PURINES & PURINE ANTIMETABOLITES IN MALARIA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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。以尿酸为对照。必要时，我们是合成此类化合物的专家。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。我们合成了一种功能强大的恶性疟原虫PNP过渡态类似物--5？-甲硫代康福霉素。在接下来的一年里，我们希望在正常细胞和被特定ADA或PNP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。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A

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. 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. Uric acid is used as the control. We are expert at the synthesis of such compounds, when necessary. 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 a powerful transition state analogue of P. falciparum PNP, 5¿-methylthio-coformycin. During the next year, we hope to follow RNA and DNA labeling in normal cells and in cells being inhibited with specific ADA or PNP inhibitors. 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. 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 A