Peroxidic antimalarials such as the semisynthetic artemisinins are critically important in the treatment of drug-resistant malaria. Nevertheless, their peroxide bond-dependent mode of action is still not well understood. Using combination experiments with cultured Plasmodium falciparum cells, we investigated the interactions of the nitroxide radical spin trap, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), and four of its analogs with artemisinin and the ozonide drug development candidate OZ277. The antagonism observed for combinations of artemisinin or OZ277 with the TEMPO analogs supports the hypothesis that the formation of carbon-centered radicals is critical for the activity of these two antimalarial peroxides. The TEMPO analogs showed a trend toward greater antagonism with artemisinin than they did with OZ277, an observation that can be explained by the greater tendency of artemisinin-derived carbon-centered radicals to undergo internal self-quenching reactions, resulting in a lower proportion of radicals available for subsequent chemical reactions such as the alkylation of heme and parasite proteins. In a further mechanistic experiment, we tested both artemisinin and OZ277 in combination with their nonperoxidic analogs. The latter had no effect on the antimalarial activities of the former. These data indicate that the antimalarial properties of peroxides do not derive from reversible interactions with parasite targets.The semisynthetic artemisinins are critically important antimalarials in the treatment of drug-resistant malaria and are recommended for use in combination with other antimalarial drugs (32) to increase efficacy and preclude or delay drug resistance. The discovery of artemisinin led to an investigation of diverse classes of synthetic peroxides as potential antimalarial agents (17,27). One such peroxide, the ozonide OZ277 (arterolane) (31), has now entered phase III clinical trials in the form of an arterolane maleate-piperaquine phosphate combination (22). A working hypothesis (16,19,23) put forth to account for the antimalarial specificity (18) of natural-product and synthetic peroxides is that the pharmacophoric peroxide bond undergoes reductive activation by heme released by parasite hemoglobin digestion (9, 11). The irreversible redox reaction between antimalarial peroxides and heme produces carbon-centered radicals or carbocations that alkylate heme (5, 20, 24, 25) and proteins (2, 3, 8, 33), leading to the perturbation of lipid components of the parasite digestive vacuole (6, 13). Although artemisinin and OZ277 are nearly equipotent inhibitors of Plasmodium falciparum growth in vitro (18), their very different 50% inhibitory concentrations (IC 50 s) (79 and 7,700 nM) (30) against one putative target enzyme, the Sarcoendoplasmic reticulum Ca 2ϩ -ATPase PfATP6 (8, 12), reveal that the precise mechanism of action of antimalarial peroxides is still not well understood.In this study, we report the results of two different types of in vitro P. falciparum combination experiments with artemisi...
