PURINES & PURINE ANTIMETABOLITES IN MALARIA

嘌呤

• 批准号: 7602406
• 负责人: Vern L. Schramm
• 金额: $ 2.4万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602406
• 关键词: Adenine; Adenosine; Antimetabolites; Blood; Carbon; Cells; Clinical Trials; Computer Retrieval of Information on Scientific Projects Database; Culicidae; Culture Media; DNA; Dose; Enzymes; Erythrocytes; Funding; Gene Expression; Genetic; Glycine; Grant; Guanosine; Human; Hypoxanthine; Hypoxanthines; Immucillin-H; Inhibitory Concentration 50; Inosine; Institution; Knock-in Mouse; Label; Malaria; Metabolic; Normal Cell; Nucleic Acids; Parasites; Pathway interactions; Pattern; Phosphotransferases; Polyamines; Precipitation; Purine Nucleoside Phosphorylase Inhibitor; Purine-Nucleoside Phosphorylase; Purines; RNA; Research; Research Personnel; Resources; Sampling; Site; Source; Testing; Time; United States National Institutes of Health; Work; Xanthines; adenosine deaminase; analog; feeding; inhibitor/antagonist; killings; prevent; purine; purine metabolism; research study; uptake; 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. 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。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 研究中心，而研究中心不一定是研究者所在的机构。 项目描述 疟疾寄生虫是嘌呤营养缺陷型，但在人类红细胞内生长，其中嘌呤浓度比寄生虫吸收的量高出数百至数千倍。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。 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。 5- 甲基硫代肌苷通过恶性疟原虫腺苷脱氨酶对5-甲基硫代肌苷的作用在寄生虫中特异性产生。- 甲硫基肌苷。 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。 我们已经合成了三种酶的强过渡态类似物，它们都是恶性疟原虫PNP的必需嘌呤补救酶。 在接下来的一年里，我们希望在正常细胞和被我们的三种特异性抑制剂抑制的细胞中跟踪RNA和DNA标记，这些抑制剂针对参与该途径的三种连续酶。 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。 其中一种酶在恶性疟原虫中是必需的，我们预计抑制剂将阻断上述任何途径的摄取。 在平行研究中，我们将在培养物中测试抑制剂的IC 50或杀伤作用，并尝试将其与AMS的14 C前体摄取相关联。 在相关工作中，我们发现Immucillin-H（而不是DADMe-Immucillin-H）（一种更强大的PNP抑制剂）可以阻止按蚊肠道中寄生虫的发育。 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AMS掺入核酸来测试的。 负责这种吸收的蚊子酶已经在我们的实验室中克隆，以将AMS研究与蚊子遗传学和基因表达途径相关联。