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

Alexander Y.F Lam,Daniel Vuong,Aaron R Jex,Andrew M Piggott,Ernest Lacey,Samantha J Emery-Corbin

doi:10.1016/j.ijpddr.2021.01.001

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.

Highlights

Trichomonas vaginalis is a microaerophilic parasitic protist that in fects the human urogenital tract causing trichomoniasis, the most prevalent non-viral sexually-transmitted disease (STD) worldwide
As the majority of trichomoniasis infections occur in lower socioeconomic populations, underinvestment in diagnostic and discovery platforms has created a deficit in upcoming effective oral treatments in clinical trials
TriTOX is a simple, cost-effective and consistent in vitro trophozoite growth assay for T. vaginalis that is compatible with medium- and highthroughput screening

Summary

Introduction

Trichomonas vaginalis is a microaerophilic parasitic protist that in fects the human urogenital tract causing trichomoniasis, the most prevalent non-viral sexually-transmitted disease (STD) worldwide. It is estimated ~170 million cases of trichomoniasis occur globally, of which 85% are in developing countries (Harp and Chowdhury, 2011; Newman et al, 2015). These rates are likely to vastly underestimate true prevalence, given the high rates of infections lacking acute sympto mology (i.e., ‘asymptomatic’), and that trichomoniasis is often. T. vaginalis infections produce advantageous urogenital environments for other STDs, including strong associated with increased viral loads and transmission of Human Immunodefi ciency Virus (HIV) (Buve et al, 2001; Kissinger, 2015; Sorvillo and Kerndt, 1998), and these coinfections are correlated with worse clinical outcomes than singular infections (Nolan et al, 2020; Van der Pol, 2007)

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