Abstract
Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2'-deoxyadenosine (F-dAdo) is a "subversive substrate." Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC(50) values of 106 and 84 nm, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2'-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 A. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 A as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.
Highlights
Trichomonas vaginalis is an anaerobic protozoan parasite that causes one of the most common sexually transmitted infections in the world
While carrying out studies to identify inhibitors of T. vaginalis purine nucleoside phosphorylase (PNP) (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2deoxyadenosine (F-dAdo) is a “subversive substrate.”
T. vaginalis is a protozoan parasite with a relatively simple scheme for purine salvage [15], albeit one that is quite distinct from those in T. fetus and G. lamblia. It relies on the functions of two pivotal enzymes, purine nucleoside phosphorylase (PNP),1 which catalyzes interconversion between purine bases and the corresponding purine nucleosides, and purine nucleoside kinase (PNK), which converts nucleosides to the nucleotides (16 –18)
Summary
Trichomonas vaginalis is an anaerobic protozoan parasite that causes one of the most common sexually transmitted infections in the world. T. vaginalis does not incorporate hypoxanthine or inosine into purine nucleotides [19] and possesses no detectable activity of purine phosphoribosyltransferase [16, 17] It apparently depends primarily on the sequential reactions catalyzed by PNP and purine nucleoside kinase to incorporate exogenous adenine and guanine into its purine nucleotide pool. Both enzymes are amenable to a specific inhibitor design that may lead to effective anti-T. vaginalis drugs if the parasitic enzymes are distinct from their mammalian counterparts in terms of substrate and inhibitor specificities.
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