High Throughput Screens for Malaria Topoisomerases

疟疾拓扑异构酶的高通量筛选

• 批准号: 8217269
• 负责人: PRADIPSINH K. RATHOD
• 金额: $ 38.1万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8217269
• 关键词: Antimalarials; Applications Grants; Area; Asia; Basic Science; Binding; Biochemical; Biological Assay; Cells; Cessation of life; Chloroplasts; DNA Gyrase; DNA Ligation; Development; Drug resistance; Dyes; Effectiveness; Enzymes; Escherichia coli; Family; Fluorescence; Genetic Transcription; Genome; Germ; Grant; Housing; Human; In Vitro; Infectious Agent; Institutes; Intervention; Laboratories; Malaria; Mission; Nuclear; Parasites; Pharmaceutical Preparations; Plasmodium; Plasmodium falciparum; Plasmodium vivax; Plastids; Preclinical Drug Evaluation; Proteins; Resistance; Screening procedure; Staging; Superhelical DNA; System; Testing; Topoisomerase; Topoisomerase II; Topoisomerase Inhibitors; Toxic effect; Translational Research; Translations; United States National Institutes of Health; Wheat; Writing; antimicrobial; base; chemotherapy; high throughput screening; innovation; miniaturize; mitochondrial genome; pathogen; prevent; public health relevance; response; small molecule; small molecule libraries; therapeutic development; tool

DESCRIPTION (provided by applicant): Malaria parasites of the genus Plasmodium are responsible for 300-500 million cases of human malaria and cause about one million deaths every year. Resistance to all traditional antimalarial drugs, and early signs of weakening effectiveness of artimesinin-based drugs in SE Asia, further emphasizes a need for basic and translational research to identify new interventions to treat and to avoid malaria. The P. falciparum and the P. vivax parasite are single cellular pathogens with multiple compartmentalized genomes: The nuclear genome, the mitochondrial genome, and the apicolplast genome. The last is a chloroplast-like relict plastid that is essential for parasite survival. The survival and development of the parasite, in all its stages, is dependent on continual management of smooth unwinding, replication, and ligations of DNA molecules. These functions rely on topoismerase enzymes, which are known to be important targets of antimicrobial chemotherapy. The development of malaria topoisomerase inhibitors has been hampered by an inability to express the proteins in functional form. The Rathod laboratory has argued and shown that toxicity of malaria proteins in functional form can limit the ability of traditional heterologous expression systems, such as E. coli, to support expression of functional protein. We have used a cell-free wheat germ translation system to successfully express a number of malaria proteins in functional for, including the first P. falciparum topoisomerase II. In this project application, we will express all P. falciparum and P. vivax candidate topoisomerase and DNA gyrase gene products, test their catalytic activity, purify them to homogeneity, and develop miniaturized efficient assays for High Throughput Screening. These studies will set the stage for direct screening of chemical libraries to ultimately yield small molecules to prevent and to treat malaria. PUBLIC HEALTH RELEVANCE: Malaria parasites infect over 500 million people and are responsible for over 1 million deaths per year, often due to drug resistant parasites. The search for new antimalarials will be greatly aided by a High Throughput Assay (HTA) for Plasmodium topoisomerases, since such enzymes are excellent targets for other infectious agents.

描述（由申请人提供）：疟原虫属的疟疾寄生虫每年导致3 -5亿例人类疟疾病例，并造成约100万人死亡。东南亚对所有传统抗疟药物的耐药性以及青蒿素类药物有效性减弱的早期迹象进一步强调需要开展基础研究和转化研究，以确定治疗和避免疟疾的新干预措施。恶性疟原虫和间日疟原虫是具有多个区隔化基因组的单细胞病原体：核基因组、线粒体基因组和顶质体基因组。最后一个是叶绿体样的残质体，对寄生虫的生存至关重要。寄生虫的生存和发展，在其所有阶段，都依赖于DNA分子的顺利展开、复制和连接的持续管理。这些功能依赖于拓扑异构酶，而拓扑异构酶是抗微生物化疗的重要靶点。疟疾拓扑异构酶抑制剂的开发一直受到无法以功能形式表达蛋白质的阻碍。Rathod实验室认为并证明了功能形式的疟疾蛋白的毒性可以限制传统的异源表达系统（如大肠杆菌）支持功能蛋白表达的能力。我们已经使用无细胞小麦胚芽翻译系统成功表达了一些具有功能的疟疾蛋白，包括第一个恶性疟原虫拓扑异构酶II。在本项目申请中，我们将表达所有恶性疟原虫和间日疟原虫候选拓扑异构酶和DNA旋切酶基因产物，测试它们的催化活性，纯化到均匀性，并开发小型化的高效检测方法用于高通量筛选。这些研究将为直接筛选化学文库奠定基础，最终产生用于预防和治疗疟疾的小分子。