Purine Metabolism in Trichomonas vaginalis

阴道毛滴虫的嘌呤代谢

• 批准号: 7061641
• 负责人: Ching Chung WANG
• 金额: $ 29.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7061641
• 关键词: Trichomonas vaginalis; X ray crystallography; adenosine kinase; antiprotozoal agents; combinatorial chemistry; computer graphics /printing; computer simulation; drug discovery /isolation; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; mass spectrometry; nucleoside analog; phosphotransferases; purine /pyrimidine metabolism; purine nucleoside phosphorylase; purine nucleosides; recombinant proteins; site directed mutagenesis

DESCRIPTION (provided by applicant): Trichomonas vaginalis is one of the most prevalent infectious pathogens with a worldwide distribution and about 5 million new cases occurring every year. It has vulnerability in lacking de novo synthesis of purine nucleotides and relying on the sequential actions of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase (PNK) for salvaging the external purine bases. Inhibition of either enzyme can result in cessation of T. vaginalis growth and could thus lead to a therapeutic gain. T. vaginalis PNP is a bacterial type hexameric protein with an overwhelming catalytic efficiency in converting adenine to adenosine, whereas human PNP is a trimeric protein not even recognizing adenine as a substrate. T. vaginalis PNK is primarily a bacterial type guanosine kinase, whereas human PNK uses only adenosine as substrate. There are thus many opportunities for designing selective inhibitors against the two parasite enzymes. The parasite PNP was cloned, expressed and had its kinetic mechanism of catalysis well analyzed by us. In the future plan, its structure will be studied by crystallography and its structure-function relationship will be dissected by site-directed mutagenesis. Computer graphic modeling will be used for selecting specific enzyme inhibitors and combinatorial library synthesis will be involved in improving the lead compounds. Well known bacterial PNP inhibitors, such as formycin A, and the subversive substrates of Escherichia coli PNP will be tested and structurally modified for improved potency against the parasite enzyme. T. vaginalis PNK will be cloned sequenced and expressed. The kinetic mechanism of its catalytic function will be examined and compared with that of human adenosine kinase (AK). Following crystallographic structural analysis and site-directed mutagenesis to verify the mechanisms of substrate binding, product release, etc., computer graphic modeling and combinatorial library synthesis will be applied for selection of potent and specific enzyme inhibitors. Many well-known inhibitors of human AK with demonstrated bioavailability will be structurally modified to convert them from human AK inhibitors into potentially selective T. vaginalis PNK inhibitors for anti-Trichomoniasis chemotherapy. The prospect in a successful outcome from this research plan appears good.

描述(申请人提供)：阴道毛滴虫是最流行的传染病病原体之一，全球分布，每年约有500万新发病例。它的弱点是缺乏嘌呤核苷酸的从头合成，依赖嘌呤核苷磷酸化酶(PNP)和嘌呤核苷激酶(PNK)的顺序作用来挽救外部嘌呤碱基。抑制任何一种酶都可以导致阴道毛滴虫停止生长，从而可能导致治疗收益。阴道毛滴虫PNP是一种细菌型六聚体蛋白，在将腺嘌呤转化为腺苷的过程中具有压倒性的催化效率，而人PNP是一种甚至不识别腺嘌呤为底物的三聚体蛋白。阴道毛滴虫PNK主要是一种细菌型鸟苷激酶，而人PNK只使用腺苷作为底物。因此，有很多机会设计针对这两种寄生虫酶的选择性抑制剂。我们克隆并表达了寄生虫PNP，并对其催化动力学机制进行了深入的分析。在未来的计划中，它的结构将通过结晶学进行研究，其结构与功能的关系将通过定点突变进行解剖。计算机图形建模将用于选择特定的酶抑制剂，组合文库合成将用于改进先导化合物。将对众所周知的细菌PNP抑制剂，如甲霉素A和大肠杆菌PNP的颠覆性底物进行测试和结构修改，以提高对寄生虫酶的效力。阴道毛滴虫PNK基因将被克隆、测序和表达。对其催化作用的动力学机制进行了研究，并与人腺苷激酶(AK)进行了比较。在晶体结构分析和定点突变以验证底物结合、产物释放等机制之后，将应用计算机图形建模和组合文库合成来选择有效和特定的酶抑制剂。许多已知的具有生物利用度的人AK抑制剂将被结构修饰，以将它们从人AK抑制剂转化为具有潜在选择性的阴道毛滴虫PNK抑制剂，用于抗滴虫病化疗。这项研究计划取得成功的前景似乎很好。