Inhibition of Recombination DNA Repair in Pancreatic Cancer Cells

描述(申请人提供)：胰腺癌仍然是美国癌症死亡的第四大原因。这些肿瘤似乎对标准可用的治疗方法相对耐药。一种能够使胰腺癌细胞对肿瘤治疗敏感的试剂可能会改善这些患者的预后。该项目的一个中心假设是，同源重组(HR)DNA修复水平的提高可以导致人类胰腺肿瘤对某些化疗和放射治疗产生耐药性，而对HR的特异性抑制可能有助于克服这种耐药性。拟议的研究计划将开发一种多步骤筛选，以确定人类RAD51的小分子抑制物，RAD51是参与启动HR的中心蛋白质。该项目最初提交的一个目标是开发一种高通量(HT)分析方法，并筛选一个天然小分子文库，以寻找能够抑制单链DNA上RAD51微丝形成的化合物。初步工作的一部分已经完成，现已在初步研究/进展报告一节中总结。10k文库的筛选结果表明，72个化合物对RAD51丝形成的抑制率为50%。这项工作成功地验证了微丝形成实验作为HT筛查的有效性，并提供了重要的信息，包括分析技术的改进和数据分析。利用这些信息，现在将使用更大的起始库(130k化合物)和机器人液体处理来重复筛选，这可能会产生具有更高活性和特异性的化合物(目标1)。为了确定哪些化合物具有特定的活性，两个筛选得到的化合物将在第二次和第三次化验中进行进一步测试。AIM 2将采用一套体外二次分析方法，旨在识别那些能够特异性阻断纯化的RAD51蛋白特定功能的化合物。在第三个目标中，化合物将进一步用基于第三级细胞的分析来表征，以确定能够通过HR特异性机制使胰腺癌细胞对DNA损伤治疗的致命影响敏感的亚群。最终目标是确定至少一种适合作为肿瘤学药物在临床试验中进一步开发的最佳化合物。胰腺癌仍然是美国癌症死亡的第四大原因。这些肿瘤似乎对目前可用的治疗方法相对耐药。一种能够克服细胞对放疗和/或化疗药物耐药性的药物可能会改善这些患者的预后。这项提议的目标是开发一种能够使胰腺癌细胞对这些常见肿瘤学疗法敏感的试剂。

DESCRIPTION (provided by applicant): Pancreatic cancer remains the 4th leading cause of cancer death in the United States. These tumors appear to be relatively resistant to standard available therapies. An agent capable of sensitizing pancreatic cancer cells to oncology therapies may improve the outcomes of these patients. A central hypothesis of this project is that elevated levels of homologous recombinational (HR) DNA repair can cause human pancreatic tumors to be resistant to some chemotherapies and radiotherapy, and that specific inhibition of HR may help overcome this resistance to therapy. The proposed research plan will develop a multi-step screen for identifying small molecule inhibitors of human RAD51, which is the central protein involved in initiating HR. The initial submission of this project included an aim proposing to develop a high-throughput (HT) assay and to screen a na¿ve library of small molecules in search of compounds that can inhibit the formation of RAD51 filaments on single-stranded DNA. A portion of initial work has been completed and is now summarized in the Preliminary Studies / Progress Report section. The screen of a 10k library identified 72 compounds that inhibit RAD51 filament formation by = 50%. This work successfully validated the filament formation assay as an HT screen, and it provided important information including refinement of assay techniques and data analysis. Using this information, the screen will be now be repeated using a larger starting library (130k compounds) and robotic liquid handling, which will likely yield compounds with even greater activities and specificities (Aim 1). To determine which have specific activities, the compounds resulting from both screens will be tested further in secondary and tertiary assays. Aim 2 will employ a set of in-vitro secondary assays aimed at identifying those compounds that can specifically block particular functions of purified RAD51 protein. In the third aim, compounds will be further characterized with tertiary cell-based assays, to identify the subset capable of sensitizing pancreatic cancer cells to the lethal effects of DNA damaging therapies via HR-specific mechanisms. The ultimate goal is to identify at least one optimal compound suitable for further development in clinical trials as an oncology drug. Pancreatic cancer remains the 4th leading cause of cancer death in the United States. These tumors appear to be relatively resistant to presently available therapies. A drug capable of overcoming the cellular resistance to radiotherapy and/or chemotherapeutic drugs may improve on the outcomes of these patients. The goal of this proposal is to develop an agent capable of sensitizing pancreatic cancer cells to these common oncology therapies.