Abstract
Trichomoniasis is the most common non-viral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoan Trichomonas vaginalis. The emergence of parasite strains resistant to current therapies necessitates the need for novel treatment strategies. Since T. vaginalis is an obligate parasite that requires nucleoside salvage pathways, essential nucleoside ribohydrolase enzymes are promising new drug targets. Fragment screening and X-ray crystallography have enabled structure-guided design of inhibitors for two of these enyzmes, uridine nucleoside ribohydrolase and adenosine/ guanosine preferring nucleoside ribohydrolase. Linkage of enzymatic and antitrichomonal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents. While a correlation with inhibition of purified enzyme would be mechanistically suggestive, a correlation with inhibition of in-cell enzyme activity would be definitive. To demonstrate this linkage, we translated our NMR-based activity assays for purified enzymes to direct measurement of enzyme activity in T. vaginalis cells. The 19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. The spectral editing afforded by 19F observation results in the detection of only the desired nucleoside substrate and nucleobase product signals. However, the 1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13Cediting to resolve the substrate 1H signals from cell and growth media background signals. The in-cell NMR assays are robust, and have been demonstrated to provide inhibition data on test compounds. Potent inhibition of in-cell enzymes will simultaneously establish the mechanism of action and demonstrate cell permeability.