PURINES & PURINE ANTIMETABOLITES IN MALARIA

嘌呤

• 批准号: 7724080
• 负责人: Vern L. Schramm
• 金额: $ 2.48万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724080
• 关键词: Adenine; Adenosine; Antimetabolites; Blood; Carbon; Computer Retrieval of Information on Scientific Projects Database; Culture Media; DNA; Enzymes; Erythrocytes; Funding; Glycine; Grant; Guanosine; Human; Hypoxanthine; Hypoxanthines; Inosine; Institution; Label; Malaria; Parasites; Pathway interactions; Polyamines; Precipitation; Purine Nucleoside Phosphorylase Inhibitor; Purine-Nucleoside Phosphorylase; Purines; RNA; Research; Research Personnel; Resources; Sampling; Source; Time; United States National Institutes of Health; Xanthines; adenosine deaminase; analog; feeding; killings; purine; purine metabolism; research study; xanthine

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project Description Malaria parasites are purine auxotrophs, but grow inside human red blood cells where the concentration of purines is hundreds to thousands of time greater than the amount taken up by the parasites. We therefore need a specific and sensitive way to establish the pathways by which precursors from the blood (or culture medium) are incorporated into the parasites. We are using 14C precursors to label the purine pool in parasites growing in human erythrocytes. The purine precursors include inosine, adenosine, guanosine, 5'-methylthioadenosine, hypoxanthine, adenine, xanthine, glycine, and a newly discovered metabolite of purine metabolism in P. falciparum, 5'-methylthioinosine. These RNA and DNA precursors are fed to cultures at levels appropriate for AMS and the RNA and DNA from the parasites isolated by extraction or precipitation. Samples from these experiments are converted into carbon for AMS analysis. Immucillins, powerful inhibitors of purine nucleoside phosphorylase (PNP) are added to establish which precursors flow through this enzyme to be incorporated in RNA and DNA. Recently we found that the malarial PNP is unique in participating in the salvage of inosine, guanosine and 5'-methylthioinosine, a metabolite that arises from the polyamine pathway in P. falciparum, but not its human host. 5'-methylthioinosine arises specifically in the parasite by the action of P. falciparum adenosine deaminase on 5'-methylthioinosine. This provides an adenine salvage function. Our current hypothesis is that parasite PNP and ADA function in two purine salvage cycles. Blocking either enzyme is productive in killing parasites in the absence of added hypoxanthine. We have synthesized powerful transition state analogues for three enzymes, all of which are in the essential purine salvage of P. falciparum PNP.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 项目描述 疟疾寄生虫是嘌呤营养缺陷型动物，但在人类红细胞内生长，其中嘌呤浓度比寄生虫吸收的量高数百至数千倍。因此，我们需要一种特定且敏感的方法来建立将血液（或培养基）中的前体掺入寄生虫的途径。 我们正在使用 14C 前体来标记人类红细胞中生长的寄生虫中的嘌呤池。 嘌呤前体包括肌苷、腺苷、鸟苷、5'-甲硫腺苷、次黄嘌呤、腺嘌呤、黄嘌呤、甘氨酸以及恶性疟原虫中新发现的嘌呤代谢代谢物5'-甲硫肌苷。 这些 RNA 和 DNA 前体以适合 AMS 的水平加入培养物中，并且通过提取或沉淀从寄生虫中分离出 RNA 和 DNA。 这些实验的样品被转化为碳用于 AMS 分析。 添加 Immucillins（嘌呤核苷磷酸化酶 (PNP) 的强效抑制剂）来确定哪些前体流过该酶并掺入 RNA 和 DNA。 最近我们发现，疟疾 PNP 在参与肌苷、鸟苷和 5'-甲硫基肌苷的挽救方面具有独特性，5'-甲硫基肌苷是恶性疟原虫中多胺途径产生的代谢产物，但不是其人类宿主。 通过恶性疟原虫腺苷脱氨酶对 5'-甲硫肌苷的作用，5'-甲硫肌苷在寄生虫中特异性产生。 这提供了腺嘌呤挽救功能。 我们目前的假设是寄生虫 PNP 和 ADA 在两个嘌呤回收周期中发挥作用。 在不添加次黄嘌呤的情况下，阻断任一酶都可以有效杀死寄生虫。 我们合成了三种酶的强大过渡态类似物，所有这些酶都存在于恶性疟原虫 PNP 的必需嘌呤补救措施中。