Trichomoniasis, the most prevalent non-viral sexually transmitted infection in the world, is caused by the parasitic protozoan Trichomonas vaginalis. Studies have indicated an association between T. vaginalis and a higher susceptibility to various other infections including chlamydia, HIV, and syphilis. The parasite has shown increasing resistance to the current treatment of 5-nitroimidazole drugs such as metronidazole. T. vaginalis is incapable of de novo synthesis of purine and pyrimidine rings, so it must rely on salvage pathway enzymes such as adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH) to scavenge nucleobases. Both enzymes have been screened to identify fragment inhibitors with high ligand efficiencies to use as starting points for drug design. AGNH and UNH can be validated as antitrichomonal targets by demonstrating a correlation between enzyme inhibition and antitrichomonal activity. Escherichia coli cells with endogenous nucleoside ribohydrolase gene expression of rihA (ybeK), rihB (yieK) and rihC (yaaF) were used as a surrogate to develop protocols for observing in vitro enzyme activity. A 1H NMR-based activity assay for AGNH using adenosine as the substrate and an 19F NMR-based activity assay for UNH using 5-fluorouridine as the substrate were previously developed for compound screening. These assays proved remarkably robust for observing nucleoside hydrolase activity in cell extracts and in whole cells. Signals for substrate and product are clearly distinguishable from background signals arising from the cell contents. Reactions have been shown to be cell-dependent, indicating that both enzymes are intracellular and that substrate can rapidly enter the cells. Similar experiments are now in progress using T. vaginalis strains to validate the molecular mechanisms of inhibition.

Translation of 1H and 19F NMR-based activity assays to in vitro characterization of nucleoside hydrolase activity in cell extracts and whole cells

2019 Spring National Meeting

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