Development of High-Throughput screening assays for identification of small molecule inhibitors of the Mcm2-7 replicative helicase

项目摘要 该提案的长期目标是鉴定新型药物靶点Mcm 2 -7的小分子抑制剂， 并将这些抑制剂开发成实验探针和人类治疗剂。DNA复制 利用许多类似酶学的蛋白质;然而，很少有实验性抑制剂可用于帮助 区分其功能。此外，尽管传统上使用该过程的抑制剂， 大多数此类化疗药物的作用是非特异性的，并且经常引起显著的DNA损伤。 对正常细胞和癌细胞都有影响与这两个问题相关，我们的实验室研究了一个重要的复制 蛋白，Mcm 2 -7复制解旋酶，目前没有可用的抑制剂。我们最近 研究确定了Mcm 2 -7和其他复制因子之间的各种新的遗传相互作用， 一种鉴定信息性抑制剂以及特异性靶向异常DNA复制的方法。因为我们使用 芽殖酵母，我们建议首先在这种生物体中开发筛选方法， 向人类细胞系统过渡。以下目标将与匹兹堡会议一起实现： Drug Discovery Institute拥有完善的检测设计和文库筛选资源： 1)我们建议开发高通量的基于细胞的测定来鉴定抑制以下任一种的测试化合物： 提出了两种基本的芽殖酵母筛选方法：一种是基于 针对查询菌株中特定Mcm突变的合成致死性，以及第二个鉴定 抑制过表达Mcm 2 -7的菌株的致死性的分子。成功发展 这两种分析将在小化学文库的试验性筛选中达到高潮， 验证，将产生少量的候选化合物进行额外的筛选。 2)我们提出了二次筛选，以确定抑制剂的目标和它们的抑制机制。鉴于 为了便于使用芽殖酵母，将开发几种遗传筛选来确定芽殖酵母的分子靶点。 抑制剂的此外，我们计划对其中一些进行额外的生化和遗传分析， 我们实验室以前开发的检测方法，以确定其抑制机制。 3)目标1中提出的筛选方法依赖于非常特定的芽殖酵母突变。作为 DNA复制在人类细胞中的研究要少得多，我们建议测试我们的上述方法是否会 工作，并因此建立人类细胞查询菌株为未来的筛选目的。

Project Summary The long-term goal of this proposal is to identify small molecule inhibitors of a novel drug target, Mcm2-7, and develop these inhibitors into both experimental probes and human therapeutic agents. DNA replication utilizes many proteins of similar enzymology; however, few experimental inhibitors are available to help distinguish their function. In addition, although inhibitors of this process have traditionally been used against cancer, most such chemotherapeutics act non-specifically, and often cause significant DNA damage to both normal and cancerous cells. Relevant to both issues, our laboratory studies an essential replication protein, the Mcm2-7 replicative helicase, for which no useful inhibitors are currently available. Our recent studies identify various novel genetic interactions between Mcm2-7 and other replication factors, providing a means to identify informative inhibitors as well as specifically target abnormal DNA replication. As we use budding yeast, we propose to first develop screening approaches in this organism while working to transition to a human cell system. The following aims will be pursued in conjunction with the Pittsburgh Drug Discovery Institute, which has well-developed resources for assay design and library screening: 1) We propose to develop high-throughput cell-based assays to identify test compounds that inhibit either Mcm2-7 or proteins that interact with it. Two basic budding yeast screens are proposed: one based upon synthetic lethality against a specific Mcm mutation in the query strain, and a second that identifies molecules that suppress the lethality of a strain that over-expresses Mcm2-7. Successful development of these two assays will culminate in pilot screening of a small chemical library, which, with additional validation, will generate a small number of candidate compounds for additional screening. 2) We propose secondary screens to identify inhibitor targets and their mechanism of inhibition. Given the ease of use of budding yeast, several genetic screens will be developed to define the molecular target of the inhibitors. In addition, we plan to subject a number of these hits to additional biochemical and genetic assays previously developed in our lab to determine their mechanism of inhibition. 3) The screening approaches proposed in Aim 1 depend upon very specific budding yeast mutations. As DNA replication is much less well studied in human cells, we propose to test if our above approaches will work and it so to build human cell query strains for future screening purposes.