进行作图。 7a 活性肽将 p53 上的共有 hdm2 结合位点定义为 Thr18-Phe-Ser-Asp-Leu-Trp23，但该六肽对 hdm2 的结合亲和力较低 (IC50) 700 µM）。 8 为了寻找能够阻断 hdm2 与 p53 相互作用的 hdm2 新型高亲和力配体，我们筛选了噬菌体展示肽库。 9 获得的最具活性的肽（肽 2；表 1）对 p53/hdm2 相互作用的抑制作用比野生型 p53 衍生肽（肽 1；表 1）高 28 倍。通过合成截短的肽 2 并在我们的体外测定中测试这些肽，获得了有关有效肽抑制剂所需氨基酸的更详细信息。 8 在此竞争测定中，ELISA 板涂有 GST-hdm2。与不同量的肽预孵育后，添加全长 p53，并使用一系列抗体检测与 hdm2 结合的 p53 部分（图 1；有关其他实验详细信息，请参阅支持信息）。截短系列包括 6 至 11 聚体的序列。虽然所有 6 聚体和 7 聚体肽都是 p53/hdm2 相互作用的较差抑制剂，但 8 聚体肽（肽 3；表 1）被确定为对 hdm2 保留微摩尔亲和力的最小序列。该肽是我们优化过程的起点。hdm2 的 N 端结构域与 15 聚体野生型 p53 衍生的 X 射线结构结合......
Introduction. The p53 tumor suppressor gene is a multifunctional protein that regulates cell proliferation by induction of growth arrest or apoptosis in response to DNA damage and/or stress stimuli. 1 Despite the high frequency and susceptibility with which this protein is mutated during neoplastic transformation, a substantial proportion of cancer cells express wild-type p53, but it seems likely that in these cells p53 is inactivated. Among the known mechanisms by which the tumor suppressor functions of p53 can be abrogated, 2 we are interested in the regulation of p53 by the human double minute 2 (hdm2) oncoprotein. 3 The hdm2 protein binds to the transactivation domain of p534 and downregulates its ability to activate transcription; in turn, p53 activates the expression of the hdm2 gene in an autoregulatory negative feedback loop. 2-4 Inhibition of p53 by hdm2 has been observed in tumors where gene amplification and other alterations can result in elevated hdm2 (eg, one-third of soft tissue sarcomas). 3c, 5 Recently, an additional mechanism by which hdm2 can regulate p53 response has been reported. hdm2 targets p53 for degradation by the ubiquitin pathway. 6 If this pathway operates in tumor cells containing low levels of p53, then disruption of the p53/hdm2 protein-protein interaction should lead to accumulation of p53 and activation of p53 responsive reported genes. The disruption of the p53/hdm2 protein-protein interaction is therefore an attractive approach for cancer therapy because it provides the possibility to regulate the threshold of the p53 response with therapeutic agents. As part of our drug discovery program to identify antagonists of the p53/hdm2 protein-protein interaction, we have attempted to determine the amino acid specificities of hdm2’s binding pockets in order to establish a pharmacophore model for this proteinprotein interaction. This work has resulted in the identification of highly potent peptide antagonists. Results and Discussion. The p53/hdm2 complex was initially studied with a series of monoclonal antibodies to identify the region of p53 that interacts with hdm2 and vice versa. Mapping of the hdm2-binding site on p53 was carried out using synthetic peptide libraries derived from the N-terminal part of p53. 7a The active peptides defined the consensus hdm2-binding site on p53 to be Thr18-Phe-Ser-Asp-Leu-Trp23, but this hexapeptide had a low binding affinity for hdm2 (IC50) 700 µM). 8 To find novel high-affinity ligands for hdm2 that are able to block the interaction of hdm2 with p53, we screened phage display peptide libraries. 9 The most active peptide obtained (peptide 2; Table 1) showed a 28-fold greater inhibition of the p53/hdm2 interaction than the wild-type p53-derived peptide (peptide 1; Table 1). More detailed information on the amino acid requirements for a potent peptide inhibitor was obtained by synthesizing truncated versions of peptide 2 and testing these peptides in our in vitro assay. 8 In this competition assay, the ELISA plates were coated with GST-hdm2. After preincubation with different amounts of peptide, full-length p53 was added and the fraction of p53 bound to hdm2 was detected with a series of antibodies (Figure 1; see Supporting Information for additional experimental details). The truncation series included sequences from 6-to 11-mers. While all 6-and 7-mer peptides were poor inhibitors of the p53/hdm2 interaction, an 8-mer peptide (peptide 3; Table 1) was identified as the minimal sequence retaining micromolar affinity for hdm2. This peptide was our starting point in the optimization process.The X-ray structure of the N-terminal domain of hdm2 bound to a 15-mer wild-type p53-derived …