An HTS Assay for Inhibitors of NBS1-ATM Interactions

NBS1-ATM 相互作用抑制剂的 HTS 测定

• 批准号: 7427147
• 负责人: BO XU
• 金额: $ 21.12万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7427147
• 关键词: 1-Phosphatidylinositol 3-Kinase; ATM gene; ATM wt Allele; Affinity; Ataxia Telangiectasia; Basic Cancer Research; Binding; Biological Assay; C-terminal; Cancer Patient; Cell Cycle Arrest; Cells; Chromosomal Breaks; Class; Complex; Consensus Sequence; Conserved Sequence; DNA Damage; DNA Repair; DNA-dependent protein kinase; Development; Dimethyl Sulfoxide; Disease; Drosophila genus; Effectiveness; Elements; Evaluation; Family; Fluorescein; Fluoresceins; Gene Family; Gene Mutation; Genes; Genetic; Glutamine; Goals; Heating; Hereditary Disease; Human; Hypersensitivity; Immunologic Deficiency Syndromes; In Vitro; Indium; Ionizing radiation; KRP protein; Lead; Malignant Neoplasms; Metabolism; Mutation; NBS1 gene; Names; Nijmegen Breakage Syndrome; Pathway interactions; Peptides; Phosphotransferases; Predisposition; Process; Protein Kinase; Proteins; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Radiosensitization; Research; Series; Serine; Signal Transduction; Structure; Targeted Radiotherapy; Testing; Texas; Therapeutic Agents; Threonine; Vertebrates; Yeasts; ataxia telangiectasia mutated protein; base; cancer therapy; chemosensitizing agent; chemotherapy; cytotoxic; development of lymphoid malignancy; high throughput screening; human FAT protein; improved; inhibitor/antagonist; kinase inhibitor; mei-41; member; molecular assembly/self assembly; novel; novel strategies; novel therapeutics; protein protein interaction; response; scaffold; sensor; size; small molecule; tool

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.

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，以确定治疗药物，通过靶向关键的DNA损伤反应途径，使细胞毒性癌症治疗（如放疗和化疗）的有效性最大化。虽然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，但很少有蛋白质-蛋白质相互作用的有效靶点被确定用于开发放射增敏剂。我们最近发现NBS1-ATM相互作用是激活ATM激酶和促进电离辐射存活的关键步骤，是放射致敏的新靶点。为了确定能够抑制NBS1-ATM相互作用的小分子，我们建议利用NBS1-ATM在HTS中的体外结合试验来开发荧光素极化（FP）试验。目的1将侧重于FP测定的发展。我们将生成GST-ATM片段和Texas Redlabeled NBS1 c端短肽。利用这些肽，我们将测试NBS1- 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设置下的分析参数，用小规模试点筛选测试分析格式，并开发用于详细评估HTS命中的二次分析。本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，以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成为DNA损伤反应信号机制研究的有力工具。

项目成果

ATM-mediated Mad1 Serine 214 phosphorylation regulates Mad1 dimerization and the spindle assembly checkpoint.

• DOI: 10.1093/carcin/bgu087
• 发表时间: 2014-09
• 期刊: Carcinogenesis
• 影响因子: 4.7
• 作者: Chunying Yang;Jianwei Hao;D. Kong;Xiaoli Cui;Wei Zhang;Haibo Wang;Xiaojing Guo;Shumei Ma;Xiaodong Liu;P. Pu;Bo Xu

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage.

• DOI: 10.1093/nar/gks707
• 发表时间: 2012-10
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Yang C;Lee M;Hao J;Cui X;Guo X;Smal C;Bontemps F;Ma S;Liu X;Engler D;Parker WB;Xu B
• 通讯作者: Xu B

The kinetochore protein Bub1 participates in the DNA damage response.

• DOI: 10.1016/j.dnarep.2011.10.018
• 发表时间: 2012-02-01
• 期刊: DNA REPAIR
• 影响因子: 3.8
• 作者: Yang, Chunying;Wang, Haibo;Xu, Yiran;Brinkman, Kathryn L.;Ishiyama, Hiromichi;Wong, Stephen T. C.;Xu, Bo
• 通讯作者: Xu, Bo