Development of a HTS Assay Targeting End Protection of Telomeres

针对端粒末端保护的 HTS 测定的开发

• 批准号: 7289370
• 负责人: DEBORAH S. WUTTKE
• 金额: $ 18.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-05 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7289370
• 关键词: Aging; Animal Model; Area; Binding; Biochemical; Biological; Biological Assay; Biological Models; Cell Aging; Cell Proliferation; Cells; Cessation of life; Chemicals; Chromosomal Stability; Chromosome abnormality; Chromosomes; Collaborations; Cultured Cells; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA biosynthesis; Data; Defect; Depth; Development; Disruption; Dominant-Negative Mutation; Drug Delivery Systems; Eligibility Determination; Family; Fission Yeast; Fluorescence Resonance Energy Transfer; Follow-Up Studies; Foundations; Genes; Genetic; Genome Stability; Genomic Instability; Health; Homologous Gene; Human; Inhibitory Concentration 50; Knock-out; Lead; Left; Length; Libraries; Link; Malignant Neoplasms; Maps; Measures; Mediating; Methods; Mus; Nucleoproteins; Phenotype; Play; Process; Protein Family; Protein Overexpression; Proteins; RNA Interference; RNA Splicing; Range; Reagent; Research; Resolution; Role; S pombe SPAC26H5.06 protein; Screening procedure; Series; Signal Transduction; Standards of Weights and Measures; Structure; Surface; System; Telomerase; Telomere Capping; Telomere Maintenance; Telomere Shortening; Testing; Therapeutic; Time; Transfection; Universities; Validation; Variant; base; cancer cell; chemical genetics; design; follow-up; high throughput screening; in vivo; inhibitor/antagonist; member; novel; programs; research study; small hairpin RNA; small molecule; small molecule libraries; telomere; tool

DESCRIPTION (provided by applicant): The ends of linear chromosomes are unique structures that require special handling by the cell. If left unattended, these ends are inappropriately processed impacting both genomic stability and cellular proliferation. Telomeres, the specialized nucleoprotein structures that cap and protect the ends of chromosomes, restore chromosomal stability and allow continued proliferation. These functions are mediated by the action of the Pot1 (Protection of Telomeres 1) family of proteins, which targets the 3' single-stranded overhang region of the telomere via a specialized single-stranded DNA binding domain (DBD). Pot1 is required for normal cellular proliferation in the model organisms S. pombe and the mouse, playing an integral role in telomere maintenance by providing a capping function as well as regulating the action of telomerase at the telomere. However, our understanding of human Pot1 function in vivo is less clear, primarily due to the caveats associated with RNAi/shRNA and transient transfection approaches. Therefore, a novel method for targeting Pot1 would greatly facilitate mechanistic studies of Pot1 function at the telomere. Association of Pot1 with telomeric ssDNA is required for its function. One unexplored method for studying Pot1 function is a "chemical genetics" approach that uses small molecule inhibitors that target its ssDNA-binding activity. Examination of high-resolution structures bound to ssDNA reveal a protein/DNA interface that is chemically rich and forms a detailed and unique surface, suggesting that a small molecule could specifically disrupt this interaction. We propose to develop and implement a high-throughput screening (HTS) assay to identify small molecules that target the ssDNA-binding activity of Pot1 proteins from S. pombe and humans. In our first specific aim, we will devise and validate a time-resolved FRET-based assay targeting Pot1/DNA binding. This "mix and measure" type assay is highly sensitive, robust, and reproducible, making it ideal for a successful HTS study. In the second specific aim, we will configure the assay for entry into the HTS format by testing two chemically distinct pilot libraries. A strategy for validation and prioritization of hit compounds is presented. Finally, we propose a series of follow up studies with "hit" compounds from the pilot screening to (a) characterize the mode and mechanism of ssDNA-binding inhibition using biochemical and structural tools and (b) study their impact on proper telomere function in vivo. Our proposed research program will be greatly aided through a collaboration with Prof. Charles McHenry, a leader in the field who uses HTS to identify inhibitors of bacterial replication and has the tools and expertise in place to assist with the design and implementation of the screen. Telomeres play key roles in cancer and aging due to their roles in discriminating natural DNA ends from damaged DNA and compensating for the inability of the standard replication machinery to fully copy the chromosomal terminus. As a result of these activities, proper telomere function is integral to human health. Small molecules that specifically target telomeres will provide valuable tools for dissecting the mechanism of telomere function and lay the groundwork for the discovery of cancer therapeutics.

描述（由申请人提供）：线性染色体的末端是需要细胞特殊处理的独特结构。如果不加注意，这些末端会被不适当地加工，影响基因组稳定性和细胞增殖。端粒是一种特殊的核蛋白结构，它覆盖并保护染色体的末端，恢复染色体的稳定性并允许继续增殖。这些功能由Pot 1（端粒1的保护）蛋白家族的作用介导，其通过专门的单链DNA结合结构域（DBD）靶向端粒的3'单链突出区。Pot 1是模式生物S中正常细胞增殖所必需的。pombe和小鼠，通过提供加帽功能以及调节端粒的端粒酶作用，在端粒维持中发挥不可或缺的作用。然而，我们对人类Pot 1在体内功能的理解还不太清楚，主要是由于与RNAi/shRNA和瞬时转染方法相关的警告。因此，靶向Pot 1的新方法将极大地促进Pot 1在端粒功能的机制研究。Pot 1与端粒ssDNA的结合是其功能所必需的。研究Pot 1功能的一种未探索的方法是“化学遗传学”方法，该方法使用靶向其ssDNA结合活性的小分子抑制剂。结合到ssDNA的高分辨率结构的检查揭示了一个蛋白质/DNA界面，该界面化学性质丰富，并形成了一个详细而独特的表面，这表明小分子可以特异性地破坏这种相互作用。我们建议开发和实施高通量筛选（HTS）检测，以确定小分子的目标的ssDNA结合活性的Pot 1蛋白从S。粟酒和人类在我们的第一个具体目标中，我们将设计并验证一种基于时间分辨FRET的检测方法，靶向Pot 1/DNA结合。这种“混合和测量”类型的测定具有高度灵敏度、稳健性和重现性，使其成为成功HTS研究的理想选择。在第二个具体目标中，我们将通过测试两个化学上不同的试点文库来配置检测试剂盒以进入HTS格式。提出了一种用于验证和优先化命中化合物的策略。最后，我们提出了一系列的后续研究与“命中”化合物从试点筛选（a）表征的模式和机制的ssDNA结合抑制使用生化和结构的工具和（B）研究它们的影响适当的端粒功能在体内。我们提出的研究计划将通过与Charles McHenry教授的合作得到极大的帮助，Charles McHenry教授是该领域的领导者，他使用HTS来识别细菌复制的抑制剂，并拥有适当的工具和专业知识来协助设计和实施屏幕。端粒在癌症和衰老中起着关键作用，因为它们在区分天然DNA末端与受损DNA和补偿标准复制机制无法完全复制染色体末端中起作用。作为这些活动的结果，适当的端粒功能是人类健康不可或缺的。特异性靶向端粒的小分子将为剖析端粒功能机制提供有价值的工具，并为发现癌症治疗方法奠定基础。