Alternative Oxidase Inhibitors Potentiate the Activity of Atovaquone against Plasmodium falciparum

Recent evidence suggests that the malaria parasite Plasmodium falciparum utilizes a branched respiratory pathway including both a cytochrome chain and an alternative oxidase. This branched respiratory pathway model has been used as a basis for examining the mechanism of action of two antimalarial agents, atovaquone and proguanil. In polarographic assays, atovaquone immediately reduced the parasite oxygen consumption rate in a concentration-dependent manner. This is consistent with its previously described role as an inhibitor of the cytochrome bc1 complex. Atovaquone maximally inhibited the rate of P. falciparum oxygen consumption by 73% 6 10%. At all atovaquone concentrations tested, the addition of the alternative oxidase inhibitor, salicylhydroxamic acid, resulted in a further decrease in the rate of parasite oxygen consumption. At the highest concentrations of atovaquone tested, the activities of salicylhydroxamic acid and atovaquone appear to overlap, suggesting that at these concentrations, atovaquone partially inhibits the alternative oxidase as well as the cytochrome chain. Drug interaction studies with atovaquone and salicylhydroxamic acid indicate atovaquone’s activity against P. falciparum in vitro is potentiated by this alternative oxidase inhibitor, with a sum fractional inhibitory concentration of 0.6. Propyl gallate, another alternative oxidase inhibitor, also potentiated atovaquone’s activity, with a sum fractional inhibitory concentration of 0.7. Proguanil, which potentiates atovaquone activity in vitro and in vivo, had a small effect on parasite oxygen consumption in polarographic assays when used alone or in the presence of atovaquone or salicylhydroxamic acid. This suggests that proguanil does not potentiate atovaquone by direct inhibition of either branch of the parasite respiratory chain. We recently presented evidence that the Plasmodium falciparum respiratory chain is branched and contains an alternative oxidase as well as a cytochrome chain (21). The alternative oxidases of plants, fungi, and trypanosomatids transfer electrons directly from ubiquinone to oxygen in a cyanide-insensitive reaction (19). In systems containing both an alternative oxidase and the cytochrome pathway, the alternative oxidase does not appear to contribute directly to the mitochondrial membrane potential or the energy balance of the cell. It can, however, contribute indirectly by accepting electrons from enzymes which donate electrons to ubiquinone. Alternative oxidase has been shown to contribute to the survival of plant cells under conditions in which the cytochrome chain is overloaded or blocked (25). The respiratory pathway of P. falciparum appears to be more important for pyrimidine biosynthesis than for energy generation (12, 22). Interestingly, the activity of P. falciparum dihydroorotate dehydrogenase, the enzymatic link between electron transport and pyrimidine biosynthesis, is inhibited by both alternative oxidase and cytochrome chain inhibitors (12, 14, 15). Atovaquone, a hydroxynaphthoquinone, is a potent antimalarial agent which is known to inhibit dihydroorotate dehydrogenase activity (13, 14). At concentrations selective for P. falciparum, atovaquone has been shown to act specifically on the cytochrome bc1 complex (complex III of the cytochrome chain) (10). Clinical trials in which atovaquone was used alone to treat P. falciparum malaria resulted in an initial clearance of parasites from the blood followed by recrudescence in 25 to 75% of the patients (5, 18). The model of a branched respiratory pathway in P. falciparum suggests that an alternative oxidase in these parasites could enable the survival of some parasites in the presence of atovaquone. This could explain the high recrudescence rate seen when atovaquone is used singly to treat P. falciparum malaria in clinical trials.