A High Throughput Screen for Telomerase Assembly (RMI)

描述（由申请人提供）：端粒酶是一种核糖核蛋白复合物，专门用于合成每条染色体末端3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则不存在。此外，抑制端粒酶活性可抑制培养癌细胞的生长，并阻止人类肿瘤异种移植小鼠模型的生长。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作用被发现。这些作用是独立于DNA合成的，这表明仅端粒酶活性抑制剂不能达到端粒酶靶向治疗的最大潜在效果。本文将介绍一种抑制端粒酶活性的新技术平台。实验的中心假设是，在原位产生的显性负端粒酶复合体将提供一种机制来破坏所有依赖端粒酶的细胞功能。我们通过观察端粒酶活性所需的端粒酶复合物的特定部分的关联，开发了一种检测来跟踪端粒酶的组装。具体目标1描述了使用闪烁接近试验产生高通量筛选的努力，以报告这些基本的大分子相互作用。具体目标2概述了进一步评估命中的次要筛选。这些研究的完成将提供一种新的HTS，它将允许识别特定阻断端粒酶全酶复合物正确组装的化合物。这些化合物将为抗癌药物的发现和阐明端粒酶在介导致瘤性中的生理作用的生物学研究提供线索。

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.