An HTS Assay for Inhibitors of NBS1-ATM Interactions

描述(由申请人提供)：我们的长期目标是开发一种可行的高通量筛查(HTS)方法，以确定能够通过靶向关键DNA损伤反应通路来最大化细胞毒性癌症治疗(如放射和化疗)有效性的治疗剂。虽然传统的筛选放射增敏剂和化学增敏剂的方法是酶分析，但很少有关于蛋白质-蛋白质相互作用的有效靶点被确定用于开发放射增敏剂。我们最近鉴定了NBS1-ATM相互作用是辐射增敏的新靶点，它是激活ATM激酶并促进对电离辐射反应的生存的关键步骤。为了确定可以抑制NBS1-ATM相互作用的小分子，我们建议利用NBS1-ATM与HTS的体外结合试验来建立一种荧光素偏振(FP)分析方法。目标1将侧重于FP分析的发展。我们将生成GST-ATM片段和德克萨斯州红色标记的NBS1 C末端短肽。利用这些多肽，我们将测试NBS1与ATM的结合亲和力、FP分析稳定性和FP DMSO耐受性。在目标2中，我们将优化HTS环境中的检测参数，用小规模中试屏幕测试检测格式，并开发二次检测以详细评估HTS HITS。这项申请中提出的研究旨在开发一类新的关键DNA损伤反应的抑制剂，目的是帮助对放射治疗反应不佳的癌症患者。这些经过验证的抑制剂也可以成为DNA损伤反应信号机制研究的有力工具。

DESCRIPTION (provided by applicant): Our long term goal is to develop a feasible assay for High Throughput Screening (HTS) to identify therapeutic agents that can maximize the effectiveness of cytotoxic cancer therapies, such as radiotherapy and chemotherapy, by targeting critical DNA damage response pathways. While traditional approaches available to screen radiosensitizers and chemosensitizers are enzymatic assays for kinase inhibitors, few validated targets on protein-protein interactions have been identified for developing radiosensitizers. We have recently characterized that the NBS1-ATM interaction, a critical step to activate the ATM kinase and promote survival in response to ionizing radiation, is a novel target for radiosensitization. To identify small molecules that can inhibit the NBS1-ATM interaction, we propose to develop a Fluorescein Polarization (FP) assay utilizing an in vitro binding assay of NBS1-ATM for HTS. Aim 1 will focus on the development of the FP assay. We will generate GST-ATM fragments and Texas Redlabeled NBS1 C-terminal short peptides. Utilizing these peptides, we will test NBS1- ATM binding affinity, FP assay stability, and FP DMSO tolerance. In Aim 2 we will optimize assay parameters in the HTS setting, test the assay format with small-scale pilot screens, and develop a secondary assay for detailed evaluation of HTS hits. The research proposed in this application aims to develop a novel class of inhibitors of critical DNA damage responses with a goal to help cancer patients that do not respond well to radiotherapy. Such validated inhibitors can also be powerful tools for mechanistic studies of DNA damage response signaling.