PURINES & PURINE ANTIMETABOLITES IN MALARIA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 项目说明 疟疾寄生虫是嘌呤营养缺乏症，但生长在人类红细胞内，其中的嘌呤浓度比寄生虫摄取的数量高出数百到数千倍。因此，我们需要一种特定和敏感的方法来建立来自血液(或培养介质)的前体被合并到寄生虫中的途径。我们正在使用14C前体来标记在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，以及新发现的恶性疟原虫嘌呤代谢产物5’-甲硫肌苷。这些RNA和DNA前体以适合AMS的水平被喂养到培养物中，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。 免疫粘菌素是嘌呤核苷磷酸化酶(PNP)的强大抑制剂，用来确定哪些前体通过这种酶进入RNA和DNA。最近我们发现，疟疾PNP在参与回收肌苷、鸟苷和5‘-甲硫肌苷方面是独一无二的，5’-甲硫肌苷是恶性疟原虫多胺途径的代谢物，而不是人类宿主。恶性疟原虫通过腺苷脱氨酶对5‘-甲硫肌苷的作用，在寄生虫体内特异性地产生5’-甲硫肌苷。这提供了一种腺嘌呤挽救功能。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。在没有添加次黄嘌呤的情况下，阻断这两种酶中的任何一种都能有效地杀死寄生虫。我们已经合成了三种酶的功能强大的过渡态类似物，它们都是恶性疟原虫PNP必需的嘌呤残留物。

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.