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.

Figures

FIG. 1

Illustration of the procedures involved in the in vitro selection of atovaquone-resistant mutants.

FIG. 2

Amino acid sequence alignment of chicken, bovine, and P. falciparum CYT b with their SCR elements represented by bars on the top, second top, and bottom, respectively. Putative residues involved in atovaquone binding are shown in boldface type. Residues associated with atovaquone resistance are marked as: P. falciparum (●), P. yoelii (□), P. carinii (○), and P. berghei (■). The putative Qo sites are labeled.

FIG. 3

(A) Putative P. falciparum CYT b contact residues involved in atovaquone binding and the residues that were mutated in the atovaquone-resistant parasites. Atovaquone is represented in green stick form, with its lone electron pairs shown in light blue. The electron densities of all the atovaquone atoms are represented by thin lines. All amino acid residues shown as thick or thin sticks in this figure, except for T142, K272, P275, and G280, are predicted contact residues. The thin sticks represent the contact residues, and the thick sticks represent the contact residues that were mutated in the resistant parasite lines. Residues that were mutated in P. falciparum are shown in purple, those in P. yoelii are shown in yellow, and those in P. carinii are shown in orange. The red sticks represent residues that were predicted to be involved in differential atovaquone binding between P. falciparum and chicken CYT b. (B) Putative P. falciparum CYT b contact residues involved in atovaquone binding with the mutated residues from P. falciparum (purple), P. yoelii (yellow), and P. carinii (orange) that are associated with atovaquone resistance. (C) Putative chicken CYT b contact residues involved in atovaquone binding.

FIG. 3

(A) Putative P. falciparum CYT b contact residues involved in atovaquone binding and the residues that were mutated in the atovaquone-resistant parasites. Atovaquone is represented in green stick form, with its lone electron pairs shown in light blue. The electron densities of all the atovaquone atoms are represented by thin lines. All amino acid residues shown as thick or thin sticks in this figure, except for T142, K272, P275, and G280, are predicted contact residues. The thin sticks represent the contact residues, and the thick sticks represent the contact residues that were mutated in the resistant parasite lines. Residues that were mutated in P. falciparum are shown in purple, those in P. yoelii are shown in yellow, and those in P. carinii are shown in orange. The red sticks represent residues that were predicted to be involved in differential atovaquone binding between P. falciparum and chicken CYT b. (B) Putative P. falciparum CYT b contact residues involved in atovaquone binding with the mutated residues from P. falciparum (purple), P. yoelii (yellow), and P. carinii (orange) that are associated with atovaquone resistance. (C) Putative chicken CYT b contact residues involved in atovaquone binding.