5.3.1. Activation of the Endoperoxide to Generate ROS

The endoperoxides are converted into carbon-centered radicals, which mediate most of their effects. Activation is performed by the transfer of electrons from transition metals, mainly iron (Box 2), leading to the homolytic cleavage of the endoperoxide bridge [106–109]. The carbon-centered radical formed can alkylate heme, preventing its polymerization into hemozoin and thus inducing the formation of toxic ROS. They also alkylate several essential biomolecules, rendering them dysfunctional and consequently leading to plasmodial death. The iron could be heme (nonchelatable iron) (Figure 9) or freely circulating iron (chelatable iron).

Available evidence shows that artemisinin reacts more efficiently with heme than other forms of iron [110], thus pointing to the huge role played by heme in the pharmacological activity of endoperoxides. The dependence on iron is affected by the stage of the parasite. However, artemisinin activation at the early ring stages depends more on chelatable iron than on the trophozoite stage (35% vs. 15%), for which artemisinin activation depends more on heme generation from hemoglobin digestion [111]. This could be explained by a hemoglobinase system that is not fully developed at the ring stage but has a low level of falcipain 2 and 3 activity sufficient to sustain hemoglobin degradation that permits normal growth [111]. The molecular structure of an endoperoxide also determines its level of dependence on heme for activation [111]. Although the most studied source of heme for artemisinin activation is hemoglobin, the debate continues on whether other hemecontaining proteins may not do the same [107,112]. Some of these proteins are cytochrome p450, catalases, peroxidases, and other heme-containing proteins [113–116]. The use of chelators of free iron and inhibitors of hemoglobin digestion revealed that artemisinin activity at the early ring and trophozoite stages depends more than 80% on heme activation, while, at the mid-ring stage, it is 40% dependent. This means that artemisinin and, by extension, other endoperoxides may have other mechanisms of action that may not be iron-dependent [111].
