A High-Throughput Screen Targeting Malaria Transmission Stages Opens New Avenues for Drug Development

Abstract

A major goal of the worldwide malaria eradication program is the reduction and eventual elimination of malaria transmission. All currently available antimalarial compounds were discovered on the basis of their activity against the asexually reproducing red blood cell stages of the parasite, which are responsible for the morbidity and mortality of human malaria. Resistance against these compounds is widespread, and there is an urgent need for novel approaches to reduce the emergence of resistance to new antimalarials as they are introduced. We have established and validated the first high-throughput assay targeting the red blood cell parasite stage required for transmission, the sexually reproducing gametocyte. This assay will permit identification of compounds specifically targeting the transmission stages in addition to the asexual stage parasites. Such stage-specific compounds may be used in a combination therapy, reducing the emergence of resistance by blocking transmission of resistant parasites that may be selected in a patient.

Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue

“…Given our emphasis on drugs that are used in first-line therapy or are candidate transmission-blocking agents, we did not include the former gold standard antimalarial drug, chloroquine. Other studies have clearly established that chloroquine exerts activity on early stage gametocytes (6,10,13,22).…”

“…To produce P. falciparum reporter lines expressing GFP-luciferase under the control of gametocytespecific promoters, we used Bxb1 mycobacteriophage integrasemediated recombination to deliver transgenes into the P. falciparum genome (14). Prior strategies have used transgenes on episomally replicating plasmids (13,15), where the absence of selection during gametocyte development can lead to the loss of plasmids and signal heterogeneity between parasites. Here, transgene integration was applied to the NF54 strain, which produces highly infectious gametocytes and is being used in human malaria vaccine trials (16).…”

“…Earlier in vitro evaluations of drug gametocytocidal activity typically relied on laborious, subjective microscopic observations of parasite morphology to assess cell viability (6). Recent advances include the use of hydroethidine or alamarBlue as fluorescent markers of metabolic activity (10,11) or GFP reporter lines to assess commitment to gametocytogenesis (12,13), notably after antimalarial drug treatment of asexual forms (10). These methods, however, have yet to be exploited to assess dose-dependent drug effects over time or define rates of action with purified, mature gametocyte populations.…”

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials

“…They are based on : 1) the discrimination by flow cytometry of asexual and sexual forms using hydroethine that is taken up by the parasite and metabolized into ethidium, a nucleic acidbinding fluorochrome (Chevalley et al, 2010); 2) the use of transgenic P. falciparum parasites expressing a green fluorescent protein chimera of the early sexual blood stage (protein Pfs16) as a marker for commitment to gametocytogenesis; this marker associated to hydroethine allows also to measure the direct activity of drugs against the late-stages gametocytes (Peatey et al, 2009). In a same way, the stage II or later stage marker (PF10_0164) fused to the green fluorescent protein was used associated with the nuclear dye Hoescht 33342 to quantify the drug effects on the asexual stages and on the sexual conversion and the gametocyte maturation in a same assay (Buchholz et al, 2011). In a general way, the application of transfection technology to malaria parasites paves the way to a new generation of assays targeting specific pathways or parasite stages.…”

In vitro and In vivo Antimalarial Activity of Amphiphilic Naphthothiazolium Salts with Amine-Bearing Side Chains

“…To test the effect of drugs on gametocyte stages, P. falciparum NF54 cultures were initiated in 24-well plates at 0.5% asexual parasitemia and 4% hematocrit. 18 Medium was changed daily up to day 18, without addition of fresh erythrocytes. Continuous cultivation without dilution leads to concomitant crash of asexual parasitemia and induction of gamteocytogenesis by day 5.…”