Mutations in Plasmodium falciparum Cytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

Abstract

Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYT bc 1 ) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25-to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.The widespread resistance of malaria parasites to standard antimalarial drugs is a serious global health problem. The urgent need for new antimalarial drugs has led to the development of atovaquone (566C80) which, combined with proguanil, has been licensed as Malarone. There is some concern that parasites may develop resistance to Malarone. In one study, 33% of patients treated with atovaquone alone experienced a recrudescence of parasitemia after treatment. These parasites tolerated up to 1,000-fold higher concentrations of atovaquone than did the pretreated parasites (16). Atovaquone-resistant parasites have been readily selected in vitro. Up to 1 in 10 5 parasites became resistant to the drug after having been cultured in the presence of 10 Ϫ8 M atovaquone for 5 weeks (21, 23).Atovaquone has potent blood schizonticidal activity and is also effective against the preerythrocytic (2, 4, 5) and sexual stages (8, 9) of the malaria parasite. It acts by inhibiting mitochondrial electron transport (10) and collapsing mitochondrial membrane potential (25). From these observations and on the basis of its structural similarity to ubiquinol, it has been postulated that atovaquone binds to parasite cytochrome b (CYT b) (31). The inhibitors stigmatellin and 5-n-undecyl-4,7-dioxobenzoxythiazol (UHDBT), which are structurally similar to atovaquone, have been shown to bind at the ubihydroquinone (Q o ) site of CYT b and inhibit electron transport. Single point mutations within the Q o site confer resistance to these inhibitors in a variety of microorganisms (7). Two mutations in close proximity to the Q o site in Pneumocystis carinii are associated with atovaquone prophylaxis failure (33). Atovaquone-resistant Plasmodium yoelii lines have been derived from infected mice treated with suboptimal doses of atovaquone. All resistant lines ...

Molecular Basis for Atovaquone Binding to the Cytochrome bc 1 Complex

“…The resulting modeled structure accounted for the enzymatic and spectroscopic results and was more stable than the structure lacking the water-mediated hydrogen bond. This computed structure differs significantly from two previously postulated structures for atovaquone bound to the bc 1 complex (12,13). In those structures, the hydroxyl group of atovaquone was oriented toward Glu 272 in both cases, and one of the structures failed to account for atovaquone interaction with the Rieske protein (13).…”

Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action

“…Yet, substitutions without major loss of enzyme activity can lower inhibition efficiency, either by deleting interactions or by introducing sterical hindrance. The most prevalent mutation that is associated with acquired atovaquone resistance in P. falciparum is Y268S 30,33 (Y279S in yeast numbering). The yeast mutation Y279S 28,32,35 showed a B40-fold increased IC 50 (B1.7 mM) for atovaquone as compared with the wild type 35 .…”

“…Similar potencies are described for other parasitic and fungal targets, but the drug also inhibits human cyt bc 1 , although with lower submicromolar affinity 27 . The molecular basis for broad target spectrum on one hand and species-specific efficacies on the other was addressed with several molecular modelling studies 25,[28][29][30][31] and genetic approaches 29,[32][33][34][35] , but was not resolved so far. Characterization of the binding mode is also essential to understand mutation-induced resistance.…”

Antimalarial 4(1H)-pyridones bind to the Q i site of cytochrome bc 1

“…Several mutations, giving rise to atovaquone-resistant strains of P. falciparum and Toxoplasma gondii, have been reported since the turn of the century (23)(24)(25)(26)(27). These mutations arise in the Q o pocket and prevent atovaquone from binding (28).…”