A High Throughput Screen for Telomerase Assembly (RMI)

描述(申请人提供)：端粒酶是一种核糖核蛋白复合体，专门用于合成位于每条染色体末端的3条链上的DNA。端粒酶活性在大多数癌细胞中升高，而在大多数未患病组织中缺失。此外，抑制端粒酶活性可以抑制培养的癌细胞的生长，并防止在小鼠模型中移植的人类肿瘤的生长。因此，端粒酶抑制剂具有普遍适用于抗癌治疗的潜力，并且端粒酶抑制作为抗癌方法的潜力已被几种方法验证。最近，端粒酶在促进肿瘤发生和抑制细胞凋亡方面的更多作用已被发现。这些作用是独立于DNA合成的，这表明仅抑制端粒酶活性的药物不能达到端粒酶靶向治疗的最大潜在效果。在这项提案中，将描述一种抑制端粒酶活性的新技术平台。实验的中心假设是，原位产生显性的负端粒酶复合体将提供一种机制，扰乱所有端粒酶依赖的细胞功能。我们已经开发了一种通过观察端粒酶复合体中端粒酶活性所需的特定部分的关联来跟踪端粒酶组装的方法。具体目标1描述了使用闪烁邻近分析产生高通量筛查以报告这些基本的大分子相互作用的努力。《特定目标2》概述了一个辅助屏幕，以进一步评估热门歌曲。这些研究的完成将提供一种新型的HTS，它将使特定阻止端粒酶全酶复合体正确组装的化合物得以鉴定。这些化合物将作为抗癌药物发现和生物学研究的线索，阐明端粒酶在介导肿瘤发生中的生理作用。

DESCRIPTION (provided by applicant): Telomerase is a ribonucleoprotein complex that is specialized for the synthesis of the DNA found on the 3 strand at the end of each chromosome. Telomerase activity is elevated in most cancerous cells while it is absent in most undiseased tissues. Furthermore, inhibiting telomerase activity inhibits growth of cultured cancer cells and prevents growth of human tumors xenografted in mouse models. Thus, telomerase inhibitors have the potential of universal applicability as anticancer therapeutics, and the potential of telomerase inhibition as an anticancer approach has been validated by several approaches. Recently, additional roles for telomerase in promoting tumorigenicity and inhibiting apoptosis have been identified. These roles are independent of DNA synthesis, suggesting that inhibitors of only telomerase enzymatic activity will not achieve the maximum potential effect of telomerase-targeted therapeutics. In this proposal, a new technology platform for inhibiting telomerase activity will be described. The central hypothesis for the experiments is that the in situ generation of a dominant negative telomerase complex will provide a mechanism to disrupt all telomerase-dependent cellular functions. We have developed an assay to follow assemblage of telomerase by observing the association of specific parts of the telomerase complex that are required for telomerase activity. Specific Aim 1 describes efforts to generate a high throughput screen using scintillation proximity assay to report on these essential macromolecular interaction. Specific Aim 2 outlines a secondary screen to further evaluate hits. The completion of these studies will provide a novel HTS that will allow compounds that specific block proper assemblage of the telomerase holoenzyme complex to be identified. Such compounds will serve as leads for anticancer drug discovery and biological studies that illuminate the physiological role of telomerase in mediating tumorigenicity.