The proteasome inhibitor epoxomicin has potent Plasmodium falciparum gametocytocidal activity

Abstract

Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester and N-acetyl-L-leucyl-L-leucyl-L-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum.

FIG. 1.

Effect of cysteine protease inhibitors on erythrocytic-stage parasites. P. falciparum cultures containing asexual (Asex) stages (A and B) or gametocytes (C and D) were incubated with MLHF (100 μM), E64d (100 μM), ALLM (100 μM), ALLN (10 or 100 μM), or the equivalent amount of DMSO. The next day Giemsa-stained smears were prepared, and the number of asexual parasites (▪, □), stage II gametocytes (▪, □), and stage III to V gametocytes (, ░⃞) per 1,000 RBC that were morphologically normal (▪,), had enlarged food vacuoles (□), or were abnormally thin (░⃞) were counted. A statistically significant difference from the DMSO control was determined by a Fisher's exact two-sided test (Prism 4, Graphpad software) (*, P ≤ 0.025; **, P ≤ 0.008). (B and D) Representative images of the parasites treated with the indicated inhibitor. The parasite stage is indicated on the right, and enlarged food vacuoles are indicated by an arrow.

FIG. 2.

Stage-specific effect of protease inhibitors on parasite morphology. Eight days after setup, P. falciparum gametocyte cultures were incubated with E64d (100 μM) or an equivalent amount of DMSO for 24 h. For the next 3 days, the cultures were fed daily with fresh medium without drug. Giemsa-stained smears were prepared on day 9 (d9), 24 h after the addition of drug, and again 3 days later on day 12 (d12). The number of morphologically normal (▪) and abnormal (□) stage II, III, IV, and V gametocytes were counted per 1,000 RBC. For each stage, the number of normal parasites was compared to that with the DMSO control, and statistical significance was determined using a Fisher's exact test (Prism 4, Graphpad software). *, P ≤ 0.0001.

FIG. 3.

Effect of proteasome inhibitors on intraerythrocytic asexual (Asex) and sexual stages (G'cyte). P. falciparum cultures containing both asexual- and sexual-stage parasites were incubated with 10 nM, 100 nM, or 1 μM epoxomicin or the equivalent amount of DMSO for 24 h and then fed daily with fresh medium for the next 3 days. Giemsa-stained smears were made 24 and 72 h after the addition of drug, and the numbers of asexual- and sexual-stage parasites with normal morphology were counted. (A) The total parasitemia (asexual and sexual stages combined) at 24 (▪) and 72 h (□) after each treatment was calculated and plotted as a percentage of the result for the DMSO control. (B) Representative images of the parasites 24 and 72 h after the indicated treatment. (C) The asexual-stage (O) and sexual-stage (▪) parasitemia and the number of viable mouse (3T3) (▴) and human (A549) (x) cells were measured and plotted as a percentage of the results for the DMSO control.

FIG. 4.

Differential inhibition of asexual (Asex) parasites and mature stage V gametocytes. P. falciparum cultures containing asexual parasites and stage V gametocytes were treated for 24 h with quinine (10 and 1 μM), artemisinin (1 and 0.1 μM), epoxomicin (1 and 0.1 μM), or an equivalent amount of DMSO. Twenty-four hours later, Giemsa-stained smears were prepared and the number of asexual parasites (▪) and stage V gametocytes (▪) in 1,000 RBC were counted and plotted. For each stage, the number of normal parasites was compared to the result with the DMSO control, and statistical significance was determined using Fisher's exact two-sided test (Prism 4, Graphpad software). *, P ≤ 0.0001. Images representative of the parasites prior to (PreRx) or 24 h after the indicated treatment with DMSO (control), quinine (10 μM), artemisinin (1 μM), or epoxomicin (1 μM and 0.1 μM) are shown below the graph.

FIG. 5.

Proteolytic processing of the gamete surface antigen Pfs230 during gametogenesis. P. falciparum cultures containing stage V gametocytes were incubated with ALLN (A) (100 μM), epoxomicin (E) (1 and 10 μM), or the equivalent amount of DMSO (D) for 1 h. The parasites were pelleted, resuspended in emergence medium, and incubated at room temperature for 30 min to induce gametogenesis. The parasites were pelleted, and the supernatant was tested for the presence of the 37-kDa fragment of Pfs230 that is released following proteolysis.