Development of Anti-malarial Drug and it's Molecular Target

该研究项目涉及多个研究领域的科学家，通过开发新的抗疟疾药物，已经为疟疾研究做出了巨大贡献。1)我们正在筛选4635份含有据称具有抗疟疾活性的天然产品、有机化合物、微生物衍生产品、组合生物化合物和海洋产品的样品。结果表明，利用体外测定系统获得了165个对恶性疟原虫具有高选择性抗疟活性（选择性细胞毒性大于100）的化合物。体外和体内研究表明，1,2,6,7-四氧阿斯匹罗[7.11]壬烷是一类新型廉价过氧化物抗疟药的理想原型。2)我们构建了表达p - atp酶（PfATP6在恶性疟原虫中）和鸡ca - atp酶嵌合蛋白的嵌合基因载体CMC，以证明这些功能对疟原虫至关重要。PfATPase在滋养体发育阶段表达。3)我们在恶性疟原虫（pfNSF）中发现了一个NSF同源物，该同源物输出到细胞外空间并定位于红细胞内囊泡。在疟原虫红细胞发育过程中，囊泡转运机制参与了蛋白质向红细胞的输出。4)我们在线粒体呼吸链中分离到了琥珀酸-泛醌还原酶（复合体II）的Ip亚基，这是一个很好的化疗靶点。Western blot和northern分析显示，疟原虫复合体II在滋养体/分殖体晚期表达最高。5)硫代磷酸酯（PS）反义寡核苷酸（AS）已被广泛用作鉴定靶蛋白作用的工具。然而，我们测试了几种psas - odn及其感知odn对恶性疟原虫几种酶的作用，没有明显差异。另一方面，具有磷酸二酯（PO）键的odn对内核酸酶和外核酸酶具有抗性，表明as - odn比它们的感觉odn更有效。利用该ODN，我们发现琥珀酸脱氢酶在恶性疟原虫中具有功能活性，是寻找抗寄生虫药物的靶酶。我们的目标是建立人类疟疾感染的动物模型，以便评估抗疟疾药物的候选药物。我们也在试图了解寄生虫侵入红细胞的分子机制。我们已经利用基因靶向技术构建了破坏Duffy抗原编码基因的突变小鼠。7)基于其他物种的x射线结构，预测了恶性疟原虫DHFR- ts二氢叶酸还原酶（DHFR）结构域的三维结构模型。利用该方法设计对环胍耐药菌株FCR3有效的新型抑制剂。因此，开发了高效、选择性的抑制剂。我们还发现了与已知DHFR抑制剂具有不同骨架结构的新抑制剂。少

The research project involves scientists from numerous research fields and has already contributed greately to the research of malaria by developing new antimalarial agents.1) We are screening 4,635 samples containing natural products, organic compounds, microorganism-derived products, combinatorial bio-compounds and marine products that are alleged to have antimalarial activity. As the results, 165 compounds with high selective antimalarial activity (selective cytotoxicity is over 100) for Plasmodium falciparum were obtained using in vitro assay system. The study of in vitro and in vivo demonstrated that 1,2,6,7-tetraoxaspiro[7.11]nonadecane is the attractive prototype in a new class of inexpensive peroxide antimalarial drugs.2) We have made a chimera-gene vector named CMC expressing a chimeric protein of P-ATPase (PfATP6 in P. falciparum) and chicken Ca-ATPase to demonstrate these functions essential for malaria parasites. PfATPase were expressed at the stage of trophozoites in P. fa … More lciparum.3) We have identified a NSF homologue in Plasmodium falciparum (pfNSF) which is exported to extracellular space and localized with intraerythrocytic vesicles. The vesicular transport mechanism is involved in protein export to erythrocyte during intraerythrocytic development of malaria parasite.4) We have isolated Ip subunit of Plasmodium succinate-ubiquinone reductase (complex II) in themitochondrial respiratory chains which is good targets for chemotherapy. Western blot using antibody against the recombinant Ip and northern analysis clearly showed that expression of parasite complex II is highest at late trophozoite/schizont stages of P. falciparum.5) Phosphorothioate (PS) antisense (AS) oligonucleotides(ODNs) have widely been used as a tool to identify a role of a target protein. However, we tested several PSAS-ODNs and their sense ODNs against several enzymes in P. falciparum without significantly different effects. On the other hands, ODNs having phsphodiester (PO) bond, which are resistant to endo- and exo-nucleases, showed that AS-ODNs are more effective than their sense ODNs. Using this ODN, we found that succinate dehydrogenase was functionally active in P. falciparum and is a target enzyme to search anti-parasitic agents.6) Our goal is to establish animal models for human malaria infection where one can evaluate candidates for anti-malaria drugs. We are also trying to understand molecular mechanisms for the invasion of the parasite into red blood cells. We already constructed mutant mice disrupting the gene encoding Duffy antigen by gene targeting technique.7) A three-dimensional structure model of dihydrofolate reductase(DHFR) domain of P. falciparum DHFR-TS was predicted based on the X-ray structures of other species. It was used to design new inhibitors effective to cycloguanil-resistant FCR3 strain. As result, highly effective and selective inhibitors were developed. We also found new inhibitors with different skeletal stuructures from known DHFR inhibitors. Less