A Male and Female Gametocyte Functional Viability Assay To Identify Biologically Relevant Malaria Transmission-Blocking Drugs

Abstract

Malaria elimination will require interventions that prevent parasite transmission from the human host to the mosquito. Experimentally, this is usually determined by the expensive and laborious Plasmodium falciparum standard membrane feeding assay (PfSMFA), which has limited utility for high-throughput drug screening. In response, we developed the P. falciparum dual gamete formation assay (PfDGFA), which faithfully simulates the initial stages of the PfSMFA in vitro. It utilizes a dual readout that individually and simultaneously reports on the functional viability of male and female mature stage V gametocytes. To validate, we screen the Medicines for Malaria Venture (MMV) Malaria Box library with the PfDGFA. Unique to this assay, we find compounds that target male gametocytes only and also compounds with reversible and irreversible activity. Most importantly, we show that compound activity in the PfDGFA accurately predicts activity in PfSMFAs, which validates and supports its adoption into the transmission-stage screening pipeline.

Luciferase-Based, High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes

“…Some assays have focused on gamete formation after xanthurenic acid (XA)-mediated activation of gametocytes (18,20,21,41) or, alternatively, have been based on the measurement of pLDH activity from non-XA-activated gametocyte cultures (18). Functional-viability assays targeting mature gametocytes, i.e., the stage that is competent for transmission in the mosquito, have been proposed as more accurate predictors of the transmissionblocking potential of compounds (41). We therefore tested the most potent Malaria Box compounds identified in our gametocyte assay using mature stage V gametocytes on day 12 of gametocytogenesis.…”

“…The most active compound from the day 8 screening, MMV006172, was still active on day 12 at a submicromolar level. The compound was shown to be a weak male-specific gametocyte/ gamete formation inhibitor, causing 56.4% inhibition of male gametogenesis at 1 M, while being inactive against female gametocytes at the same concentration in a screen evaluating male and female gamete formation (41). More recently, an imaging-based assay reported the compound as a female gamete formation inhibitor (IC 50 ϭ 0.46 M [21]).…”

Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities

“…The Medicines for Malaria Venture (MMV) Malaria Box of 400 compounds (selectively active against asexual parasites [22]) has been screened worldwide for activity against stage IV/V gametocytes [20,[23][24][25][26][27][28][29][30][31]. Primary hit rates (>80% inhibition at 5 μM) of 4.5-24% [20,23,27,29] were achieved when screened against stage IV/V gametocytes; this increased to 33% against stage I-III gametocytes [23,26].…”

“…dual gamete formation assay) may be used to initially assess the ability of the compound to have sex-specific activity on gametocytes by measuring male/female gamete formation [30]. An assay of immature gametocytes will indicate whether the compound exhibits stage specificity, which is an important aspect since the assumption that all compounds targeting asexual parasites will target immature gametocytes does not always hold true [20,26].…”

“…An assay specific for male and female gamete formation provide an indication of compound effects on gametocyte infectivity [30], however, this adds complexity to the design of a transmission-blocking pipeline. This also raises the issue of whether there is a need for compounds to target both forms of gametes or potentially only female or male gametes.…”