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

抗疟药物及其分子靶点的开发

• 批准号: 08281105
• 负责人: WATAYA Yusuke
• 金额: $ 124.16万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08281105/
• 关键词: Malaria; Anti-malaria drug; Screening; antisense; tropical fever; protozoa; Plasmodium falciparum; target molecule; ミトコンドリア; ATPase; マラリア治療薬; スクリーニンヅ; 熱帯熱マラリア; インターネット

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

该研究项目涉及多个研究领域的科学家，并已通过开发新的抗疟疾药物为疟疾研究作出了巨大贡献。1)我们正在筛选4635个样本，其中包含据称具有抗疟疾活性的天然产物、有机化合物、微生物衍生产品、组合生物化合物和海洋产品。结果表明，利用体外实验系统，共获得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，1，2，6，7-四氧螺[7.11]正十一烷是一类新型过氧化氢抗疟疾药物的诱人原型。2)我们构建了表达P-ATPase(恶性疟原虫PfATP6)和鸡Ca-ATPase嵌合蛋白的嵌合基因载体CMC，以证明这些功能对疟疾寄生虫是必要的。PfATPase在P.fa…滋养体阶段表达3)我们已经在恶性疟原虫(PfNSF)中发现了一种NSF同源物，它被输出到细胞外空间并定位于红细胞内的小泡。囊泡转运机制参与了疟疾原虫在红细胞内发育过程中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Ip亚基，这是很好的化疗靶点。免疫印迹和Northern分析清楚地表明，在恶性疟原虫滋养体/裂殖体晚期，寄生虫复合体II的表达最高。5)硫代硫酸盐(PS)反义寡核苷酸(ODN)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PSA-ODN及其正义ODN对恶性疟原虫几种酶的作用，但没有明显的差异。另一方面，具有磷二酸酯(PO)键的ODN对内切和外切核酸酶都具有抗性，表明AS-ODN比它们的正义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。6)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。我们还试图了解这种寄生虫入侵红细胞的分子机制。我们已经通过基因打靶技术构建了编码Duffy抗原基因的突变小鼠。7)根据其他物种的X射线结构，预测了恶性疟原虫二氢叶酸还原酶(DHFR-TS)结构域的三维结构模型。将其用于设计对环鸟苷耐药的FCR3菌株有效的新型抑制剂。因此，高效、选择性的抑制剂应运而生。我们还发现了与已知的DHFR抑制剂具有不同骨架结构的新抑制剂。较少

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