PURINES & PURINE ANTIMETABOLITES IN MALARIA

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。疟疾寄生虫是嘌呤营养缺陷型，但在人类红细胞内生长，其中嘌呤的浓度比寄生虫吸收的量高数百至数千倍。因此，我们需要一种特异而敏感的方法来建立血液（或培养基）中的前体被纳入寄生虫的途径。我们正在使用14 C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 '-甲硫基腺苷、次黄嘌呤、腺嘌呤、黄嘌呤、甘氨酸和新发现的恶性疟原虫中嘌呤代谢的代谢物5'-甲硫基肌苷。尿酸用作对照。必要时，我们擅长合成此类化合物。将这些RNA和DNA前体以适合AMS的水平进料至培养物，并通过提取或沉淀分离来自寄生虫的RNA和DNA。这些实验的样品被转化为碳，用于AMS分析。 Immucilins是嘌呤核苷磷酸化酶（PNP）的强效抑制剂，加入后可确定哪些前体流经该酶并掺入RNA和DNA中。最近我们发现，疟疾PNP是唯一参与救援的肌苷，鸟苷和5 '-甲硫基肌苷，一种代谢产物，产生于多胺途径的恶性疟原虫，但不是它的人类宿主。通过恶性疟原虫腺苷脱氨酶对5 <$-甲硫基肌苷的作用，5 <$-甲硫基肌苷在寄生虫中特异性产生。这提供了腺嘌呤补救功能。我们目前的假设是，寄生虫PNP和ADA的功能，在两个嘌呤补救周期。在没有添加次黄嘌呤的情况下，阻断任何一种酶都能有效地杀死寄生虫。我们已经合成了一个强大的过渡态类似物的恶性疟原虫PNP，5 <$-甲硫基-coformycin。在接下来的一年里，我们希望在正常细胞和被特异性ADA或PNP抑制剂抑制的细胞中进行RNA和DNA标记。如果14 C掺入的模式在敲除中和存在ADA和PNP抑制剂的正常寄生虫中是相同的，我们将有证据表明，抑制剂的代谢抑制的唯一位点是在这些酶上。 在相关的工作中，我们发现Immucillin-H，而不是DADMe-Immucillin-H，一种更强大的PNP抑制剂，喂养给按蚊可以防止寄生虫在蚊子肠道中发育。我们还发现，更高剂量的Immucillin-H可以杀死蚊子。从临床试验中我们知道，这些剂量对人类没有毒性。我们的假设是，蚊子含有一种激酶，该激酶使Immucillin-H 5 '-磷酸化，然后掺入核酸中。这是通过给蚊子喂微量的14 C-Immucillins并通过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