Imaging-Based High-Throughput Screening Assay To Identify New Molecules with Transmission-Blocking Potential against Plasmodium falciparum Female Gamete Formation

Abstract

In response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z= factor, 0.7 ؎ 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 M concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential. D espite the efforts made over decades of scientific research, malaria still remains a major health problem in tropical and subtropical areas, with more than 220 million cases and 600,000 deaths being registered per year (1). This parasitic disease is caused by Plasmodium infection through the bite of infected Anopheles female mosquitoes, with Plasmodium falciparum being responsible for the highest mortality rates (2).Traditionally, pharmacological antimalarial treatments have targeted parasite asexual reproduction inside erythrocytes, which leads to the clinical symptoms of malaria. However, a small proportion of these asexual blood stages (0.2 to 1%) are committed to develop into sexual stages: male and female gametocytes (3, 4). Their differentiation process inside erythrocytes takes 8 to 12 days for P. falciparum, and inside erythrocytes they undergo a series of morphological and metabolic changes classically categorized into five stages of maturation (5, 6). While most schizonticides, such as chloroquine, affect young gametocytes (stages I, II, and III), gametocytes at late stages of maturation are not sensitive to them (7). These insensitive stage V gametocytes, which are responsible for ...

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

“…Two of the five compounds tested from the amino alcohol class, namely, halofantrine and mefloquine (racemic and the R and S isomers), killed late-stage gametocytes with limited potency, showing IC 50 s ranging between 2.31 and 3.39 M. These levels of activity are lower than what has been reported in other late-stage (stage IV and V) gametocytocidal assays based on imaging and the measurement of metabolic activity (19,30) and are in better agreement with recent chemiluminescence-and fluorescence-labeled antibody assays on functionally mature stage V gametocytes (18,20).…”

“…Members of the 8-aminoquinoline compound class, especially primaquine, have been shown to possess gametocytocidal activity in P. falciparum-infected patients. Their activities against either early-or late/mature-stage gametocytes in vitro, however, have been moderate at best (15,19,20,28). This discrepancy has been attributed to the requirement for the compounds to be metabolically transformed in order to exert activity, a process that does not occur in vitro (29).…”

“…Among the seven antibiotics tested, only thiostrepton showed moderate inhibition of late-stage gametocytes (IC 50 Ϯ SEM ϭ 1.00 Ϯ 0.07 and 2.31 Ϯ 0.13 M, respectively). Thiostrepton has previously been described as a late-stage gametocytocidal (19) and has exhibited activity against parasite gamete formation in other assays (18,20,27).…”

“…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).…”

“…Gametocytocidal assays with high throughput capability (i.e., utilizing 384-or 1,536-well plates) for late-stage and mature (stage III to V) P. falciparum gametocytes utilizing fluorescent indicators of metabolic activity (17,18), fluorescence imaging (19)(20)(21), or chemiluminescence (18) have been previously described. We have recently developed and validated an HTS assay for early-stage (stage I to III) P. falciparum gametocytes (15).…”