The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites

Abstract

Background: Malaria remains a disease of devastating global impact, killing more than 800,000 people every year-the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance.

Methods and findings: Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony.

Conclusions: These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle-wide analyses of drugs for other pathogens with complex life cycles.

Figure 1. The main classes of antimalarials.

The chemical structures of all the main classes of antimalarials and other therapeutic and control molecules are assembled according to either the chemical classes they belong to (endoperoxides, 4- and 8- AQs, amino-alcohols) or their function (antifolate, antibiotics), or both (e.g., sulfonamides, a chemical class of antibiotic used in combined antimalarial therapies). The colour code associated with each class is consistent in all the figures in this report.

Figure 2. The potencies of selected antimalarials against asexual blood stages.

Main classes of antimalarials were tested against the blood stage of seven Plasmodium falciparum strains in the [³H]hypoxanthine incorporation assay as described in Vennerstrom et al. . The strains tested here (obtained from MR4) were two drug-sensitive strains NF54 and D6 and five drug-resistant strains: K1, resistant to chloroquine (CQ) and pyrimethamine (PYR), origin Thailand, carries mutations in genes pfmdr1, pfcrt, pfdhfr, pfdhps; W2, resistant to CQ, PYR, quinine, cycloguanil, and sulfadoxine, origin Vietnam; 7G8, resistant to CQ and PYR, origin Brazil; TM90C2A, resistant to CQ, PYR, and MFQ, origin Thailand; and V1/S, resistant to CQ, PYR and cycloguanil, origin Vietnam. Results are expressed as the concentration resulting in 50% growth inhibition (IC₅₀).Values are means of ≥3 independent experiments. (A) Endoperoxides. (B) 4-AQs. (C) 8-AQs and amino alcohols. (D) Antifolates, antibiotics, and others.

Figure 3. The transmission-blocking potential of selected antimalarials in three bioassays that cover different phases of Plasmodium vector stage development.

(A) Assays examined exflagellation (P. falciparum), ookinete formation (P. berghei), or oocyst formation (P. falciparum). All antimalarials were screened at 10 µM. (B) The biological content of the in vitro P. berghei ookinete assay spans gamete formation, fertilization, zygote development, and ookinete formation. Ookinete formation was insensitive to most of the antimalarials tested. Atovaquone, cycloheximide, pyronaridine, pyrimethamine, and thiostrepton all strongly inhibited ookinete formation (p<0.03 by Student's t-test), while tafenoquine, cis-mirincamycin, and fosmidomycin gave an enhancement of ookinete formation that was not statistically significant. (C) The medium throughput fluorescent ookinete assay determined IC₅₀ values for pyronaridine, thiostrepton, cycloheximide, and atovaquone of 6 µM, 8 µM, 25 nM, and 65 nM, respectively. (D) The in vitro P. falciparum exflagellation assay exposes mature gametocytes to antimalarials for 24 h before triggering exflagellation. 16 out of 29 antimalarials tested, including all but one of the endoperoxides and 4-AQs, showed a statistically significant >50% inhibition of exflagellation (p<0.05). Pyronaridine, tert-butyl isoquine, NPC-1161B, OZ277, and cycloheximide all inhibited exflagellation totally at 10 µM. All experiments in triplicate. (E) The in vivo P. falciparum oocyst assay differs from the P. berghei oocyst assay in that mature gametocytes were exposed to the antimalarials in culture for 24 h before feeding to mosquitoes. The endoperoxides all strongly reduced transmission. NPC1161-B, lumefantrine, halofantrine, and mefloquine +RS isomer were also active. (n = 4–61 observations; average ± standard error of the mean [SEM]).

Figure 4. Validation and results of anti-infectives tested against P. yoelii liver stage parasites.

(A) Opera-generated images from a time point experiment using a 20× objective lens. P. yoelii parasites (red) were visualized using a mouse polyclonal HSP70 antibody, and the HepG2 cells (green) were stained with Hoechst 33342 nucleic acid dye. Median parasite areas for the 15-h, 27-h, and 51-h time points were calculated as 16, 52, and 158 µm², respectively, using a custom Acapella script. (B) A dose response plot with atovaquone from data generated from the Opera imaging system. The median areas of compound-treated parasites were compared to untreated DMSO controls to determine the degree of inhibition. The IC₅₀ value of atovaquone is 22 nM. (C) From the Acumen ^eX3 imaging system, a 384-well heat map plate image of P.yoelii fluorescence intensities from a dose response experiment with the most active anti-infectives. The three wells at the top left corner were not infected with sporozoites and served as a negative control. Wells were treated with a 1∶3 dilution of compounds starting at 30 µM (0.1% DMSO). In the experiments where the highest compound concentration tested was 10 µM, the entry is labelled 10. Compounds used are: naphthoquine (naph.), pyrimethamine (pyri.), trimethoprim (trim.), deferoxamine (defe.), artemisone (artem.), atovaquone (ato.), artesunate (artes.), cycloguanil (cyclo.), methylene blue (met. bl.), quinidine (quin.), dihydroartemisinin (DHA). Triangles represent the dilution steps of the drugs as described above. Stars designate the three wells with no sporozoite. (D) IC₅₀ results from a compound dose response experiment performed in a 384-well plate format. Results were generated from the Opera using median parasite area to determine level of inhibition.

Figure 5. Summary of the activity of the most widely used antimalarials throughout the life cycle of Plasmodium.

The three main phases, i.e., liver stage, blood stage, and vector stage, of the life cycle of Plasmodium are shown. The two key entry points leading to transmission of the parasites from vector to host and from host to vector are indicated (green circles). Parasite forms specific to each stage are highlighted and drugs identified as inhibitors of development of these forms are listed in boxes and coloured as described in Figure 1. Stars highlight components of the main artemisinin combination therapies: green, coartem; red, pyramax; orange, eurartesim; blue, ASAQ.