Development of Assays for DNA Damage Checkpoints

描述(由申请人提供)： DNA损伤检查点是一种细胞监视机制，允许 细胞来监测基因组的完整性并发出DNA存在的信号 对细胞的损害。检查点通路在肿瘤中经常被打乱 细胞，证明了这种反应对维持 基因组。多条证据表明，DNA损伤的抑制剂 检查点可以更有效地使癌细胞对DNA损伤敏感 比正常细胞更强的药物。因此，DNA损伤检查点的抑制剂 可能是临床上有用的抗癌药物，以及临床前研究支持 这个假说。 这项工作广泛的长期目标是确定和验证新的 癌症治疗的靶点。研究表明，ATR，一种蛋白激酶 与脂蛋白激酶家族成员相关，是 哺乳动物细胞中的DNA损伤检查点。通过以下途径抑制ATR 显性-阴性形式的蛋白质的表达选择性地增敏 Gl/S调节异常的细胞对DNA损伤剂的作用。更有甚者 咖啡因，ATR及其相关的ATM的抑制剂，选择性地 使P53缺陷细胞对几种DNA损伤剂敏感。这些数据 提示ATR可能是癌症治疗的有用靶点。 本申请中提出的项目是开发两种检测方法来筛选 用于ATR和其他检查点蛋白的小分子抑制剂。ATR是一种 DNA结合蛋白和研究表明ATR与DNA的相互作用 是由另一种蛋白质(S)介导的。一种化验方法将被设计用于筛查 在体外抑制这种相互作用的小分子。因为多个 蛋白质参与了这种相互作用，ATR的抑制剂及其 可以同时寻找相关的DNA结合活性。在第二个 化验，直接与ATR结合的化合物将使用 小分子微阵列。一旦分子被识别为这些屏幕， 这些分子的目标及其对检查站的影响将是 下定决心。通过这些分析发现的分子可能被证明是有用的工具 探讨ATR的生理功能及其DNA的意义 结合活性。这些分子也可能在临床上对 抗癌疗法，特别是当与 激活DNA损伤检查点或抑制DNA的化疗药物 复制。

DESCRIPTION (provided by applicant): The DNA damage checkpoints are cellular surveillance mechanisms that allow the cell to monitor the integrity of the genome and to signal the presence of DNA damage to the cell. Checkpoint pathways are frequently disrupted In tumor cells, demonstrating the importance of this response to the maintenance of the genome. Several lines of evidence suggest that inhibitors of the DNA damage checkpoint can be more effective at sensitizing cancer cells to DNA damaging agents than normal cells. Therefore, inhibitors of the DNA damage checkpoint could be clinically useful anti-cancer agents, and preclinical studies support this hypothesis. The broad long-term objectives of this work are to identify and validate new targets for cancer therapy. Studies suggest that ATR, a protein kinase related to members of the lipid kinase family, is a central component of the DNA damage checkpoint in mammalian cells. Inhibition of ATR through expression of a dominant-negative form of the protein selectively sensitizes cells with abnormal Gl/S regulation to DNA damaging agents. Moreover caffeine, an inhibitor of ATR and the related kinase ATM, selectively sensitizes p53-deficient cells to several DNA damaging agents. These data suggest ATR may be a useful target for cancer treatment. The project proposed in this application is to develop two assays to screen for small molecule inhibitors of ATR and other checkpoint proteins. ATR is a DNA binding protein and studies suggest that the interaction of ATR with DNA is mediated by another protein(s). One assay will be designed to screen for small molecule that inhibit this interaction in vitro. Because multiple proteins are involved in this interaction, inhibitors of ATR and its associated DNA binding activity can be sought simultaneously. In the second assay, compounds that bind directly to ATR will be sought using a small-molecule microarray. Once molecules are identified with these screens, the targets of these molecules and their cffects on the checkpoint will be determined. Molecules found with these assays could prove to be useful tools to probe the physiological functions of ATR and the significance of its DNA binding activity. These molecules may also be clinically useful for anti-cancer therapy, particularly when used in combination with chemotherapeutic agents that activate the DNA damage checkpoint or inhibit DNA replication.