Purine Metabolism in Trichomonas vaginalis

描述（申请人提供）：阴道毛滴虫是全球最流行的感染性病原体之一，每年约有500万新发病例。它缺乏嘌呤核苷酸的从头合成，并且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，因此具有脆弱性。任何一种酶的抑制都可以导致阴道梭菌生长的停止，从而可能导致治疗增益。阴道T. PNP是一种细菌型六聚体蛋白，在将腺嘌呤转化为腺苷方面具有压倒性的催化效率，而人类PNP是一种三聚体蛋白，甚至不识别腺嘌呤作为底物。阴道T. PNK主要是细菌型鸟苷激酶，而人类PNK仅使用腺苷作为底物。因此，有许多机会设计针对这两种寄生虫酶的选择性抑制剂。对寄生虫PNP进行了克隆、表达，并对其催化动力学机制进行了分析。在未来的计划中，将通过晶体学研究其结构，并通过定点诱变剖析其结构-功能关系。计算机图形建模将用于选择特定的酶抑制剂，组合文库合成将涉及改进先导化合物。众所周知的细菌PNP抑制剂，如formycin A和大肠杆菌PNP的颠覆性底物将被测试和结构修饰，以提高对寄生虫酶的效力。对阴道绦虫PNK进行克隆、测序和表达。研究了其催化作用的动力学机制，并与人腺苷激酶（AK）进行了比较。在晶体结构分析和定点诱变验证底物结合、产物释放等机制之后，计算机图形建模和组合文库合成将用于选择有效和特异性的酶抑制剂。许多已知的具有生物利用度的人类AK抑制剂将在结构上进行修饰，使其从人类AK抑制剂转化为具有潜在选择性的阴道滴虫PNK抑制剂，用于抗滴虫化疗。这个研究计划取得成功的前景看来很好。

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.