Development of Novel Topoisomerase and Replication Initiator Assays

描述(申请人提供)：酶活性的可靠测量是生物化学的基石。生化分析几乎用于所有的蛋白质研究，以回答有关蛋白质/配体相互作用、催化机制和生物过程控制的基本问题。分析还包括高通量筛选的关键组成部分，用于干扰或调节功能的小分子制剂。从这些努力中分离或重新设计的化合物使研究人员能够分析酶在体外和体内的作用，并为疾病的治疗提供必要的治疗药物。作为高通量筛查的记者，化验必须满足严格的技术要求。不能满足这些要求--例如低成本、高灵敏度和可靠性、易用性以及可移植到96孔或更高格式--可能会严重阻碍小分子激动剂和拮抗剂的识别。如果不使用凝胶电泳或放射性标记等经典方法，目标酶催化的反应类型很难表征，这个问题就特别严重。许多负责复制和包装基因组信息的蛋白质都属于这类具有挑战性的靶标。DNA复制是细胞增殖中的一个决定性事件，长期以来一直受到基础研究部门的关注。复制因子也是具有有益的抗生素、抗病毒或抗肿瘤特性的抑制剂的有价值的靶点。不幸的是，对这些药物的耐药性和/或毒副作用需要找到新药。同样迫切需要开发新的复制酶小分子效应器来探索分子机制。这类试剂的鉴定需要通过高通量方法根据适当的报告分析来筛选化学文库。这项与项目公告PA-07-320协同开发的应用程序的目标是利用我的实验室和该领域最近的结构和概念突破，设计一套新的高通量分析方法，用于两种基本细菌酶：II型拓扑异构酶和复制启动子DNAA。我们预计，这一努力不仅将为研究这些蛋白质提供急需的新工具，还将为小分子筛选寻找生化功能的新探针和抗生素发现工作的新线索铺平道路。 公共卫生相关性：确定治疗疾病的药物的途径需要专门的生化分析，报告特定的酶活性及其被临床药物抑制的情况。这项工作将开发几种新的分析方法，监测两种基本细菌蛋白进行的关键反应，以支持DNA复制和细胞生长。这些化验将成为高通量筛查项目的宝贵工具，旨在确定治疗急性和慢性感染的新抗生素。

DESCRIPTION (provided by applicant): The reliable measurement of enzyme activity is a cornerstone of biochemistry. Biochemical assays are used in nearly all studies of proteins to answer fundamental questions about protein/ligand interactions, catalytic mechanism, and the control of biological processes. Assays also comprise a critical component of high-throughput screens for small molecule agents that disrupt or modulate function. Compounds isolated or re-engineered from such efforts allow researchers to analyze enzyme action in vitro and in vivo, and provide essential therapeutics for the treatment of disease. To serve as a reporter for high-throughput screens, an assay must satisfy stringent technical demands. An inability to meet these requirements - e.g., low cost, high sensitivity and reliability, ease of use, and portability to 96-well or higher formats - can significantly impede the identification of small molecule agonists and antagonists. This problem is particularly acute if the type of reaction catalyzed by a target enzyme is difficult to characterize without the use of classic methods such as gel electrophoresis or radiolabeling. Many proteins responsible for copying and packaging genomic information fall into this class of challenging targets. DNA replication is a defining event in cell proliferation that has long attracted the attention of the basic research sector. Replication factors also have served as valuable targets for inhibitors with beneficial antibiotic, antiviral, or anti-tumorigenic properties. Unfortunately, resistance to and/or toxic side effects from these agents require that new drugs be found. There is likewise a pressing need to develop new small molecule effectors of replication enzymes for probing molecular mechanism. The identification of such agents requires screening chemical libraries against appropriate reporter assays via high-throughput approaches. The goal of this application, developed in synergy with program announcement PA-07-320, is to take advantage of recent structural and conceptual breakthroughs from my lab and the field to design and benchmark a suite of new high-throughput assays for two essential bacterial enzymes: type II topoisomerases and the replication initiator, DnaA. We expect that this effort will not only provide much-needed new tools for studying these proteins, but also will pave the way for small molecule screens to identify new probes for biochemical function and novel leads for antibiotic-discovery effort. PUBLIC HEALTH RELEVANCE: The pathway to identifying drugs for the treatment of disease requires specialized biochemical assays that report on specific enzymatic activities and their inhibition by clinical agents. This effort will develop several new assays that monitor key reactions carried out by two essential bacterial proteins in support of DNA replication and cell growth. These assays will serve as valuable tools for high-throughput screening programs aimed at identifying new antibiotics for the treatment of acute and chronic infections.