Development of a HTS Assay Targeting End Protection of Telomeres

描述（由申请人提供）：线性染色体的末端是独特的结构，需要细胞进行特殊处理。如果不加注意，这些末端被不恰当地加工，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持续的增殖。这些功能是由Pot1（端粒保护1）蛋白家族的作用介导的，它通过一个特殊的单链DNA结合域（DBD）靶向端粒的3'单链悬空区。Pot1是模式生物S. pombe和小鼠正常细胞增殖所必需的，通过提供封顶功能和调节端粒酶在端粒上的作用，在端粒维持中起着不可或缺的作用。然而，我们对人类体内Pot1功能的了解尚不清楚，主要是由于与RNAi/shRNA和瞬时转染方法相关的警告。因此，一种新的靶向Pot1的方法将极大地促进Pot1在端粒功能的机制研究。Pot1与端粒ssDNA的关联是其功能所必需的。一种尚未开发的研究Pot1功能的方法是“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。对与ssDNA结合的高分辨率结构的检查显示，蛋白质/DNA界面具有丰富的化学成分，并形成了一个详细而独特的表面，这表明一个小分子可以特异性地破坏这种相互作用。我们建议开发并实施一种高通量筛选（HTS）试验，以鉴定来自S. pombe和人类的Pot1蛋白的ssdna结合活性的小分子。在我们的第一个具体目标中，我们将设计并验证一种针对Pot1/DNA结合的时间分辨的基于fret的检测方法。这种“混合和测量”型分析具有高灵敏度、鲁棒性和可重复性，使其成为成功的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先导文库来配置进入HTS格式的分析。提出了一种对命中化合物进行验证和排序的策略。最后，我们提出了一系列针对“命中”化合物的后续研究，从中试筛选开始(a)利用生化和结构工具表征ssdna结合抑制的模式和机制，以及(b)研究它们对体内适当端粒功能的影响。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筛选。端粒在癌症和衰老中发挥着关键作用，因为它们可以区分自然DNA末端和受损DNA，并补偿标准复制机制无法完全复制染色体末端的缺陷。由于这些活动，适当的端粒功能对人类健康是不可或缺的。专门针对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。

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.