Study for pyrimidine de novo biosynthetic pathway of Toxoplasma gondii(application for development of anti-protozoan drug)

弓形虫嘧啶从头生物合成途径研究（抗原虫药物开发申请）

• 批准号: 13670255
• 负责人: ASAI Takashi
• 金额: $ 1.86万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670255/
• 关键词: Parasitic protozoa; Toxoplasma gondii; Pyrimidine de novo synthesis; CPS-II

The aim of this study is to find out a primary factor that regulates a flow of de novo pyrimidine biosynthetic pathway in Toxoplasma gondii within term of the project. To perform a study of the project, the cellular contents of nucleic acid precursors, purine and pyrimidine nucleotides, were investigated by examining an analytical method to measure the compounds. The nucleotide concentrations in T.gondii cell were measurable by a high performance liquid chromatography and the sensitivity of the measurement was within an experimental condition. For another approach to the project, a cloning of the gene encoding carbamylphosphate synthetase-II (CPS-II) that is the first step enzyme and the most important enzyme of pyrimidine de novo biosynthetic pathway, was attempted. Consequently, a partial cDNA clone encoding a substrate binding site of CPS-II was obtained, however, full length of CPS-II gene had been reported by a group of Dartmouth University in the United States before our success. … More Anyway, it was clearly identified that there is no PRPP, the most potent activator of CPS-II, binding site in the T.gondii CPS-II from our results and the data from the group of Dartmouth University. We started a collaboration project with the group of Dartmouth University to study an enzyme regulation of T.gondii CPS-II and tried to make a full size of recombinant CPS-II. Unfortunately, the expression of the recombinant CPS-II has not been successful because T.gondii CPS-II is a huge protein. Although an enzymological study has not been performed, the data of full amino acid sequence of CPS-II strongly suggests that the most important factor that contributes to the flow of de novo pyrimidine biosynthetic pathway is a supply of ATP, a substrate of CPS-II. An accurate assay of ATP concentration in T.gondii cell is very hard because of the ATP contamination from host cell, nevertheless, the estimated concentration of ATP in growing tachyzoite cell (〜5 mM) is quite higher than that of dormant cell (〜1 mM). Considering the km value of T.gondii CPS-II for ATP (〜10 mM), almost no enzyme activity is probably present under dormant condition. The results strongly suggest that the supply of energy is the most important factor for the flow of de novo pyrimidine biosynthesis. The aim of this project was almost achieved, though many facts should be further confirmed. Less

本研究的目的是在项目期限内找出调节刚地弓形虫从头合成嘧啶生物合成途径流动的主要因素。为了进行该项目的研究，通过检查测量化合物的分析方法，研究了核酸前体嘌呤和嘧啶核苷酸的细胞含量。采用高效液相色谱法测定了弓形虫细胞中核苷酸的浓度，其灵敏度在一定的实验条件下。对于该项目的另一种方法，试图克隆编码氨基甲酰磷酸合成酶- ii （CPS-II）的基因，这是嘧啶从头合成途径的第一步酶和最重要的酶。因此，我们获得了一个编码CPS-II底物结合位点的部分cDNA克隆，然而，在我们成功之前，美国达特茅斯大学的一个研究小组已经报道了CPS-II基因的全长。无论如何，根据我们的结果和达特茅斯大学研究小组的数据，我们清楚地发现弓形虫的CPS-II中没有最有效的激活剂PRPP结合位点。我们启动了与达特茅斯大学团队的合作项目，研究弓形虫CPS-II的酶调控，并尝试制作全尺寸的重组CPS-II。不幸的是，由于弓形虫CPS-II是一个巨大的蛋白质，重组CPS-II的表达一直没有成功。虽然没有进行酶学研究，但CPS-II的全氨基酸序列数据强烈表明，促进从头合成嘧啶生物合成途径流动的最重要因素是CPS-II的底物ATP的供应。由于寄主细胞对ATP的污染，弓形虫细胞中ATP的浓度很难准确测定，但生长的速殖子细胞（~ 5 mM）中ATP的估计浓度远高于休眠细胞（~ 1 mM）。考虑到弓形虫CPS-II对ATP的km值（~ 10 mM），在休眠状态下可能几乎不存在酶活性。结果表明，能量的供应是新合成嘧啶生物流动的最重要因素。该项目的目的已基本实现，但仍有许多事实有待进一步证实。少

项目成果

Asai T, Takeuchi T, Diffenderfer J, Sibley LD: "Identification of small-molecule inhibitors of nucleoside triphosphate hydrolase in Toxoplasma gondii"Antimicrobial Agents and Chemotherapy. 46(8). 2393-2399 (2002)

Asai T、Takeuchi T、Diffenderfer J、Sibley LD：“弓形虫中核苷三磷酸水解酶小分子抑制剂的鉴定”抗菌剂和化疗。

Saito T, Maeda T, Nakazawa M, Takeuchi T, Nozaki T, Asai T.: "Characterisation of hexokinase in Toxoplasma gondii tacyzoite"Int.J.Parasitol.. 32(8). 961-967 (2002)

Saito T、Maeda T、Nakazawa M、Takeuchi T、Nozaki T、Asai T.：“弓形虫tacyzoite 中己糖激酶的表征”Int.J.Parasitol.. 32(8)。

Saito T, Maeda T, Nakazawa M, Takeuchi T, Nozaki T, Asai T: "Characterization of hexokinase in Toxoplasma gondii"International Journal for Parasitology. (In press). (2002)

Saito T、Maeda T、Nakazawa M、Takeuchi T、Nozaki T、Asai T：“弓形虫中己糖激酶的表征”国际寄生虫学杂志。

Saito T, Maeda T, Nakazawa M, Takeuchi T, Nozaki T, Asai T: "Characterization of hexokinase in Toxoplasma gondii tachyzoite"International Journal for Parasitology. 32. 961-967 (2002)

Saito T、Maeda T、Nakazawa M、Takeuchi T、Nozaki T、Asai T：“弓形虫速殖子中己糖激酶的表征”国际寄生虫学杂志。