Rapid kill of malaria parasites by artemisinin and semi-synthetic endoperoxides involves ROS-dependent depolarization of the membrane potential

Objectives: Artemisinin and artemisinin semi-synthetic derivatives (collectively known as endoperoxides) are first-line antimalarials for the treatment of uncomplicated and severe malaria. Endoperoxides display very fast killing rates and are generally recalcitrant to parasite resistance development. These key pharmacodynamic features are a result of a complex mechanism of action, the details of which lack consensus. Here, we report on the primary physiological events leading to parasite death. Methods: Parasite mitochondrial (δΨ_m) and plasma membrane (δΨ_p) electrochemical potentials were measured using real-time single-cell imaging following exposure to pharmacologically relevant concentrations of endoperoxides (artemisinin, dihydroartemisinin, artesunate and the synthetic tetraoxane RKA182). In addition, mitochondrial electron transport chain components NADH:quinone oxidoreductase (alternative complex I), bc₁ (complex III) and cytochrome oxidase (complex IV) were investigated to determine their functional sensitivity to the various endoperoxides. Results: Parasite exposure to endoperoxides resulted in rapid depolarization of parasite δΨ_m and δΨ_p. The rate of depolarization was decreased in the presence of a reactive oxygen species (ROS) scavenger and Fe. ³⁺ chelators. Depolarization of δΨ_m by endoperoxides is not believed to be through the inhibition of mitochondrial electron transport chain components, owing to the lack of significant inhibition when assayed directly. Conclusions: The depolarization of δΨ_m and δΨ_p is shown to be mediated via the generation of ROS that are initiated by iron bioactivation of endoperoxides and/or catalysed by iron-dependent oxidative stress. These data are discussed in the context of current hypotheses concerning the mode of action of endoperoxides.