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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 项目描述 疟疾寄生虫是嘌呤的合子，但在人类红细胞内生长，其中嘌呤的浓度比寄生虫所吸收的数量大数百到数千倍。因此，我们需要一种特定和敏感的方式来建立从血液（或培养基）中掺入寄生虫中的前体的途径。 我们正在使用14C前体来将嘌呤池标记在人类红细胞中生长的寄生虫中。 嘌呤的前体包括肌苷，腺苷，鸟苷，5'-甲基硫代腺苷，低黄嘌呤，腺嘌呤，黄嘌呤，甘氨酸，以及在5'-甲基硫代氨酸5'-甲基硫代氨酸的恶性疟原虫中新发现的嘌呤代谢的代谢。 这些RNA和DNA前体以适合AMS的水平以及通过提取或沉淀分离的寄生虫的RNA和DNA喂食。 这些实验的样品转化为碳进行AMS分析。 添加了嘌呤核苷磷酸化酶（PNP）的强大抑制剂，以确定要在RNA和DNA中掺入该酶的前体流过哪些前体。 最近，我们发现疟疾PNP在参与肌苷，鸟嘌呤和5'-甲基噻氨基氨酸的拯救方面是独一无二的，这是一种代谢物，它来自恶性疟原虫的多胺途径，但不是其人类宿主。 5-甲基噻氨基氨酸是通过恶性疟原虫腺苷脱氨酶对5-甲基噻氨基胺的作用而特别在寄生虫中产生的。 这提供了腺嘌呤救助功能。 我们目前的假设是两个嘌呤挽救周期中的寄生虫PNP和ADA功能。 在没有添加的低黄嘌呤的情况下，阻断任何一种酶在杀死寄生虫方面都是有效的。 我们已经合成了三种酶的强大过渡状态类似物，所有这些酶都是在恶性疟原虫PNP的必需嘌呤拯救中。 在第二年，我们希望在正常细胞中跟随RNA和DNA标记，并在该途径中涉及的三种顺序酶的三种特定抑制剂中抑制细胞中的RNA和DNA标记。 如果在ADA和PNP抑制剂的存在下，在敲除和正常寄生虫中的14C掺入模式是相同的，我们将有证据表明，抑制剂的代谢抑制作用唯一位于这些酶处。 这些酶之一在恶性疟原虫中似乎是必不可少的，我们希望抑制剂会阻止上述任何途径的吸收。 在平行研究中，我们将测试IC50培养物或杀死的抑制剂，并尝试将其与AMS的14C前体摄取相关。 在相关工作中，我们发现不可或缺的PNP抑制剂DADME-IMMUCILLIN-H，而不是DADME-IMMUCILLIN-H，喂入蚊子的蚊子可以防止寄生虫在蚊子中发育。 我们还发现，较高剂量的无限林-H会杀死蚊子。 从临床试验中，我们知道这些剂量对人类无毒。 我们的假设是蚊子含有一种激酶，该激酶5'-磷酸化，然后掺入核酸。 这是通过喂养14c-污染物蛋白的蚊子痕迹和AMS掺入核酸中的。 拟议的负责这种吸收的蚊子已被克隆在我们的实验室中，以将AMS研究与蚊子遗传学和基因表达途径相关联。