Study on inhibitors of vacuolar-type H^+-pyrophosphatase to search specific medicines for malaria disease

最致命的疟疾是由原生动物恶性疟原虫引起的，它通过蚊子的叮咬传播给人类。抗击这一毁灭性疾病的关键挑战是抗疟疾药物耐药性的传播。我们注意到一个事实，即H^+-焦磷酸酶(H^+-PPase)存在于这些原生动物中，而不存在于动物细胞中。因此，H^+-PPase抑制剂有望成为抗疟疾药物的候选药物。在本研究中，我们从145种海洋生物中制备了水溶组分和有机溶剂可溶组分，并研究了它们对H~+-PPase的影响。我们发现，从软珊瑚中提取的水溶性部分中，有几种化合物对H^+-PPase有抑制作用。这些是酰基亚精胺衍生物，它们是N‘，N“，N”-三甲基亚精胺，它们被甲基支链不饱和脂肪酸酰化。酰基亚精胺抑制PPI水解酶活性和依赖于PPI的H~+转运(半数抑制浓度，1μM)。我们还获得了以下关于H^+-PPase的新信息：(1)缺失H+-PPase基因的突变体植株非常小。这表明H^+-PPase是植物正常生长所必需的。(2)通过绿豆H~+-PPase的定点突变，确定了参与PPI水解的两个必需结构域。(3)通过膜片钳分析直接确定了依赖于PPI的H~+-PPase的分子性质。(4)建立了链霉菌H~+-PPase在大肠杆菌中高效表达的实验条件，研究了该酶的结构与功能的关系。

The most virulent form of malaria is caused by the protozoan, Plasmodiumfalciparum, which is transmitted to humans by the bite of Anopheles mosquito. Key challenges to combating this devastating disease are the spread of resistance to antimalarial drugs. We pay attention to a fact that H^+-pyrophosphatase (H^+-PPase) exists in these protozoa but not in animal cells. Thus, inhibitors of H^+-PPase are expected as a good candidate of antimalarial drug. During this study, we prepared water-soluble and organic-solvent soluble fractions from 145 marine organisms, and examined their effects on H^+-PPase. We found that a few compounds in the water-soluble fraction prepared from a soft coral inhibited H^+-PPase. These are acylspermidine derivatives, which are N',N",N"-trimethylspermidines athat are acylated by a methylbranched unsaturated fatty acid. Acylspermidines inhibited PPi hydrolysis activity and PPi-dependent H^+ translocation (half inhibition concentrations, 1 μM). These compounds actually inhibited H^+-PPase and prevent acidification of vacuoles in living protoplasts.We also obtained the following new information on H^+-PPase.(1) Mutant Arabidopsis plants that lack the H^+-PPase gene were extremely small. This observation indicates that H^+-PPase is essential for normal growth of plants.(2) Two essential domains, which are involved in PPi hydrolysis, were determined by site-directed mutagenesis of mung bean H^+-PPase.(3) Molecular properties of the PPi-dependent H^+ current through H^+-PPase were directly determined by patch clamp analysis.(4) We established experimental conditions for expressing Streptomyces H^+-PPase in E.coli cells with a high efficiency to investigate the structure-function relationship of the enzyme.