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Accelerated Communication

Acridinediones: Selective and Potent Inhibitors of the Malaria Parasite Mitochondrial bc₁ Complex

Liverpool School of Tropical Medicine, Liverpool, United Kingdom (G.A.B., N.F., P.A.S., P.G.B., S.A.W.). Departments of Chemistry (N.B., R.B.-L., P.M.O.) and Pharmacology and Therapeutics (D.P.W., A.O.), University of Liverpool, Liverpool, United Kingdom; and Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France (B.M.)

Received January 10, 2008; accepted February 29, 2008

	Abstract

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The development of drug resistance to affordable drugs has contributed to a global increase in the number of deaths from malaria. This unacceptable situation has stimulated research for new drugs active against multidrug-resistant Plasmodium falciparum parasites. In this regard, we show here that deshydroxy-1-imino derivatives of acridine (i.e., dihydroacridinediones) are selective antimalarial drugs acting as potent (nanomolar K_i) inhibitors of parasite mitochondrial bc₁ complex. Inhibition of the bc₁ complex led to a collapse of the mitochondrial membrane potential, resulting in cell death (IC₅₀ 15 nM). The selectivity of one of the dihydroacridinediones against the parasite enzyme was some 5000-fold higher than for the human bc₁ complex, significantly higher (200 fold) than that observed with atovaquone, a licensed bc₁-specific antimalarial drug. Experiments performed with yeast manifesting mutations in the bc₁ complex reveal that binding is directed to the quinol oxidation site (Q_o) of the bc₁ complex. This is supported by favorable binding energies for in silico docking of dihydroacridinediones to P. falciparum bc₁ Q_o. Dihydroacridinediones represent an entirely new class of bc₁ inhibitors and the potential of these compounds as novel antimalarial drugs is discussed.

Acridine-based drugs have a long history in malaria chemotherapy. Mepacrine was the first synthetic antimalarial blood schizontocide used clinically (Wernsdorfer and Payne, 1991); the related drug pyronaridine has been used for nearly 20 years as a monotherapy to treat malaria in China (Shao, 1990). Pyramax, a pyronaridine-artesunate combination treatment, is currently undergoing phase III clinical trials (http://www.mmv.org). In addition, acridine congeners, including the acridones (Basco et al., 1994; Winter et al., 2006) and dihydroacridinediones (Dürckheimer et al., 1980; Dorn et al., 2001), have also demonstrated potent antimalarial activity, in some cases with good in vitro therapeutic indices (Winter et al., 2006).

Many acridine-based compounds can bind to heme (e.g., Chou and Fitch, 1993; Dorn et al., 1998, 2001; Auparakkitanon et al., 2003, 2006), the by-product of parasite hemoglobin digestion. Clinically relevant acridines, such as quinacrine and pyronaridine, are believed to confer almost all of their antimalarial activity through this interaction by preventing the crystallization of heme (Dorn et al., 1998; Auparakkitanon et al., 2006). However, not all acridine-based inhibitors kill the parasite via this route. Some 9-aniloacridines, for example, have been shown to exert their antimalarial activity through the inhibition of DNA topoisomerase II (Gamage et al., 1994; Auparakkitanon and Wilairat, 2000). Although some dihydroacridinediones have been reported to inhibit the malaria parasite respiratory pathway, causing a reduction in whole-cell O₂ consumption (Suswam et al., 2001).

View larger version (7K): [in this window] [in a new window]   Fig. 1. Chemical structure of key bc1 inhibitors used in this study.

In this study, we have investigated the antimalarial mode of action of two dihydroacridinediones, floxacrine and WR249685 (the S-enantiomer of WR243246), developed by the Walter Reed Army Institute of Research (Fig. 1) (Raether and Fink, 1979, 1982; Schmidt, 1979; Kesten et al., 1992; Dorn et al., 2001). Both of these compounds show heme binding and bc₁ inhibitory properties; however, whereas floxacrine kills parasites via a heme-mediated process, WR249685 is shown here to be a highly selective inhibitor of the Q_o of the Plasmodium falciparum bc₁ complex. The molecular nature of the selectivity of these drugs and their potential as novel antimalarial drugs is discussed.

	Materials and Methods

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Parasite, Culture, and Drug Sensitivity Assays. P. falciparum (3D7 strain) cultures consisted of a 2% suspension of O+ erythrocytes in RPMI 1640 medium (glutamine, and NaHCO₃) supplemented with 10% pooled human AB+ serum, 25 mM HEPES, pH 7.4, and 20 µM gentamicin sulfate (Trager and Jensen, 1976). Cultures were grown under a gaseous headspace of 4% O₂, 3%CO₂ in N₂ at 37°C. Parasite growth was synchronized by treatment with sorbitol (Lambros and Vanderberg, 1979). The sensitivity of P. falciparum-infected erythrocytes to various drugs was determined using the [³H]hypoxanthine incorporation method (Desjardins et al., 1979) with an inoculum size of 0.5% parasitemia (ring stage) and 1% hematocrit. IC₅₀ values were calculated by using the four-parameter logistic method (Grafit program; Erithacus Software, Horley, Surrey, UK). To determine whether the antimalarial activity of two drugs is additive, antagonistic, or synergistic, parasite growth was tested by titration of the two drugs at fixed ratios proportional to their IC₅₀ values. The fractional inhibitory concentrations of the resulting IC₅₀ values were plotted as isobolograms (Berenbaum, 1978).

Inhibition of in Vitro Hemozoin Formation. Assays were performed based on the methods by Bray et al. (1999) and Stead et al. (2001). In brief, an aliquot of ghost erythrocyte membrane (100 µl) and FPIX (100 µl of 3 mM in 0.1 M NaOH) were mixed with an aliquot of 1 M HCl (10 µl) and sodium acetate (500 mM, pH 5.2) was added to give a volume of 900 µl in each tube. A series of drug concentrations were prepared in water, and 100 µl of each was added to the appropriate samples. Samples were mixed and incubated for 48 h at 37°C, with occasional mixing. After incubation, samples were centrifuged (15,000g, 15 min, 21°C), and the hemozoin pellet was repeatedly washed with 2% (w/v) SDS in 0.1 M sodium bicarbonate, pH 9.0, until the supernatant was clear (usually 3 to 4 times). After the final wash, the supernatant was removed, and the pellet was resuspended in 1 ml of 0.1 M NaOH and incubated for a further 1 h at room temperature. The hemozoin content was determined by measuring the absorbance at 400 nm. The concentration of drug required to produce 50% inhibition of hemozoin production (IC₅₀) was determined graphically as described for the drug sensitivity assays.

Determination of Heme-Drug Dissociation Constants. Heme-drug equilibrium constants were determined based on a UV-visible spectroscopic method (Egan et al., 1997). To provide a strictly monomeric heme (ferriprotoporphyrin IX) species in solution, heme (6 µM) was prepared in a HEPES (20 mM, pH 7.2)-buffered solution of 40% (v/v) DMSO (Egan et al., 1997). UV-visible titrations of antimalarial drugs chloroquine, amodiaquine, floxacrine, and WR249685 were performed monitoring the Soret band of the porphyrin (390-460 nm). The resulting titration curves were analyzed using a nonlinear curve-fitting program (Pro-Fit) and thermodynamic parameters were derived from modeling, assuming a 1:1 complex of drug and heme (Marques et al., 1996; Egan et al., 1997).

Preparation of P. falciparum Cell-Free Extracts. Free parasites were prepared from aliquots of infected erythrocytes (approximately 8 x 10⁹ cells/ml) by adding 5 volumes of 0.15% (w/v) saponin in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 1.76 mM K₂HPO₄, 8.0 mM Na₂HPO₄, and 5.5 mM D-glucose, pH 7.4) for 5 min, followed by three washes in HEPES (25 mM)-buffered RPMI containing a protease inhibitor cocktail (Complete Mini; Roche, Mannheim, Germany). A cell extract was prepared by repeated freezethawing in liquid N₂, followed by disruption with a sonicating probe.

Human Liver Microsome Preparation. Histological normal liver was obtained from white transplant donors. The certified cause of death was traumatic injury due to a automobile accident. The liver samples were transferred to the laboratory within 30 min of death. They were portioned, frozen in liquid nitrogen, and stored at -80°C. Approval was granted by the Liverpool local research Ethics Committee, and prior consent was obtained from the donors' relatives.

Liver tissue was washed briefly in ice-cold isolation buffer (0.154 M KCl and 50 mM Tris-HCl, pH 7.4). The tissues were homogenized in 4x volume of isolation buffer and then centrifuged (10,000g, 20 min, 4°C). The pellet was discarded and the supernatant was then centrifuged (105,000g, 60 min, 4°C). The microsomal pellet was washed by resuspension in fresh buffer and centrifuged again (105,000g, 60 min, 4°C). Microsomes were resuspended in 2x volume of 0.12 M Tris, pH 7.4, and stored frozen (-80°C) in 1-ml aliquots at 80°C.

Preparation of Yeast Cytochrome b Mutants. Generation of mutant strains and preparation of crude mitochondrial membranes was performed as described previously (Fisher et al., 2004b).

Bovine Mitochondrial Membrane Preparation. Bovine mitochondrial membranes (Keilin-Hartree particles) were prepared as described by Kuboyama et al. (1972)

Rat Liver Microsome Preparation. Adult male Wistar rats were obtained from Charles River Laboratories (Margate, Kent, UK). Wistar rat liver microsomes were prepared from male rats (125-170 g) as described by Gill et al. (1995).

Preparation of Decylubiquinol. The artificial quinol electron donor was prepared based on the method of Fisher et al. (2004b). In brief, 2,3-dimethoxy-5-methyl-n-decyl-1,4-benzoquinone (decylubiquinone), an analog of ubiquinone, was dissolved (10 mg) in 400 µl of nitrogen-saturated hexane. An equal volume of aqueous 1.15 M sodium dithionite was added, and the mixture shaken vigorously until colorless. The upper, organic phase was collected, and the decylubiquinol recovered by evaporating the hexane under N₂. The decylubiquinol was dissolved in 100 µl of 96% ethanol (acidified with 10 mM HCl) and stored in aliquots at -80°C. Decylubiquinol concentration was determined spectrophotometrically from absolute spectra, using _288-320 = 4.14 mM^-1 cm^-1.

Measurement of bc₁ Activity. Cytochrome c reductase activity measurements were assayed in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 10 mM KCN, and 30 µM equine cytochrome c (Sigma Chemical, Poole, Dorset, UK) at room temperature (Fisher et al., 2004b). Cytochrome c reductase activity was initiated by the addition of decylubiquinol (50 µM). Reduction of cytochrome c was monitored in a Cary 4000 UV-visible spectrophotometer (Varian, Inc., Palo Alto, CA) at 550 versus 542 nm. Initial rates (computer-fitted as zero-order kinetics) were measured as a function of decylubiquinol concentration. The cytochrome b content of membranes was determined from the dithionite-reduced minus ferricyanide-oxidized difference spectra, using _562-575 = 28.5 mM^-1 cm^-1 (Vanneste, 1966). Turnover rates of cytochrome c reduction were determined using _550-542 = 18.1 mM^-1 cm^-1 (Margoliash and Walasek, 1967).

Inhibitors of bc₁ activity were added without prior incubation. DMSO in the assays did not exceed 0.3% (v/v). IC₅₀ values were calculated using the four-parameter logistic method (Grafit). The equilibrium dissociation constant (K_i) of inhibitor binding to bc₁ was determined using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

Real-Time Single-Cell Monitoring of Membrane Potential. The rhodamine derivative tetramethylrhodamine ethyl ester (TMRE) was used to monitor the mitochondrial membrane potential (_m) of the plasma membrane and mitochondria of malaria-infected red blood cells (Biagini et al., 2006). TMRE is cationic and reversibly accumulates inside energized membranes according to the Nernst equation. For experimentation, suspensions (1%) of infected erythrocytes in HEPES-buffered RPMI 1640 medium (no serum) were loaded with TMRE (250 nM; Invitrogen, Carlsbad, CA) for 10 min at 37°C. For imaging, malaria parasite-infected erythrocytes were immobilized using polylysine-coated coverslips in a perfusion chamber (FCS2; Bioptechs, Butler, PA) and maintained at 37°C in growth medium (no serum). Inhibitors were added to the perfusate, and the membrane potential-dependent fluorescence responses were monitored in real time. During all manipulations, the concentration of TMRE in the perfusate was kept at 250 nM. The fluorescence signals from malaria-infected erythrocytes were collected on a confocal laser scanning microscope (Pascal; Zeiss, Welwyn Garden City, UK) through a Plan-Apochromat 63 x 1.2 numerical aperture water objective. Excitation of TMRE was performed using the HeNe laser line at 543 nm. Emitted light was collected through a 560-nm long pass filter from a 543-nm dichroic mirror. Photobleaching (the irreversible damage of TMRE producing a less fluorescent species) was assessed by continuous exposure (5 min) of loaded cells to laser illumination. For each experiment, the laser illumination and microscope settings (e.g., laser power both voltage settings and attenuation [%], scan speed, pinhole diameter, number of scan sweeps, and degree of magnification) that gave no reduction in signal were used. Data capture and extraction were carried out with Zeiss Pascal software and Photoshop.

Dihydroacridinedione Docking into P. falciparum Cytochrome b (Q_o). A predicted model of the P. falciparum cytochrome b of the bc₁ complex was constructed with SWISS-MODEL using bovine cytochrome b Protein Data Bank coordinate sets 1ntmC, 1sqxC, 1l0nC, 1ntkC, and 1be3c as the structural templates. In silico docking was performed using Autodock 3.05 (Morris et al., 1998) and associated suite of programs. Autodock uses an empirical scoring function to estimate the free energy of binding. This function contains five terms: A Lennard_Jones 12-6 dispersion/repulsion term; a directional 12-10 hydrogen bonding term; a screened Coulombic electrostatic potential; unfavorable entropy of binding due to restricted conformations; and a desolvation energy term. For the calculations, the protein atoms were kept fixed with the inhibitors allowed full flexibility. A combination of a Lamarckian genetic algorithm and pseudo-Solis and Wets local search was used to generate docking poses for each molecule. The docking was performed using a grid much larger than either the Q_o or Q_i binding sites. The parameters used in this blind docking procedure were those that have been shown to reproduce the binding mode of drugs within known structures of drug-crystal complexes with no prior knowledge of the binding site (Hetenyi and van der Spoel, 2002). The most favorable docking pose for each molecule was identified by the scoring function.

	Results

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Dihydroacridinedione Binding to Heme. Assays were performed to determine the affinity of dihydroacridinediones for heme as well as to determine their ability to inhibit heme crystallization relative to known heme-binding drugs. Floxacrine and chloroquine (CQ) were shown to inhibit heme crystallization with IC₅₀ values of 63 and 56 µM, respectively. WR249685 inhibited hemozoin formation by 50% at 130 µM; however, complete inhibition was not measurable because this was the highest concentration achievable in the assay (values are means of three independent determinations).

Heme-drug equilibrium constants were determined for CQ, amodiaquine (AQ), floxacrine, and WR249685 by measuring the shift of the heme Soret band on titration of drugs. In buffered DMSO [40% (v/v)] solutions, the heme-drug dissociation constants (K_i) were calculated to be; 1.38 µM for CQ, 1.55 µM for AQ, 1.87 µM for floxacrine, and 31.74 µM for WR249685 (values means of two independent determinations).

The relative poor heme binding affinity of WR249685 was in contrast to its potent in vitro antimalarial activity (IC₅₀ 15 nM), which was comparable with that for CQ (IC₅₀ 7.4 nM), AQ (IC₅₀ 4.5 nM), and significantly better than that for floxacrine (IC₅₀ 140 nM, Table 1).

Dihydroacridinediones Inhibit P. falciparum bc₁ Activity. The ability of floxacrine and WR249685 to inhibit bc₁ complex activity was determined in a number of species and compared with that of well known bc₁ inhibitors (Fig. 1). For all species, bc₁ activity was determined by monitoring the reduction of cytochrome c with decylubiquinol (QH₂) as electron donor.

P. falciparum bc₁ activity exhibited Michaelis-Menten kinetics with an apparent concentration of substrate leading to half-maximal velocity (K_m) for QH₂ of 6.2 ± 2 µM reaching a maximum/limiting velocity (V_max) of 576 ± 88 nmol of cytochrome c reduction s^-1 mg^-1 free parasite protein (Fig. 2A). Human bc₁ displayed similar saturation kinetics with a K_m for QH₂ of 7.8 ± 2 µM and a V_max turnover of 3 s^-1 (Fig. 2B). Note that turnover (seconds^-1) for P. falciparum cell-free extracts could not be determined as a result of interference of free heme and hemozoin with cytochrome b determinations. Both P. falciparum and human liver bc₁ activities could be stimulated 10-fold by addition of surfactant [0.025% (w/v) dodecyl maltoside]. However, to compare data with similar studies (Fry and Pudney, 1992), all inhibitory assays were performed in the absence of surfactant.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Ubiquinol (QH2) concentration dependent cytochrome c reduction of (A) P. falciparum cell-free extracts and (B) human liver microsomes. Data points are means from observations from three individual experiments. Data were fitted to a function describing simple ligand binding at a single site by nonlinear regression analysis (Marquart method) using an iterative procedure to generate the best fit (2) of the curve to the data.

Inhibition of Yeast bc₁ Complex by Dihydroacridinediones Is Specific to the Quinol Oxidation Site (Q_o). The inhibitory profile of the dihydroacridinediones was determined against wild type and genetically engineered Saccharomyces cerevisiae harboring cytochrome b mutations Y279S and G143A. The Y279S mutation corresponds to the Y268S mutation in P. falciparum cytochrome b Q_o exhibiting an atovaquone-resistant phenotype (Srivastava et al., 1999; Syafruddin et al., 1999; Korsinczky et al., 2000; Fisher and Meunier, 2005). The G143A mutation confers dramatic resistance to heme-proximal Q_o inhibitors such as myxothiazol (Fisher et al., 2004a; Fisher and Meunier, 2005). As expected, atovaquone was shown to have potent bc₁ inhibitory activity against wild-type yeast (IC₅₀, 3 nM) and G143A mutants (IC₅₀, 27 nM), whereas the Y279S mutation conferred significant resistance (IC₅₀, 2689 nM, Table 2). Note that the Y279S mutation was also associated with a moderate increase in tolerance to the pyridone GW844520, floxacrine, and WR249685 (Table 2). Taken together, these data indicate that all of these inhibitors target the Q_o site of the bc₁ complex.

Dihydroacridinediones Collapse Mitochondrial Membrane Potential. The measurement of _m was based on the accumulation of the cationic fluorescence probe TMRE according to the Nernst equation. Addition of TMRE to P. falciparum-infected erythrocytes resulted in a strong fluorescence signal originating from the parasite cytosol, denoting the existence of a high _m. This phenomenon has been observed previously (Biagini et al., 2006) and is the result of the high _m (-100 mV) of the plasma membrane (Allen and Kirk, 2004). Upon addition of the V-type H⁺ ATPase inhibitors bafilomycin A₁ or concanamycin (200 nM), approximately 70 to 80% of the fluorescence signal was lost (data not shown), leaving a small but strong signal originating from the parasite mitochondrion (Biagini et al., 2006).

Because both the plasma membrane and the mitochondrion _m contribute to the accumulation of TMRE, we could not accurately quantify the finite _m values. Therefore, for all experiments, the fluorescence dynamic range was set up so that untreated TMRE-loaded cells were regarded as having complete fluorescence (100%), whereas the baseline (0%) was set by addition of the protonophore FCCP (10 µM). For mitochondrial-dependent fluorescence, bafilomycin A₁-treated cells were normalized to 100%; again, the baseline (0%) was set by FCCP (10 µM).

Addition of the dihydroacridinediones WR249685 (Fig. 3A) and floxacrine (data not shown) was observed to partially reduce (20%) the total cellular _m-dependent TMRE fluorescence, possibly indicating an effect on the mitochondrial contribution. This view was confirmed by the reduction of _m-dependent fluorescence from bafilomycin A₁-treated parasites with WR249685 (Fig. 3B) and floxacrine (not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of WR249685 on m. Time course of TMRE-dependent fluorescence after the addition of WR249685 (10 µM) to untreated (A) and bafilomycin A1 (200 nM)-treated (B) P. falciparum-infected erythrocytes. Data were normalized to 100% in untreated (A) or bafilomycin-treated cells (B) and to 0% in FCCP (10 µM)-treated cells. Graphs show means from experiments performed independently ± standard errors (n 3).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Isobole analysis of the fractional inhibitory concentrations (FIC) of IC50 values for atovaquone versus WR249685 (figure is representative of three independent experiments).

	Discussion

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As concluded by Dorn et al. (2001), it seems that heme does not play a major role in the mode of action of all dihydroacridinediones. Given that related dihydroacridinediones have been shown to affect respiration of P. falciparum (Suswam et al., 2001), we next investigated whether the mode of action of WR249685 was related to the inhibition of respiratory components.

Mild cross-resistance (4- to 9-fold) of a dihydroacridinedione (WR243251) has been described in P. falciparum strains displaying 8700- to 23,000-fold resistance increase in atovaquone (Suswam et al., 2001). Although these parasite lines also displayed an increase in resistance to other antimalarials such as CQ, because the site of action of atovaquone is the bc₁ complex (Fry and Pudney, 1992), this respiratory component was chosen for investigation.

The bc₁ complex is a membrane-bound enzyme catalyzing the transfer of electrons from ubiquinol to cytochrome c coupled with the concomitant vectorial translocation of protons across the inner mitochondrial membrane (Crofts, 2004). The catalytic core of the enzyme is made up of cytochrome b, cytochrome c₁, and the Rieske iron-sulfur protein (ISP). The catalytic mechanism, known as the Q-cycle (Mitchell, 1975; Crofts, 2004), involves two distinct quinone binding sites within cytochrome b, the quinol oxidation site Q_o and the quinone reduction site Q_i (Crofts, 2004). These two sites are situated on opposite sides of the membrane linked by a transmembrane electron pathway via hemes b_l and b_h (Crofts, 2004). A number of inhibitors selective for bc₁ Q_o and Q_i sites have been developed over recent years, most notably to control crop and human pathogens (Crofts et al., 1999; Esser et al., 2004; Fisher et al., 2004a).

In our study, stigmatellin, which binds in the b_l distal domain of Q_o (close to the docking site of ISP) (Crofts et al., 1999) and myxothiazol, which binds in the b_l-proximal position (Crofts et al., 1999), were both shown to be potent broad spectrum bc₁ inhibitors (Table 1). It is noteworthy, however, that inhibition of bc₁ activity by the dihydroacridinediones, pyridone, and naphthoquinone was highly species selective (Table 1).

Species selectivity was most notably demonstrated by WR249685, which displayed a K_i for P. falciparum of 0.3 nM and an in vitro TI against human bc₁ of >4600 (Table 1). Yeast carrying the Y279S mutation in cytochrome b (corresponding to the Y268S mutation in P. falciparum conferring atovaquone resistance (Srivastava et al., 1999; Syafruddin et al., 1999; Korsinczky et al., 2000; Fisher and Meunier, 2005)) were observed to be less sensitive to WR249685, suggesting that Q_o is the binding site for this inhibitor (Table 2). So what is it about the P. falciparum bc₁ Q_o site that lends itself to inhibition by WR249685?

X-ray crystallography has shown that the overall fold of the -carbon backbone of cytochrome b is highly conserved in prokaryotic and eukaryotic organisms (Fig. 5A). However, despite the high degree of sequence and structural conservation, there are notable differences in key regions of the malaria parasite Q_o site. Significantly, a homology model of the P. falciparum cytochrome b (constructed with SWISS-MODEL using bovine cytochrome b atomic coordinates as the structural template) suggests that the four-residue deletion in the cd2 helix results in a 13-Å displacement of this structural element compared with the mammalian enzyme (Fig. 5B). Likewise, the -carbon atom of the N-terminal proline of the ef helix (containing the catalytically essential `PEWY' motif) is predicted to be displaced by 2 Å compared with the mammalian enzyme. Other important differences include the replacement of lysine (269) by valine and alanine (277) by phenylalanine in the P. falciparum ef helix, and the exchange of phenylalanine (140) for tyrosine in the cd1 helix. Docking of WR249685 (and floxacrine; data not shown) to the P. falciparum bc₁ Q_o model was energetically favorable (binding energy, -8.1 kcal/mol; Fig. 6B); in addition, the model demonstrated selectivity in the docking of traditional Q_o and Q_i inhibitors (e.g., famoxadone and antimycin; data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 5. A, sequence alignment of Qo site regions from bovine (Bt), human (Hs), P. falciparum (Pf), rat (Rn), and Baker's yeast (Sc) cytochrome b. Helices are identified by arrows above the alignment. below the sequence data identify residues in close contact (<3.5 Å separation, hydrogen bonding or 40 Å2 surface contact) with stigmatellin in 1SQX.pdb (Esser et al., 2004). , residues in close contact with myxothiazol in 1SQP.pdb (Esser et al., 2004); , residues associated with atovaquone resistance mutations in Plasmodium sp., Toxoplasma gondii and Pneumocystis jirovecii. The gray shaded area identifies residues located within a 4-Å radius of WR249685 docked into the P. falciparum homology model of cytochrome b. B, structural alignment of selected Qo site regions from bovine cytochrome b (1SQX.pdb, red) and the homology model of P. falciparum cytochrome b (green). Highlighted residues are discussed in the text.

View larger version (36K): [in this window] [in a new window]   Fig. 6. A, SWISS-PROT homology model of P. falciparum cytochrome b in cartoon representation showing WR249685 docked at the Qo site region. The protein backbone is represented in orange, with the hemes in red wireframe. WR249685 and associated liganding residues are represented in white and green wireframe, respectively (B). Detail of WR249685 (white wireframe) docked at Qo in the P. falciparum homology model showing residues within a 4-Å radius of the bound inhibitor (CPK wireframe).

Figure 6B shows Q_o site residues predicted to be within 4 Å of the bound WR249685 (most energetically favorable conformation). The interactions are predominantly hydrophobic, although a backbone hydrogen bond from Ser241 to the aromatic secondary amine of WR249685 is likely to be important for the positioning of the compound at Q_o. The glutamyl side chain of Glu261 shows considerable mobility and may also be involved in weak dipolar interactions with the chlorine atoms of WR249685. The most striking feature of the model for WR249685 binding to P. falciparum cytochrome b is the putative association between the inhibitor and the E-ef linker region (residues 236-241) of the cytochrome (Figs. 5A and 6B), a region of low sequence identity between P. falciparum and mammalian cytochrome b. The E-ef linker has not previously been recognized as a component of the Q_o site in the elucidated bc₁ crystal structures, and thus may explain the very high degree of selectivity of WR249685 for the P. falciparum enzyme.

It is necessary, however, to be circumspect in the interpretation of the modeling data. It should be noted that "structural" water molecules at Q_o were not included in the modeling process, and these may influence the binding energy and positioning of WR249685. In addition, the Rieske ISP headgroup was omitted during the modeling process, which has two important consequences. First, the loss of a potential hydrogen-bond donor to the Q_o site via [2Fe-2S] cluster ligand His-161 (Esser et al., 2004); second, the steric volume occupied by the ISP is absent, which may allow for nonphysiological but otherwise energetically favorable in silico docking of bulky inhibitors at Q_o.

The 5.6-fold increase in IC₅₀ for atovaquone in rat liver microsome preparations compared with the human equivalent (Table 1) is, at first sight, surprising given the sequence homology between these species in the cd1 and ef regions of cytochrome b (Fig. 5A). It is possible that this difference is due to slight variation in the local fold and protein environment around Q_o, but a minor change in hydrogen-bonding capacity in the C-terminal region of transmembrane helix C may also weaken the interaction with atovaquone, raising the binding energy required for a stable association. It is noteworthy that bovine and human cytochrome b possess a potential Q_o-site hydrogen bond donor in the forms of Thr121 and Thr122, respectively, residues that are absent in rat. In addition, there is conservative variation in the aliphatic composition of the F1 helix between these three species, which may result in an unfavorable steric environment for atovaquone binding in rat cytochrome b.

In a similar fashion, the pyridone GW844520, shown to be specific for the bc₁ Q_o site (Table 2), also displayed a 2-fold selectivity for human bc₁ over the rat enzyme (Table 1), with an in vitro TI against human bc₁ of only 5 (Table 1). The drug development of this particular pyridone was terminated in late 2005 by the Medicines for Malaria Venture (MMV) because of toxicity issues (http://www.mmv.org). Currently a new pyridone (GSK932121A) is being developed with reduced toxicity. It will be interesting to establish whether this compound has an improved TI against human bc₁.

To our knowledge, this study is the first to report human liver bc₁ activity. At this stage, we have no idea of interpatient variation of bc₁ activities; nonetheless, our data indicate that rat liver enzyme is a poor model for human bc₁, and therapeutic indices generated from rat liver data should be treated with a degree of caution.

Addition of dihydroacridinediones to malaria-infected erythrocytes was shown to cause the depolarization of _m (Fig. 3). We hypothesize that the depolarization of _m leads to a loss mitochondrial function and parasite death. Given that during the intraerythrocytic stage of the malaria life cycle, the parasite relies mainly on fermentation of glucose, the essential role(s) of the mitochondrion is not known, but it probably includes orotate production for pyrimidine biosynthesis (Gutteridge et al., 1979; Hammond et al., 1985) and Ca²⁺ homeostasis (Uyemura et al., 2000; Gazarini and Garcia, 2004). Furthermore, the close juxtaposition of the mitochondrion with the plastid suggests an interdependence for essential metabolism (Goodman et al., 2007; Kobayashi et al., 2007).

It has recently been reported that addition of atovaquone does not cause a depolarization of parasite _m, because _m is generated by the ATP synthase and adenine nucleotide translocator operating in reverse (Painter et al., 2007). We have questioned these conclusions (Fisher et al., 2008), however, and maintain that targeting the proton pumping bc₁ complex leads to a depolarization of _m resulting in a loss of mitochondrial function and parasite death. Thus in our opinion targeting the mitochondrial ETC leading to a depolarization of _m remains a viable chemotherapeutic strategy. The merit of this strategy is supported by recent evidence showing an up-regulation of parasite expression of mitochondrial ETC components during in vivo growth compared with in vitro culture (Daily et al., 2007; van Dooren and McFadden, 2007).

This study has described a new class of highly selective P. falciparum inhibitors predicted to target the Q_o site of the bc₁ complex. The ability of these compounds to additionally disrupt hemozoin formation makes them attractive inhibitors that merit further drug development. This view is strengthened by the potent picomolar antimalarial activity displayed by the recently synthesized haloalkoxyacridones (Winter et al., 2006). Furthermore, we predict that by assessing the inhibitory activity of these molecules against human bc₁, it may be possible to circumvent toxicological issues previously encountered during the development of other dihydroacridinediones such as floxacrine (Raether and Fink, 1982).

	Acknowledgements

 
We thank GlaxoSmithKline for providing the pyridone GW844520 and Dr. Jonathan Vennerstrom (Nebraska Medical Center, Omaha, NE) for supplying the floxacrine and WR249685. We thank the staff and patients of Ward 7Y and the Gastroenterology Unit, Royal Liverpool Hospital, for their generous donation of blood.

	Footnotes

 
This work was supported by the Leverhulme Trust.

ABBREVIATIONS: WR243246, 7-chloro-3-(2,4-dichlorophenyl)-3,4-dihydro-1,9 (2H, 10H)-acridinedione; DMSO, dimethyl sulfoxide; Q_o, quinol oxidation site; TMRE, tetramethylrhodamine ethyl ester; _m, mitochondrial membrane potential; CQ, chloroquine; AQ, amodiaquine; TI, therapeutic index; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone; ISP, iron-sulfur protein.

Address correspondence to: Giancarlo A. Biagini, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. E-mail: biagini{at}liv.ac.uk

	References

Top Abstract Materials and Methods Results Discussion References

 
Allen RJ and Kirk K (2004) The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum. J Biol Chem 279: 11264-11272.[Abstract/Free Full Text]

Auparakkitanon S, Chapoomram S, Kuaha K, Chirachariyavej T, and Wilairat P (2006) Targeting of hematin by the antimalarial pyronaridine. Antimicrob Agents Chemother 50: 2197-2200.[Abstract/Free Full Text]

Auparakkitanon S, Noonpakdee W, Ralph RK, Denny WA, and Wilairat P (2003) Antimalarial 9-anilinoacridine compounds directed at hematin. Antimicrob Agents Chemother 47: 3708-3712.[Abstract/Free Full Text]

Auparakkitanon S and Wilairat P (2000) Cleavage of DNA induced by 9-anilinoacridine inhibitors of topoisomerase II in the malaria parasite Plasmodium falciparum. Biochem Biophys Res Commun 269: 406-409.[CrossRef][Medline]

Basco LK, Mitaku S, Skaltsounis AL, Ravelomanantsoa N, Tillequin F, Koch M, and Le Bras J (1994) In vitro activities of furoquinoline and acridone alkaloids against Plasmodium falciparum. Antimicrob Agents Chemother 38: 1169-1171.

Berenbaum MC (1978) A method for testing for synergy with any number of agents. J Infect Dis 137: 122-130.[Medline]

Biagini GA, O'Neill PM, Bray PG, and Ward SA (2005) Current drug development portfolio for antimalarial therapies. Curr Opin Pharmacol 5: 473-478.[CrossRef][Medline]

Biagini GA, O'Neill PM, Nzila A, Ward SA, and Bray PG (2003) Antimalarial chemotherapy: young guns or back to the future? Trends Parasitol 19: 479-487.[CrossRef][Medline]

Biagini GA, Viriyavejakul P, O'Neill PM, Bray PG, and Ward SA (2006) Functional characterization and target validation of alternative complex I of Plasmodium falciparum mitochondria. Antimicrob Agents Chemother 50: 1841-1851.[Abstract/Free Full Text]

Bray PG, Janneh O, Raynes KJ, Mungthin M, Ginsburg H, and Ward SA (1999) Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum. J Cell Biol 145: 363-376.[Abstract/Free Full Text]

Cheng Y and Prusoff WH. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099-3108.[CrossRef][Medline]

Chou AC and Fitch CD (1993) Control of heme polymerase by chloroquine and other quinoline derivatives. Biochem Biophys Res Commun 195: 422-427.[CrossRef][Medline]

Crofts AR (2004) The cytochrome bc1 complex: function in the context of structure. Annu Rev Physiol 66: 689-733.[CrossRef][Medline]

Crofts AR, Barquera B, Gennis RB, Kuras R, Guergova-Kuras M, and Berry EA (1999) Mechanism of ubiquinol oxidation by the bc(1) complex: different domains of the quinol binding pocket and their role in the mechanism and binding of inhibitors. Biochemistry 38: 15807-15826.[CrossRef][Medline]

Daily JP, Scanfeld D, Pochet N, Le Roch K, Plouffe D, Kamal M, Sarr O, Mboup S, Ndir O, Wypij D, et al. (2007) Distinct physiological states of Plasmodium falciparum in malaria-infected patients. Nature 450: 1091-1095.[CrossRef][Medline]

Desjardins RE, Canfield CJ, Haynes JD, and Chulay JD (1979) Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710-718.[Abstract/Free Full Text]

Dorn A, Scovill JP, Ellis WY, Matile H, Ridley RG, and Vennerstrom JL (2001) Short report: floxacrine analog WR 243251 inhibits hematin polymerization. Am J Trop Med Hyg 65: 19-20.[Abstract]

Dorn A, Vippagunta SR, Matile H, Jaquet C, Vennerstrom JL, and Ridley RG (1998) An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials. Biochem Pharmacol 55: 727-736.[CrossRef][Medline]

Dürckheimer W, Raether W, Seliger H, and Seidenath H (1980) 10-Hydroxy-3,4-dihydroacridine-1,9(2H,10H)-diones, a new group of malaricidal and coccidiostatic compounds. Arzneimittelforschung 30: 1041-1046.[Medline]

Edwards G and Biagini GA (2006) Resisting resistance: dealing with the irrepressible problem of malaria. Br J Clin Pharmacol 61: 690-693.[CrossRef][Medline]

Egan TJ, Mavuso WW, Ross DC, and Marques HM (1997) Thermodynamic factors controlling the interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX. J Inorg Biochem 68: 137-145.[CrossRef][Medline]

Esser L, Quinn B, Li YF, Zhang M, Elberry M, Yu L, Yu CA, and Xia D (2004) Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex. J Mol Biol 341: 281-302.[CrossRef][Medline]

Fisher N, Bray PG, Ward SA, and Biagini GA (2008) Malaria-parasite mitochondrial dehydrogenases as drug targets: too early to write the obituary. Trends Parasitol 24: 9-10.[CrossRef][Medline]

Fisher N, Brown AC, Sexton G, Cook A, Windass J, and Meunier B (2004a) Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b. Eur J Biochem 271: 2264-2271.[Medline]

Fisher N, Castleden CK, Bourges I, Brasseur G, Dujardin G, and Meunier B (2004b) Human disease-related mutations in cytochrome b studied in yeast. J Biol Chem 279: 12951-12958.[Abstract/Free Full Text]

Fisher N and Meunier B (2005) Re-examination of inhibitor resistance conferred by Qo-site mutations in cytochrome b using yeast as a model system. Pest Manag Sci 61: 973-978.[CrossRef][Medline]

Fry M and Pudney M (1992) Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4- (4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem Pharmacol 43: 1545-1553.[CrossRef][Medline]

Gamage SA, Tepsiri N, Wilairat P, Wojcik SJ, Figgitt DP, Ralph RK, and Denny WA (1994) Synthesis and in vitro evaluation of 9-anilino-3,6-diaminoacridines active against a multidrug-resistant strain of the malaria parasite Plasmodium falciparum. J Med Chem 37: 1486-1494.[CrossRef][Medline]

Gazarini ML and Garcia CR (2004) The malaria parasite mitochondrion senses cytosolic Ca²⁺ fluctuations. Biochem Biophys Res Commun 321: 138-144.[CrossRef][Medline]

Gill HJ, Tingle MD, and Park BK (1995) N-Hydroxylation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity. Br J Clin Pharmacol 40: 531-538.[Medline]

Goodman CD, Su V, and McFadden GI (2007) The effects of anti-bacterials on the malaria parasite Plasmodium falciparum. Mol Biochem Parasitol 152: 181-191.[CrossRef][Medline]

Gutteridge WE, Dave D, and Richards WH (1979) Conversion of dihydroorotate to orotate in parasitic protozoa. Biochim Biophys Acta 582: 390-401.[Medline]

Hammond DJ, Burchell JR, and Pudney M (1985) Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone in vitro. Mol Biochem Parasitol 14: 97-109.[CrossRef][Medline]

Hetenyi C and van der Spoel D (2002) Efficient docking of peptides to proteins without prior knowledge of the binding site. Protein Sci 11: 1729-1737.[CrossRef][Medline]

Kesten SJ, Degnan MJ, Hung J, McNamara DJ, Ortwine DF, Uhlendorf SE, and Werbel LM (1992) Synthesis and antimalarial properties of 1-imino derivatives of 7-chloro-3-substituted-3,4-dihydro-1,9(2H,10H)-acridinediones and related structures. J Med Chem 35: 3429-3447.[CrossRef][Medline]

Kobayashi T, Sato S, Takamiya S, Komaki-Yasuda K, Yano K, Hirata A, Onitsuka I, Hata M, Mi-Ichi F, Tanaka T, et al. (2007) Mitochondria and apicoplast of Plasmodium falciparum: behaviour on subcellular fractionation and the implication. Mitochondrion 7: 125-132.[CrossRef][Medline]

Korsinczky M, Chen N, Kotecka B, Saul A, Rieckmann K, and Cheng Q (2000) Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrob Agents Chemother 44: 2100-2108.[Abstract/Free Full Text]

Kuboyama M, Yong FC, and King TE (1972) Studies on cytochrome oxidase. 8. Preparation and some properties of cardiac cytochrome oxidase. J Biol Chem 247: 6375-6383.[Abstract/Free Full Text]

Lambros C and Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418-420.[CrossRef][Medline]

Margoliash E and Walasek OF (eds) (1967) Cytochrome c from vertebrate and invertebrate sources. Methods Enzymol 10: 339-349.[Medline]

Marques HM, Voster K, and Egan TJ (1996) The interaction of the heme-octapeptide, N-acetylmicroperoxidase-8 with antimalarial drugs: solution studies and modeling by molecular mechanics methods. J Inorg Biochem 64: 7-23.[CrossRef][Medline]

Mitchell P (1975) The protonmotive Q cycle: a general formulation. FEBS Lett 59: 137-139.[CrossRef][Medline]

Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, and Olson AJ (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J Comput Chem 19: 1639-1662.[CrossRef]

Oettmeier W, Masson K, and Kalinna S (1995) [3H]7-azido-4-isopropylacridone labels Cys159 of the bovine mitochondrial ADP/ATP-carrier protein. Eur J Biochem 227: 730-733.[Medline]

Oettmeier W, Masson K, and Soll M (1992) The acridones, new inhibitors of mitochondrial NADH: ubiquinone oxidoreductase (complex I). Biochim Biophys Acta 1099: 262-266.[Medline]

Oettmeier W, Masson K, Soll M, and Reil E (1994) Acridones and quinolones as inhibitors of ubiquinone functions in the mitochondrial respiratory chain. Biochem Soc Trans 22: 213-216.[Medline]

Painter HJ, Morrisey JM, Mather MW, and Vaidya AB (2007) Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum. Nature 446: 88-91.[CrossRef][Medline]

Raether W and Fink E (1979) Antimalarial activity of Floxacrine (HOE 991) I. Studies on blood schizontocidal action of Floxacrine against Plasmodium berghei, P. vinckei and P. cynomolgi. Ann Trop Med Parasitol 73: 505-526.[Medline]

Raether W and Fink E (1982) Antimalarial activity of floxacrine (HOE 991) II: Studies on causal prophylactic and blood schizontocidal action of floxacrine and related dihydroacridinediones against Plasmodium yoelii and P. berghei. Ann Trop Med Parasitol 76: 507-516.[Medline]

Schmidt LH (1979) Antimalarial properties of floxacrine, a dihydroacridinedione derivative. Antimicrob Agents Chemother 16: 475-485.[Abstract/Free Full Text]

Shao BR (1990) A review of antimalarial drug pyronaridine. Chin Med J (Engl) 103: 428-434.[Medline]

Snow RW, Trape JF, and Marsh K (2001) The past, present and future of childhood malaria mortality in Africa. Trends Parasitol 17: 593-597.[CrossRef][Medline]

Srivastava IK, Morrisey JM, Darrouzet E, Daldal F, and Vaidya AB (1999) Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites. Mol Microbiol 33: 704-711.[CrossRef][Medline]

Stead AM, Bray PG, Edwards IG, DeKoning HP, Elford BC, Stocks PA, and Ward SA (2001) Diamidine compounds: selective uptake and targeting in Plasmodium falciparum. Mol Pharmacol 59: 1298-1306.[Abstract/Free Full Text]

Suswam E, Kyle D, and Lang-Unnasch N (2001) Plasmodium falciparum: the effects of atovaquone resistance on respiration. Exp Parasitol 98: 180-187.

Syafruddin D, Siregar JE, and Marzuki S (1999) Mutations in the cytochrome b gene of Plasmodium berghei conferring resistance to atovaquone. Mol Biochem Parasitol 104: 185-194.[CrossRef][Medline]

Trager W and Jensen JB (1976) Human malaria parasites in continuous culture. Science 193: 673-675.[Abstract/Free Full Text]

Uyemura SA, Luo S, Moreno SN, and Docampo R (2000) Oxidative phosphorylation, Ca²⁺ transport, and fatty acid-induced uncoupling in malaria parasites mitochondria. J Biol Chem 275: 9709-9715.[Abstract/Free Full Text]

van Dooren GG, and McFadden GI (2007) Malaria: differential parasite drive. Nature 450: 955-956.[CrossRef][Medline]

Vanneste WH (1966) Molecular proportion of the fixed cytochrome components of the respiratory chain of Keilin-Hartree particles and beef heart mitochondria. Biochim Biophys Acta 113: 175-178.[Medline]

Wernsdorfer WH and Payne D (1991) The dynamics of drug resistance in Plasmodium falciparum. Pharmacol Ther 50: 95-121.[CrossRef][Medline]

Winter RW, Kelly JX, Smilkstein MJ, Dodean R, Bagby GC, Rathbun RK, Levin JI, Hinrichs D, and Riscoe MK (2006) Evaluation and lead optimization of antimalarial acridones. Exp Parasitol 114: 47-56.[CrossRef][Medline]

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