Development of Novel Topoisomerase and Replication Initiator Assays

新型拓扑异构酶和复制引发剂检测的开发

• 批准号: 8010546
• 负责人: JAMES M BERGER
• 金额: $ 37.68万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010546
• 关键词: ATP Hydrolysis; Acute; Adverse effects; Agonist; Antibiotic Therapy; Antibiotics; Antiviral Agents; Attention; Bacteria; Bacterial Proteins; Basic Science; Benchmarking; Biochemical; Biochemical Reaction; Biochemistry; Biological Assay; Biological Process; Cell Proliferation; Cell Survival; Cells; Chemicals; Chemistry; Chromosomes; Chronic; Clinical; DNA; DNA biosynthesis; Development; Dimerization; Disease; Drug resistance; Engineering; Enzymes; Event; Fluorescence; Fluoroquinolones; Gene Expression; Genomics; Goals; Health; Human; In Vitro; Infection; Lead; Life; Ligands; Measurement; Methods; Molecular Probes; Monitor; NIH Program Announcements; Pathway interactions; Pharmaceutical Preparations; Play; Process; Property; Proteins; Radiolabeled; Reaction; Reader; Replication Initiation; Replication Origin; Reporter; Reporting; Research Personnel; Resistance; Role; Route; Screening procedure; Series; Staging; Structure; Superhelical DNA; System; Therapeutic; Time; Topoisomerase; Topoisomerase II; Work; bacterial resistance; base; catalyst; cell growth; cost; cytotoxic; design; enzyme activity; falls; gel electrophoresis; high throughput screening; in vivo; inhibitor/antagonist; insight; interest; meetings; melting; novel; portability; programs; protein function; public health relevance; radiotracer; self assembly; small molecule; small molecule libraries; tool; tumorigenic

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.

描述（由申请人提供）：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蛋白质的研究，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。测定还包括高通量筛选破坏或调节功能的小分子试剂的关键组成部分。从这些努力中分离或重新设计的化合物允许研究人员在体外和体内分析酶的作用，并为疾病的治疗提供必要的治疗方法。为了作为高通量筛选的报告者，测定必须满足严格的技术要求。无法满足这些要求-例如，低成本、高灵敏度和可靠性、易于使用和可移植到96孔或更高的格式可能显著阻碍小分子激动剂和拮抗剂的鉴定。如果不使用凝胶电泳或放射性标记等经典方法难以表征靶酶催化的反应类型，则该问题尤其严重。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目标。DNA复制是细胞增殖中的一个决定性事件，长期以来一直吸引着基础研究部门的注意力。复制因子也已经作为具有有益的抗生素、抗病毒或抗肿瘤特性的抑制剂的有价值的靶标。不幸的是，这些药物的耐药性和/或毒副作用要求发现新的药物，同样迫切需要开发新的复制酶的小分子效应物以探索分子机制。这些试剂的鉴定需要通过高通量方法针对适当的报告基因测定筛选化学文库。该应用程序的目标是与计划公告PA-07-320协同开发，利用我的实验室和该领域最近的结构和概念突破，设计和基准测试一套新的高通量测定两种基本的细菌酶：II型拓扑异构酶和复制引发剂DnaA。我们希望这项工作不仅为研究这些蛋白质提供急需的新工具，而且还将为小分子筛选铺平道路，以确定用于生物化学功能的新探针和用于药物发现工作的新线索。 公共卫生相关性：鉴定用于治疗疾病的药物的途径需要专门的生化测定，其报告特定的酶活性及其被临床药剂抑制。这项工作将开发几种新的检测方法，监测支持DNA复制和细胞生长的两种基本细菌蛋白质的关键反应。这些检测将作为高通量筛选项目的宝贵工具，旨在确定用于治疗急性和慢性感染的新抗生素。