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选（HTS）检测方法，以识别可以通过靶向关键DNA损伤来最大限度地提高细胞毒性癌症疗法（例如放疗和化疗）有效性的治疗药物。响应途径。虽然可用于筛选放射增敏剂和化学增敏剂的传统方法是激酶抑制剂的酶促测定，但已确定用于开发放射增敏剂的蛋白质-蛋白质相互作用的经验证的靶标很少。我们最近的特点是，NBS 1-ATM相互作用，激活ATM激酶和促进生存响应电离辐射的关键步骤，是一个新的目标放射增敏。为了鉴定可以抑制NBS 1-ATM相互作用的小分子，我们建议开发一种利用NBS 1-ATM的体外结合测定来测定HTS的黄绿素极化（FP）测定。目标1将重点关注FP检测试剂盒的开发。我们将产生GST-ATM片段和德克萨斯红标记的NBS 1 C末端短肽。利用这些肽，我们将测试NBS 1- ATM结合亲和力、FP测定稳定性和FP DMSO耐受性。在目标2中，我们将优化HTS设置中的检测参数，使用小规模中试筛选测试检测格式，并开发用于详细评估HTS命中的二次检测。该申请中提出的研究旨在开发一类新的关键DNA损伤反应抑制剂，旨在帮助对放射治疗反应不佳的癌症患者。这种有效的抑制剂也可以是DNA损伤反应信号传导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.