Synthetic and Analytical Methods Targeting Telomerase

描述（由申请人提供）：本申请的主要长期目标是建立一种抑制端粒酶的新策略。端粒酶是一种很有前途的通用抗癌靶点。它的活性在大多数癌细胞和不朽细胞系中发现，而在大多数健康体细胞中不存在。除了这种相关性之外，癌细胞需要端粒酶还有很强的机制原因。该项目的中心假设是，能够结合端粒酶催化循环过程中形成的RNA/DNA双链的分子将作为端粒酶的抑制剂，因此将成为潜在的抗癌疗法。这些分子抑制端粒酶的假设机制要么是通过阻止链解离（端粒酶催化循环的关键步骤），要么是通过双相底物的扭曲，导致催化效果差。我们的初步研究表明，已知的RNA/DNA双链结合分子能够抑制端粒酶，并且这种抑制作用与RNA/DNA双链相互作用的方式一致。这项工作的具体目的是：1)评估已知的双相结合分子，如插入物作为端粒酶抑制剂的活性。该评估将包括对抑制模式的深入动力学分析，以明确表征化合物的作用机制2)使用最成功的化合物作为使用组合化学合成的各种化合物文库的基础。这样做的目的是在分子中引入新的部分，然后引入与周围独特的端粒酶表面的特定相互作用。3)开发高通量分析，包括亲和力方法和酶分析，我们将使用这些方法从组合文库中鉴定对端粒酶具有最高亲和力的分子。这些技术将允许快速评估大量化合物，允许从大量混合物中分离出一小组高亲和力化合物。结合这三个目标，将允许验证端粒酶抑制的新方法和开发高特异性，高亲和力的铅抑制剂。

DESCRIPTION (provided by applicant): The major long term objective of this application is to establish a new strategy to inhibit the enzyme telomerase. Telomerase is a promising universal anti-cancer target. Its activity is found in a majority of cancer cells and immortal cell lines, while being absent from a majority of healthy somatic cells. In addition to this correlation there are strong mechanistic reasons why cancer cells require telomerase. The central hypothesis of this project is that molecules which can bind the RNA/DNA duplex formed during telomerase's catalytic cycle will act as inhibitors of the enzyme, and will therefore be potential anti-cancer therapeutics. The postulated mechanism by which these molecules will inhibit the enzyme is either through the prevention of strand dissociation, a key step in telomerase's catalytic cycle, or by the distortion of the duplex substrate, leading to poor catalysis. Our preliminary studies have demonstrated that known RNA/DNA duplex binding molecules are able to inhibit telomerase, and do so in a manner consistent with this inhibition being due to interaction with the RNA/DNA duplex. The specific aims of this work are: 1) Assess known duplex binding molecules such as intercalators for activity as telomerase inhibitors. This assessment will include in-depth kinetic analysis of the mode of inhibition to definitively characterize the mechanism of action of the compounds 2) Use the most successful compounds as the basis of diverse libraries of compounds synthesized using combinatorial chemistry. The purpose of this is to introduce new moieties into the molecule, which will then introduce specific interactions with the unique surrounding telomerase surfaces. 3) Develop high throughput assays, including affinity methods and enzyme assays, which we will use to identify molecules from the combinatorial libraries with the highest affinity for telomerase. These techniques will allow the rapid assessment of large numbers of compounds, allowing a small group of high affinity compounds to be isolated from a large mixture. Combined, these three aims will allow the validation of a new approach for telomerase inhibition and the development of high specificity, high affinity lead inhibitors.