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。 在接下来的一年里，我们希望在正常细胞和被我们针对这一途径中涉及的三种序列酶的三种特定抑制剂抑制的细胞中进行RNA和DNA标记。如果在基因敲除和正常寄生虫中存在ADA和PNP抑制剂，14C掺入的模式是相同的，我们将有证据表明，抑制代谢的唯一位置是在这些酶上。这些酶中的一种似乎对恶性疟原虫是必不可少的，我们预计抑制剂将阻止上述任何一种途径的摄取。在平行研究中，我们将测试培养中的抑制剂的IC50或杀伤作用，并试图将其与AMS摄取14C前体相关联。 在相关工作中，我们发现，喂给按蚊的伊莫西林-H，而不是DADMe-伊莫西林-H，一种更强大的PNP抑制剂，可以防止寄生虫在蚊子的肠道中繁殖。我们还发现，较高剂量的伊莫西林-H可以杀死蚊子。从临床试验中我们知道，这些剂量对人体无毒。我们的假设是，蚊子含有一种5‘-磷酸化免疫粘菌素-H的激酶，然后结合到核酸中。这是通过喂食蚊子微量的14C-免疫粘菌素并在AMS将14C-免疫粘菌素掺入核酸中来测试的。我们实验室已经克隆了负责这种摄取的蚊子酶，以将AMS研究与蚊子遗传学和基因表达途径相关联。

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. During the next year, we hope to follow RNA and DNA labeling in normal cells and in cells being inhibited with three of our specific inhibitors for the three sequential enzymes involved in this pathway. If the pattern of 14C incorporation is the same in knock-outs and in normal parasites in the presence of ADA and PNP inhibitors, we will have evidence that the sole site of metabolic inhibition of the inhibitor is at these enzymes. One of these enzymes appears essential in P. falciparum, and we expect that inhibitors will block uptake from any of the above pathways. In parallel studies, we will test inhibitors in culture for IC50 or killing and try and correlate this with 14C precursor uptake by AMS. In related work, we found that Immucillin-H, but not DADMe-Immucillin-H, an even more powerful PNP inhibitor, fed to Anopholes mosquitoes prevents parasites from developing in the mosquito gut. We also found that higher doses of Immucillin-H kills mosquitoes. From clinical trials we know that these doses are not toxic to humans. Our hypothesis is that mosquito contains a kinase that 5'-phosphorylates Immucillin-H followed by incorporation into nucleic acids. This is being tested by feeding mosquitoes traces of 14C-Immucillins and following incorporation into nucleic acids by AMS. The proposed mosquito enzymes responsible for this uptake have been cloned in our lab to correlate AMS studies with mosquito genetics and gene expression pathways.