Uncovering the Molecular Mode of Action of the Antimalarial Drug Atovaquone Using a Bacterial System

Abstract

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc 1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c 1 of the cytochrome bc 1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc 1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc 1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc 1 complex.Malaria is one of the most intractable human afflictions in the world. Despite intensive campaigns against the parasitic disease, an estimated 300 to 500 million cases still occur each year. Plasmodium falciparum, the causative agent of the most lethal form of malaria, is responsible for over 2 million deaths annually, largely among young children and pregnant women (1, 2). Efforts to eradicate malaria have not been successful, and the situation may be worsening due, in large part, to the emergence and spread of drug-resistant parasites. The need for new antimalarial drugs is now widely recognized. Atovaquone represents a new class of drugs having a metabolic target different from extant antimalarial drugs to which resistance is widespread. Atovaquone was found to be a very effective antimalarial compound, but unsuitable for use as a single agent because of the relatively quick emergence of resistance (3-5). However, in combination with the synergistic agent proguanil, it has been effective for both therapeutic and prophylactic uses (5-7). On the other hand, once atovaquone resistance has arisen, the combination is no longer effective against malaria parasites (8,9).Previous studies have shown that atovaquone selectively inhibits mitochondrial electron transport in the parasite, consistent with the prevalent theory that hydroxynaphthoquinones function as ubiquinone antagonists (10, 11). Additionally, atovaquone was found to colla...

Metabolic Fate of Fumarate, a Side Product of the Purine Salvage Pathway in the Intraerythrocytic Stages of Plasmodium falciparum

“…The latter generates reduced coenzyme Q (CoQH 2 ), from coenzyme Q (CoQ) along with the oxidation of malate to oxaloacetate. CoQH 2 is then oxidized directly by complex III, and the reducing equivalents are transferred to cytochrome c. Atovaquone is a specific and potent inhibitor of the enzymatic activity of P. falciparum complex III (25), and thus, treatment of P. falciparum parasites should lead to a depletion of CoQ. We examined the effect of this depletion of CoQ on the conversion of fumarate to aspartate.…”

A drug-selected Plasmodium falciparum lacking the need for conventional electron transport

“…Although these interrelationships determine the viability of the parasite and our ability to reduce that viability, they are only generally understood. Review of Plasmodial electron transport and cyt bc 1 complex function are beyond the scope of this report, but in-depth discussions are available [1][2][3].…”

“…Although these interrelationships determine the viability of the parasite and our ability to reduce that viability, they are only generally understood. Review of Plasmodial electron transport and cyt bc 1 complex function are beyond the scope of this report, but in-depth discussions are available [1][2][3].The anti-malarial drug atovaquone occupies the quinol oxidase (Q o ) site of mitochondrial cyt b, inhibiting electron flux through the cyt bc 1 complex (ubiquinol:cytochrome c oxidoreductase or complex III) and collapsing mitochondrial membrane potential with a potency 1000-fold greater in Plasmodium than mammalian cells [4,5]. Unfortunately, high rates of recrudescent infection and treatment failure were seen after anti-malarial use of atovaquone alone [6], and treatment failures after atovaquone-proguanil combination therapy (Malarone®) were soon evident [7,8] despite only limited worldwide anti-malarial use.…”

Variation among Plasmodium falciparum Strains in Their Reliance on Mitochondrial Electron Transport Chain Function

“…DHODH catalyzes the fourth step and the sole redox reaction of the de novo pyrimidine biosynthesis pathway, which is the only source of pyrimidines in malaria parasites (17). Atovaquone, one of the two drugs composing the antimalarial product Malarone, poisons the parasites by inhibiting the cytochrome bc 1 complex, thereby blocking the mtETC (13,19,30). P. falciparum DHODH itself is also a promising drug target, with recent reports of inhibitors with low-nanomolar 50% effective concentrations (EC 50 s), such as several aryl-substituted triazolopyrimidines (2,16,26).…”