Nowhere to hide: interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination

Abstract

BackgroundThe discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions.MethodsGametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable.ResultsA highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4 %. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident.ConclusionsWith this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0718-z) contains supplementary material, which is available to authorized users.

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species

“…Gametocytogenesis and gametocyte culturing was performed as described by Reader et al (2015). Contaminating asexual cultures were removed by N-acetyl glucosamine (NAG) (50 mM) treatment for 96 hours (Reader et al, 2015).…”

“…Contaminating asexual cultures were removed by N-acetyl glucosamine (NAG) (50 mM) treatment for 96 hours (Reader et al, 2015). Gametocyte development from stages I to V was monitored daily by microscopic examination of Giemsa-stained smears of cultures.…”

“…Modified protocols of the method of Makler and Hinrichs (1993) were used to set up the et al, 2015, 2013) and for examining gametocyte viability spectrophotometrically (Reader et al, 2015).…”

“…Plates were then incubated for a further 72 hrs before assessing viability by measuring pLDH activity (Reader et al, 2015). All assays were done in technical triplicates with three independent biological repeats each.…”

Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities

“…Similar approaches have been proposed to discover transmission-blocking compounds by screening against primarily mature gametocytes but this has been very difficult compared to screening asexual blood stages, particularly because the apparent quiescence of mature gametocytes has hampered the development of cell-based screening assays specific to stage V gametocytes [19]. This limited metabolic repertoire restricts the 'druggable' pool of biochemical activities in these parasites [20].…”