Atovaquone, a
coenzyme Q analogue has been indicated to specifically target the
cytochrome bc1 complex of the mitochondrial respiratory chain in the malarial parasite and other protozoan. Various mutations in the
quinone binding site of the
cytochrome b gene of Plasmodium spp. such as M133I, L144S, L271V, K272R, Y268C, Y268S, Y268N, and V284F are suggesting to associate with resistance to
atovaquone. There is no direct evidence of relation between the mutations and resistance to
atovaquone in Plasmodium parasite that has been available. Technical difficulties in isolating active assayable mitochondria in the malarial parasite hinder us to obtain direct biochemical evidence to support the relation between the mutations and drug resistance. The establishment of a mitochondrial isolation method for the
malaria parasite has allowed us to test the degree of resistance of Plasmodium berghei isolates to
atovaquone directly. We have tested the activity of
dihydroorotate (DHO)-
cytochrome c reductase in various P. berghei
atovaquone resistant clones in the presence of a wide concentration range of
atovaquone. Our results show the IC(50) of P. berghei
atovaquone resistant clones is much higher (1.5 up to 40 nM) in comparison to the
atovaquone sensitive clones (0.132-0.465 nM). The highest IC(50) was revealed in clones carrying Y268C and Y268N mutations (which play an important role in
atovaquone resistance in Plasmodium falciparum), with an approximately 100-fold increase. The findings indicate the importance of the mutation in the
quinone binding site of the
cytochrome b gene and that provide a direct evidence for the
atovaquone inhibitory mechanism in the
cytochrome bc1 complex of the parasite.