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Trichomoniasis is the most common nonviral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoan Trichomonas vaginalis . Although the disease itself is typically mild, individuals with trichomonal infections have a higher susceptibility to more serious conditions. 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. Linkage of enzymatic and antiprotozoal 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 definitively establish this linkage. To demonstrate this linkage, we have translated our NMR-based activity assays that measure the activity of purified enzymes for use in T. vaginalis cells. The 19 F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the 1 H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13 C-editing to resolve the substrate 1 H 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. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.

Trichomonas vaginalis is the causative parasitic protozoan of the disease trichomoniasis, the most prevalent, nonviral sexually transmitted disease in the world. T. vaginalis is a parasite that scavenges nucleosides from the host organism via catalysis by nucleoside hydrolase (NH) enzymes to yield purine and pyrimidine bases. One of the four NH enzymes identified within the genome of T. vaginalis displays unique specificity toward purine nucleosides, adenosine and guanosine, but not inosine, and atypically shares greater sequence similarity to the pyrimidine hydrolases. Bioinformatic analysis of this enzyme, adenosine/guanosine-preferring nucleoside ribohydrolase (AGNH), was incapable of identifying the residues responsible for this uncommon specificity, highlighting the need for structural information. Here, we report the X-ray crystal structures of holo , unliganded AGNH and three additional structures of the enzyme bound to fragment and small-molecule inhibitors. Taken together, these structures facilitated the identification of residue Asp231, which engages in substrate interactions in the absence of those residues that typically support the canonical purine-specific tryptophan-stacking specificity motif. An altered substrate-binding pose is mirrored by repositioning within the protein scaffold of the His80 general acid/base catalyst. The newly defined structure-determined sequence markers allowed the assignment of additional NH orthologs, which are proposed to exhibit the same specificity for adenosine and guanosine alone and further delineate specificity classes for these enzymes.