Parasitology assays to assess Trichomonas vaginalis nucleoside ribohydrolase inhibitors

Abstract

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.