TriTOX: A novel Trichomonas vaginalis assay platform for high-throughput screening of compound libraries.

Affiliation

Lam AYF(1), Vuong D(2), Jex AR(3), Piggott AM(4), Lacey E(5), Emery-Corbin SJ(6).
Author information:
(1)Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia.
(2)Microbial Screening Technologies, Smithfield, NSW, Australia.
(3)Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia.
(4)Department of Molecular Sciences, Faculty of Science and Engineering, Macquarie University, North Ryde, NSW, Australia.
(5)Microbial Screening Technologies, Smithfield, NSW, Australia; Department of Molecular Sciences, Faculty of Science and Engineering, Macquarie University, North Ryde, NSW, Australia.
(6)Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia. Electronic address: [Email]

Abstract

Trichomonas vaginalis is a neglected urogenital parasitic protist that causes 170 million cases of trichomoniasis annually, making it the most prevalent non-viral, sexually transmitted disease. Trichomoniasis treatment relies on nitroheterocyclics, such as metronidazole. However, with increasing drug-resistance, there is an urgent need for novel anti-trichomonals. Little progress has been made to translate anti-trichomonal research into commercialised therapeutics, and the absence of a standardised compound-screening platform is the immediate stumbling block for drug-discovery. Herein, we describe a simple, cost-effective growth assay for T. vaginalis and the related Tritrichomonas foetus. Tracking changes in pH were a valid indicator of trichomonad growth (T. vaginalis and T. foetus), allowing development of a miniaturised, chromogenic growth assay based on the phenol red indicator in 96- and 384-well microtiter plate formats. The outputs of this assay can be quantitatively and qualitatively assessed, with consistent dynamic ranges based on Z' values of 0.741 and 0.870 across medium- and high-throughput formats, respectively. We applied this high-throughput format within the largest pure-compound microbial metabolite screen (812 compounds) for T. vaginalis and identified 43 hit compounds. We compared these identified compounds to mammalian cell lines, and highlighted extensive overlaps between anti-trichomonal and anti-tumour activity. Lastly, observing nanomolar inhibition of T. vaginalis by fumagillin, and noting this compound has reported activity in other protists, we performed in silico analyses of the interaction of fumagillin with its molecular target methionine aminopeptidase 2 for T. vaginalis, Giardia lamblia and Entamoeba histolytica, highlighting potential for fumagillin as a broad-spectrum anti-protistal against microaerophilic protists. Together, this new platform will accelerate drug-discovery efforts, underpin drug-resistance screening in trichomonads, and contributing to a growing body of evidence highlighting the potential of microbial natural products as novel anti-protistals.