Artemisinin-based combination
therapies (ACTs) are highly effective for the treatment of
Plasmodium falciparum malaria, yet their sustained efficacy is threatened by the potential spread of parasite resistance. Recent studies have provided evidence that
artemisinins can inhibit the function of PfATP6, the P. falciparum ortholog of the ER
calcium pump SERCA, when expressed in Xenopus laevis oocytes. Inhibition was significantly reduced in an L263E variant, which introduced the mammalian residue into a putative
drug-binding pocket. To test the hypothesis that this single mutation could decrease P. falciparum susceptibility to
artemisinins, we implemented an allelic-exchange strategy to replace the wild-type pfatp6 allele by a variant allele encoding L263E. Transfected P. falciparum clones were screened by PCR analysis for disruption of the endogenous locus and introduction of the mutant L263E allele under the transcriptional control of a
calmodulin promoter. Expression of the mutant allele was demonstrated by
reverse transcriptase (RT) PCR and verified by sequence analysis. Parasite clones expressing wild-type or L263E variant PfATP6 showed no significant difference in 50% inhibitory concentrations (IC(50)s) for
artemisinin or its derivatives
dihydroartemisinin and
artesunate. Nonetheless, hierarchical clustering analysis revealed a trend toward reduced susceptibility that neared significance (
artemisinin, P approximately = 0.1;
dihydroartemisinin, P = 0.053 and P = 0.085; and
artesunate, P = 0.082 and P = 0.162 for the D10 and 7G8 lines, respectively). Notable differences in the distribution of normalized IC(50)s provided evidence of decreased responsiveness to
artemisinin and
dihydroartemisinin (P = 0.02 for the D10 and 7G8 lines), but not to
artesunate in parasites expressing mutant PfATP6.