Insights into the role of heme in the mechanism of action of antimalarials

Abstract

By using cell fractionation and measurement of Fe(III)heme-pyridine, the antimalarial chloroquine (CQ) has been shown to cause a dose-dependent decrease in hemozoin and concomitant increase in toxic free heme in cultured Plasmodium falciparum that is directly correlated with parasite survival. Transmission electron microscopy techniques have further shown that heme is redistributed from the parasite digestive vacuole to the cytoplasm and that CQ disrupts hemozoin crystal growth, resulting in mosaic boundaries in the crystals formed in the parasite. Extension of the cell fractionation study to other drugs has shown that artesunate, amodiaquine, lumefantrine, mefloquine, and quinine, all clinically important antimalarials, also inhibit hemozoin formation in the parasite cell, while the antifolate pyrimethamine and its combination with sulfadoxine do not. This study finally provides direct evidence in support of the hemozoin inhibition hypothesis for the mechanism of action of CQ and shows that other quinoline and related antimalarials inhibit cellular hemozoin formation.

Figure 1

Iron and heme species in untreated and drug-treated parasites. a Total Fe content of untreated and drug treated trophozoites exposed to varying concentrations of CQ, or other antimalarials at 2.5 times their respective IC₅₀ values. Parasites were synchronized with sorbitol and cultured for 32 h with or without drug. Total iron was measured using the ferrozine method of Carter.(11) None of the measurements shown are significantly different from the control value, 0 nM CQ, (P > 0.05, n = 4, 4, 3, 7, 3, 5, 7, 3, 4, 4 and 4 respectively). Fraction of total heme present as b hemozoin (fraction 4), c “free” heme (fraction 3) and d Hb (fraction 2). Asterisks indicate statistical significance relative to control (2-tailed t-test): * P < 0.05; ** P < 0.01; *** P < 0.001, n = 3 except for pyrimethamine (P) where n = 6. e Parasite survival curve (open circles, left axis) determined using the lactate dehydrogenase assay of Makler et al.(12) and percentage “free” heme (closed circles, right axis) as a function of CQ dose.

Figure 2

A schematic representation of the strategy for measuring heme species in different cellular fractions. Parasites were cultured according to the method of Trager and Jensen.(16) The spectrum of fraction 1 was characteristic of Hb. The heme content of the remaining fractions were then measured using 2% SDS and 2.5% pyridine to form a heme-pyridine complex (based on a reported method for heme quantification).(17)

Figure 3

TEM images of control and treated parasitized RBCs with iron distribution (blue) measured by ESI using EELS. a Untreated trophozoites show negligible Fe except for the strong signal from hemozoin (e.g. crystal marked by arrow). On the other hand, Fe is readily detected in the RBC cytoplasm. Note the sharp gradient in Fe signal between RBC and parasite and at the edges of hemozoin crystals indicating that Fe distribution is not affected by fixation. b Parasites treated with 30 nM CQ clearly exhibit iron in the parasite cytoplasm at the trophozoite stage, especially concentrated in regions such as that indicated by the star. Note the low Fe signal in the DV apart from hemozoin. Since almost all Fe in both control and treated parasites is present as heme species, this Fe distribution can be attributed to the heme distribution. Furthermore, since there is little Hb present in trophozoites treated with 30 nM CQ, the heme present in the parasite cytoplasm is probably the “free” heme fraction. RBCs delineated by long blue dashes, parasite by short red dashes and DV by yellow dots in the ESI images. Samples were prepared as described in a previous report.(2) (c) A thin layer chromatogram run in acetone shows that heme is completely immobilized by OsO₄ treatment. Similar results were seen in ethanol and aqueous ethanol (not shown). (d) Extracted, untreated hemozoin crystals are highly ordered, with uniform diffraction fringes throughout the crystals. An enlargement of the crystal is shown in inset in which the fringes can be more clearly seen. A measurement of intensity along the blue line in the enlargement permits measurement of the lattice spacing which corresponds closely to that expected for the {100} face of hemozoin (12.57 Å).(18) (e) Extracted hemozoin crystals from parasites treated with 30 nM CQ are less uniform. (f) Grain boundaries are clearly visible at 60 nM CQ. In (a) and (b) R = RBC, P = parasite, F = DV, H = hemozoin, N = probable nucleus, m = mitochondrion and er = possible endoplasmic reticulum. In (c) the solvent front is marked by a dashed arrow and the position of the hemin spot by a solid arrow. Scale bars in d and e are 5 nm and 200 nm, respectively. See Supporting Information.

Figure 4

Effects of single doses of antimalarial drugs on: a “free” heme; and b hemozoin in purified trophozoites cultured in synchrony after 32 h exposure. Drugs were present at 2.5 times their respective IC₅₀ values (except CQ which was present at 45 nM and SP present at 2.5 times the IC₅₀ of pyrimethamine and with 16 mol equivalents of sulfadoxine relative to pyrimethamine). Asterisks indicate statistical significance relative to untreated control (2-tailed t-test): * P < 0.05; ** P < 0.01; *** P < 0.001, n = 3 except for artesunate (n = 5), mefloquine (n = 5) and pyrimethamine (labeled P on the graph) (n = 7). The white dotted lines represent three standard deviations in the control values.