title: Direct evidence for the atovaquone action on the Plasmodium cytochrome bc1 complex.
abstract: 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.
authors.affiliation: Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia.
authors.forename: Josephine E
authors.initials: JE
authors.lastname: Siregar
authors.affiliation: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Genji
authors.initials: G
authors.lastname: Kurisu
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tamaki
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Motomichi
authors.initials: M
authors.lastname: Matsuzaki
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Sakamoto
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Fumika
authors.initials: F
authors.lastname: Mi-ichi
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yoh-ichi
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Department of Medical Zoology, Jichi Medical School, Minami-kawachi, Tochigi 329-0498, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hirai
authors.affiliation: Department of Medical Zoology, Jichi Medical School, Minami-kawachi, Tochigi 329-0498, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Matsuoka
authors.affiliation: Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia.
authors.forename: Din
authors.initials: D
authors.lastname: Syafruddin
authors.affiliation: Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia.
authors.forename: Sangkot
authors.initials: S
authors.lastname: Marzuki
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: kitak@m.u-tokyo.ac.jp.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
mesh_terms: D000962:Antimalarials / Q000378:metabolism / Q000494:pharmacology*; D053626:Atovaquone / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D003198:Computer Simulation; D045303:Cytochromes b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics; D050259:Genes, Mitochondrial; D008958:Models, Molecular; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000962:Antimalarials; D045303:Cytochromes b; D010088:Oxidoreductases; D014450:Electron Transport Complex III; D053626:Atovaquone
keywords: Atovaquone-resistance; Cytochrome bc(1) complex; Malaria; Plasmodium