Potent Acridone Antimalarial against All Three Life Stages of Plasmodium

Abstract

New antimalarial therapeutics ideally should target all three major Plasmodium life cycle stages. Here we present an acridone antimalarial chemotype that is potent against blood, liver, and mosquito stages of malaria parasites, with the potential for single-dose cure of bloodstream infections, radical cure of liver infections, and blocking of transmission to mosquitoes. Attributes of lead candidate T111 include potent in vitro activity against cultured parasites, ex vivo activity against clinical isolates, oral single dose cure in an asexual blood stage rodent model, inhibition of sexual blood stage parasites, activity against relapsing parasites in non-human primate liver cells, prevention of parasite development in mosquitoes, and synergy in combination with tafenoquine against blood and liver stage parasites. Analysis of parasites selected for resistance to T111 suggested inhibition of the mitochondrial electron transport chain, with a mechanism distinct from that of other antimalarials in use or under development. Safety profiles, including toxicology evaluations in rats, showed a favorable therapeutic index. Overall, T111 emerges as a promising candidate for treatment and prevention of malaria.

Figure 1.

Antimalarial activities of T111 against blood and mosquito stage Plasmodium parasites. A) Dose response curves for activity against different asexual blood stage P. falciparum strains. n = 3, each performed in biological triplicates; error bars represent means of 3 independent experiments ± S.E. B) Ex vivo susceptibilities of blood stage P. falciparum clinical isolates from Burkina Faso and Uganda. Mean IC₅₀ values are shown as red lines with means for control laboratory strains in red (3D7) and green (Dd2). C) In vivo efficacy in mice of a single oral dose of T111 against blood stage P. yoelii. D) In vitro inhibition of sexual blood stage P. falciparum gametocyte development. n = 2, each performed in duplicate;, one-way ANOVA with Kruskal-Wallis multiple comparison test, *P = 0.0163; error bars represent means ± S.D. E) In vivo inhibition of P. falciparum oocyst development in mosquitoes after direct membrane feeding. Values represent the means for 10–23 mosquitoes per treatment in each of 3 independent experiments. Oocyst numbers are normalized to the mean of untreated controls. Differences between treatments were significant at p = 0.011 (Friedman test). F) In vivo inhibition of P. falciparum oocyst development in mosquitoes after tarsal contact. Pie charts show prevalence of infection (proportion of mosquitoes with at least one oocyst; Fisher’s exact test, ****P<0.0001), and intensity graph shows the number of oocysts per infected mosquito (median lines indicated, Mann-Whitney test). n=number of mosquitoes, two biological replicates.

Figure 2.

Drug-drug interactions between T111 and TQ against Plasmodium parasites. A) Isobologram for asexual blood stage P. falciparum parasites in vitro. B) Isobologram for liver stage rhesus P. cynomolgi with prophylactic (PROPH; drugs administered 1 hour post sporozoite infection on day 0, then continued on day 1 and 2) and radical cure (RAD CURE; drug exposure began on day 4, and continued on day 5, 6, and 7 post sporozoite infection) dosing. C) In vivo potentiation of TQ oral efficacy by T111 vs. P. yoelii in the blood stage 4-day suppression murine model.

Figure 3.

Bioluminescence and real time in vivo imaging of parasite loads in mice with or without treatment of test drugs or test drug combinations. The bioluminescence level represents the parasite burden. Intensity of bioluminescence is indicated by the color scale.

Figure 4.

Effect of T111 and TQ on eryptosis. A) Phosphatidylserine exposure, shown as RBC annexin V binding after 48 hr incubation with drugs. B) Reactive oxygen species formation, shown as 2’,7’-dichlorodihydrofluorescenin diacetate (DCFDA) fluorescence after 48 hr exposure to drugs. C) The size/volume of RBC, shown as forward scatter after 48 hr exposure to drugs. Two-way ANOVA comparison test. *p < 0.05, **p < 0.005, ***p < 0.0005.

Figure 5.

Selection of cyt b mutations in cultures incubated with the indicated concentrations of T111 for the indicated durations. *NC: not calculated; wks: weeks.