Artemisinin exposure at the ring or trophozoite stage impacts Plasmodium falciparum sexual conversion differently

Malaria transmission is dependent on the formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on sexual conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~ fourfold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications.

Exposure to artemisinin at the trophozoite stage increases sexual conversion rates in the malaria parasite Plasmodium falciparum

ABSTRACTMalaria transmission is dependent on formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~4-fold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications.

A Metabolomic Investigation of the Effect of Eosin B on Gameto-cyte of Plasmodium falciparum using 1HNMR Spectroscopy

Background: Recently eosin B was shown to have an effect on the asexual stage of Plasmodium falciparum and in this study, its activity against gametocytes and changes in the culture medium metabolites were investigated using an1HNMR-based metabolomics approach. Methods: In the Biochemistry Department of Pasteur Institute of Iran in 2017, parasites were cultured and gametocytogenesis induced by heparin and 5% hematocrit. Sexual stage parasites were tested by eosin B in 90 well plates and IC50 determined using Lactate Dehydrogenase assay. Gametocytes were treated by IC50 dose of eosin B and the medium collected in the two groups: with eosin B and controls and sent for 1HNMR spectroscopy. The spectra were analyzed on MATLAB interface and the altered metabolites in the culture medium and eosin-affected biochemical pathways were identified by Human Metabolome Database and Metaboanalyst website. Results: The results revealed eosin B had an effective gametocytocidal activity against P. falciparum. The significant metabolites changed in the medium were thiamine, Asp, Asn, Tyr, Lys, Ala, Phenylpyruvic acid, NAD+ and lipids. The main pathways identified were aminoacyl-tRNA biosynthesis, Phenylalanine, tyrosine and tryptophan biosynthesis, Alanine, aspartate and glutamate metabolism, Phenylalanine metabolism, Nicotinate and nicotinamide metabolism, and lysine degradation. Conclusion: Eosin B exhibited substantial gametocytocidal activity and affected important drug targets in the Plasmodium.

Maduramicin Rapidly Eliminates Malaria Parasites and Potentiates the Gametocytocidal Activity of the Pyrazoleamide PA21A050

New strategies targetingPlasmodium falciparumgametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective againstP. falciparumgametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations againstP. falciparumsexual stages. In this study, we showed that maduramicin has an EC50(effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocksin vivotransmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidlyin vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na+concentration that is proposed to be due to inhibition of PfATP4, a putative Na+pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stageP. falciparumparasites and the potential of targeting this pathway for drug development.

Potent Plasmodium falciparum Gametocytocidal Activity of Diaminonaphthoquinones, Lead Antimalarial Chemotypes Identified in an Antimalarial Compound Screen

ABSTRACTForty percent of the world's population is threatened by malaria, which is caused byPlasmodiumparasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. IfPlasmodium falciparumgametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria.

Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay

ABSTRACTThe design of new antimalarial combinations to treatPlasmodium falciparuminfections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinantP. falciparumline expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the early-stage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC50s) below 2.5 μM. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of ≤5%, an average signal-to-noise ratio (S:N) of >30, and a Z′ of ∼0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy.