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.
| Rinaldo-Matthis, Agnes; Wing, Corin; Ghanem, Mahmoud et al. (2007) Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues. Biochemistry 46:659-68 |
| Zang, Yang; Wang, Wen-Hu; Wu, Shaw-Wen et al. (2005) Identification of a subversive substrate of Trichomonas vaginalis purine nucleoside phosphorylase and the crystal structure of the enzyme-substrate complex. J Biol Chem 280:22318-25 |
