High Throughput Screens for Malaria Topoisomerases

描述(申请人提供)：疟原虫属的疟疾寄生虫每年造成3亿-5亿人疟疾病例，并导致约100万人死亡。东南亚对所有传统抗疟疾药物的抗药性以及以青蒿素为基础的药物效力减弱的早期迹象，进一步强调需要进行基础和转化性研究，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单细胞病原体，具有多个区块化的基因组：核基因组、线粒体基因组和顶体基因组。最后一种是残存的叶绿体，它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，都依赖于对DNA分子的顺畅解离、复制和连接的持续管理。这些功能依赖于拓扑异构酶，而拓扑异构酶被认为是抗菌化疗的重要靶点。由于不能以功能性形式表达蛋白质，疟疾拓扑异构酶抑制剂的开发受到了阻碍。Rathod实验室论证并表明，疟疾蛋白功能形式的毒性可以限制传统的异源表达系统，如大肠杆菌，支持功能蛋白的表达能力。我们利用一个无细胞的麦胚翻译系统成功地表达了包括第一个恶性疟原虫拓扑异构酶II在内的许多疟疾蛋白。在本项目的应用中，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旋转酶基因产物，测试它们的催化活性，纯化它们到同源性，并建立小型化、高效的高通量筛选方法。这些研究将为直接筛选化学库，最终产生预防和治疗疟疾的小分子奠定基础。

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.