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

空泡型H^-焦磷酸酶抑制剂的研究寻找治疗疟疾的特效药物

• 批准号: 13556013
• 负责人: MAESHIMA Masayoshi
• 金额: $ 9.02万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13556013/
• 关键词: vacuolar membrane; H^+-pyrophosphatase; inhibitor; malaria; proton pump; acylspermidine; soft coral; マラリア原虫; 海洋生物; H^+ピロホスファターゼ; マラリア病原虫

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.

最致命的疟疾是由原生动物恶性疟原虫引起的，它通过按蚊的叮咬传播给人类。防治这一毁灭性疾病的关键挑战是抗疟药物耐药性的蔓延。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药物的良好候选者。在这项研究中，我们从145种海洋生物中制备了水溶性和有机溶剂可溶性组分，并检测了它们对H^+-PPase的影响。我们发现，从软珊瑚中提取的水溶性组分中的一些化合物抑制H^+-PPase。这些都是酰基亚精胺衍生物，它们是N '，N'，N '-三甲基亚精胺，被甲基支化不饱和脂肪酸酰化。酰基亚精胺抑制PPi水解活性和PPi依赖的H^+转运（半抑制浓度，1 μM）。这些化合物实际上抑制了H^+-PPase，并阻止了活原生质体中液泡的酸化。(1)缺乏H^+-PPase基因的突变拟南芥植株非常小。这一观察结果表明H^+-PPase对植物的正常生长是必不可少的。(2)通过对绿豆H^+-PPase进行定点突变，确定了两个参与PPi水解的关键结构域。(3)通过H^+-PPase的PPi依赖性H^+电流的分子特性可以通过膜片钳分析直接确定。(4)为了研究链霉菌H^+-PPase的结构与功能的关系，我们建立了在大肠杆菌中高效表达该酶的实验条件。

项目成果

Ikeda, M.: "Functional expression of Acetabularia acetabulum vacuolar H^+-pyrophosphatase in a yeast VMA3-deficient strain."Journal of Exerimental Botany. 53. 2273-2275 (2002)

Ikeda，M.：“酵母VMA3缺陷菌株中髋臼液泡H 2 -焦磷酸酶的功能表达。”实验植物学杂志。

Fujiwara, S.: "Molecular properties of a unique matrix attachment region-binding protein in tobacco cells."Plant and Cell Physiology. 43. 1558-1567 (2002)

Fujiwara, S.：“烟草细胞中独特的基质附着区域结合蛋白的分子特性。”植物和细胞生理学。

Suga, S., Imagawa, S., Maeshima, M.: "Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs."Planta. 212. 294-304 (2001)

Suga, S.、Imakawa, S.、Maeshima, M.：“萝卜器官中质膜和液泡膜水通道蛋白的 mRNA 和蛋白质积累的特异性。”Planta。

Nakanishi, Y., Saijo, T., Wada, Y., Maeshima, M.: "Mutagenic analysis of functional residues in putative substrate binding site and acidic regions of vacuolar H^+-pyrophosphatase."J.Biol.Chem.. 276. 7654-7660 (2001)

Nakanishi, Y.、Saijo, T.、Wada, Y.、Maeshima, M.：“空泡 H2-焦磷酸酶的推定底物结合位点和酸性区域中功能残基的诱变分析。”J.Biol.Chem.. 276

Kamiya, T., Maeshima, M.: "Residues in internal repeats of the rice cation/H^+ exchanger are involved in the transport and selection of cations."J.Biol.Chem.. 279. 812-819 (2004)

Kamiya, T., Maeshima, M.：“水稻阳离子/H^交换器内部重复序列中的残基参与阳离子的运输和选择。”J.Biol.Chem.. 279. 812-819 (2004)