Inhibition of Recombination DNA Repair in Pancreatic Cancer Cells

描述（由申请人提供）：胰腺癌仍然是美国第四大癌症死亡原因。这些肿瘤似乎对现有的标准疗法有相对的抵抗力。一种能够使胰腺癌细胞对肿瘤治疗敏感的药物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修复水平的升高可能导致人类胰腺肿瘤对某些化疗和放疗产生耐药性，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方法，用于鉴定人类RAD51的小分子抑制剂，RAD51是参与启动HR的中心蛋白。该项目最初提交的目标包括开发一种高通量（HT）测定方法，并筛选小分子na - ve文库，以寻找能够抑制单链DNA上RAD51细丝形成的化合物。部分初步工作已经完成，现将其摘要载于《初步研究/进度报告》一节。10k文库的筛选鉴定出72种抑制RAD51细丝形成的化合物，抑制率为50%。这项工作成功地验证了细丝形成分析作为高温筛选，并提供了重要的信息，包括改进分析技术和数据分析。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大活性和特异性的化合物（Aim 1）。为了确定哪些化合物具有特定的活性，将在二级和三级分析中进一步测试两种筛选产生的化合物。目标2将采用一组体外二级分析，旨在鉴定那些能够特异性阻断纯化RAD51蛋白特定功能的化合物。在第三个目标中，化合物将通过基于三级细胞的分析进一步表征，以确定能够通过hr特异性机制使胰腺癌细胞对DNA损伤治疗的致死效应敏感的亚群。最终目标是确定至少一种适合作为肿瘤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有抵抗力。一种能够克服细胞对放射治疗和/或化疗药物的耐药性的药物可以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疗法敏感的药物。

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.