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 19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the 1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13C-editing 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. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, a sexually transmitted disease that affects an estimated 275 million people worldwide. The Centers for Disease Control and Prevention recognizes trichomoniasis as a neglected parasitic infection, with an estimated 2.6 million prevalent infections in the United States in 2018. Clinical manifestations of infections are typically mild, but the immune system can be compromised resulting in higher susceptibility to more serious conditions such as pelvic inflammatory disease, HIV‐1, and other infections. Trichomonal infection has also been associated with prostate cancer and benign prostatic hyperplasia. Traditionally, compounds such as metronidazole or tinidazole have been used to treat trichomoniasis, however, strains resistant to these drugs are becoming more widespread. T. vaginalis requires nucleoside salvage pathways for its survival. Nucleoside ribohydrolase inhibitors thus represent a possible target for the development of new treatments with different mechanisms of action compared to metronidazole. Toward this goal, fragment screening identified nucleoside ribohydrolase inhibitors as starting points for medicinal chemistry efforts. Newly synthesized compounds are routinely evaluated for potency against purified enzymes. However, a better test of potency is antitrichomonal activity. Metronidazole‐sensitive strain B7RC2 (ATCC 50167) of T. vaginalis was cultured anaerobically at 37 ℃ in TYM Diamond’s media supplemented with streptomycin, penicillin and iron solution. Compounds were prepared as 25 mM DMSO stock solutions and tested at 100 µM. DMSO and metronidazole were used as vehicle and positive controls respectively. Parasite cellular viability was measured after 24 hours of incubation with compounds or controls. Cells were counted using a hemocytometer. Active compounds were then tested in a dose‐dependent manner to determine antitrichomonal IC50 values. Compounds with the best combination of enzyme inhibition and T. vaginalis activity were then prioritized for guiding the design of compounds to be synthesized. It is expected that this process will simultaneously optimize enzyme inhibition and antitrichomonal activity resulting in compounds with nM activity in enzyme assays, against metronidazole‐sensitive T. vaginalis, and ultimately against metronidazole‐resistant T. vaginalis.