HDQ, a potent inhibitor of Plasmodium falciparum proliferation, binds to the quinone reduction site of the cytochrome bc1 complex

Abstract

The mitochondrial bc(1) complex is a multisubunit enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c coupled to the vectorial translocation of protons across the inner mitochondrial membrane. The complex contains two distinct quinone-binding sites, the quinol oxidation site of the bc(1) complex (Q(o)) and the quinone reduction site (Q(i)), located on opposite sides of the membrane within cytochrome b. Inhibitors of the Q(o) site such as atovaquone, active against the bc(1) complex of Plasmodium falciparum, have been developed and formulated as antimalarial drugs. Unfortunately, single point mutations in the Q(o) site can rapidly render atovaquone ineffective. The development of drugs that could circumvent cross-resistance with atovaquone is needed. Here, we report on the mode of action of a potent inhibitor of P. falciparum proliferation, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ). We show that the parasite bc(1) complex--from both control and atovaquone-resistant strains--is inhibited by submicromolar concentrations of HDQ, indicating that the two drugs have different targets within the complex. The binding site of HDQ was then determined by using a yeast model. Introduction of point mutations into the Q(i) site, namely, G33A, H204Y, M221Q, and K228M, markedly decreased HDQ inhibition. In contrast, known inhibitor resistance mutations at the Q(o) site did not cause HDQ resistance. This study, using HDQ as a proof-of-principle inhibitor, indicates that the Q(i) site of the bc(1) complex is a viable target for antimalarial drug development.

Fig 1

Effect of HDQ on P. falciparum PfNDH2, bc₁ complex and mitochondrial membrane potential. (a) The PfNDH2 activity (●) was determined by monitoring NADH oxidation and concomitant decylquinone reduction; the bc₁ activity (○) was determined by monitoring cytochrome c reduction using decylubiquinol as an electron donor (see Materials and Methods). All data have been acquired from multiple observations from at least three separate preparations. DMSO in the assays did not exceed 0.3%. The IC₅₀s were calculated by using the four-parameter logistic method. (b) Effect of HDQ on the mitochondrial membrane potential (Ψm) of P. falciparum. The time course of TMRE-dependent fluorescence after the addition of HDQ (100 nM) to P. falciparum-infected erythrocytes is shown. The data were normalized to 100% in untreated and to 0% in CCCP (10 μM)-treated cells. Graph shows the mean data derived from experiments performed independently ± the standard errors (n ≥ 3).

Fig 2

Sensitivity of the yeast bc₁ complex activity to HDQ and to the Q_o site inhibitor azoxystobin. The quinol cytochrome c reductase activity was measured using mitochondria prepared from WT yeast cells (see Materials and Methods). The measures were repeated at least three times and averaged. The errors did not exceed 10% of the presented values. The data are presented as the ratio of the inhibitor concentration (○, azoxystrobin; ●, HDQ) to the bc₁ complex concentration. An IC₅₀ ratio of 4 to 5 (inhibitor molecules per bc₁ complex) was estimated from the plots.

Fig 3

Model of HDQ binding in the Q_i site and localization of Q_i mutations. (a) Structural model (obtained as described in Materials and Methods) showing mutations causing resistance to HDQ (red) and mutations without effect on HDQ resistance (green). (b) Comparison of the sequences of Q_i site region between bovine (Bt), yeast S. cerevisiae (Sc), and P. falciparum (Pf) cytochromes b. Residues in close contact (>4 Å) with the antimycin bound at the Q_i site in the bovine enzyme are indicated in boldface (28). The mutated residues analyzed in the present study are marked with an asterisk (*).