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Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs Diana Ortiz,a Isaac Forquer,c Jan Boitz,b Radika Soysa,b Carolyn Elya,a Audrey Fulwiler,b Aaron Nilsen,c Tamsen Polley,a Michael K. Riscoe,a,c Buddy Ullman,b Scott M. Landfeara Departments of Molecular Microbiology & Immunologya and Biochemistry & Molecular Biology,b Oregon Health & Science University, Portland, Oregon, USA; Veterans Affairs Medical Center, Portland, Oregon, USAc

Endochin-like quinolones (ELQs) are potent and specific inhibitors of cytochrome bc1 from Plasmodium falciparum and Toxoplasma gondii and show promise for novel antiparasitic drug development. To determine whether the mitochondrial electron transport chain of Leishmania parasites could be targeted similarly for drug development, we investigated the activity of 134 structurally diverse ELQs. A cohort of ELQs was selectively toxic to amastigotes of Leishmania mexicana and L. donovani, with 50% inhibitory concentrations (IC50s) in the low micromolar range, but the structurally similar hydroxynaphthoquinone buparvaquone was by far the most potent inhibitor of electron transport, ATP production, and intracellular amastigote growth. Cytochrome bc1 is thus a promising target for novel antileishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for development of enhanced therapeutics.

L

eishmania spp. are parasites of the Kinetoplastida order that are responsible for a spectrum of human diseases that include cutaneous, mucocutaneous, and visceral leishmaniasis, each caused by various species within the genus (1), and an estimated 12 million individuals worldwide suffer from these diseases. Parasites exist in several life cycle forms, including extracellular promastigotes that live in the gut of the sand fly vector and intracellular amastigotes that reside within the phagolysosomal vesicles of mammalian host macrophages. Promastigotes can be cultured readily in a variety of media and represent the most facile system for in vitro studies, and amastigotes can be grown inside cultured macrophages such as the J774.A1 cell line (2) or axenically at elevated temperatures (32 to 37°C) and at pH 5.6 to 5.7 (3). All of the drugs employed to treat the leishmaniases are suboptimal (4) and suffer from problems such as toxicity, difficulties in delivery, high expense, and development of resistance. Hence, there is an acute need for development of novel, improved, and alternative therapies. One potential target for antiparasitic therapies is the mitochondrial electron transport chain (ETC) (5) that mediates transport of electrons, derived from electron donors such as NADH and FADH2 and generated in a variety of catabolic processes, to molecular oxygen. Electron transport entails the sequential transfer of electrons via multiprotein complexes (designated complex I to complex IV) present in the inner mitochondrial membrane, with electron transfer between several complexes being mediated by ubiquinone, also known as coenzyme Q, a 1,4-benzoquinone that is a component of the inner mitochondrial membrane. In addition to its well-recognized role in generating cellular ATP in many organisms, the ETC of Plasmodium species is required for synthesis of metabolically essential pyrimidines (6) rather than for synthesis of ATP. Furthermore, blocking the ETC can generate toxic reactive oxygen species (ROS) (7) such as superoxide anion (O2-) (8), and uncoupling of the ETC in kinetoplastid parasites has been shown to increase generation of toxic ROS (7), with complex III, also known as cytochrome bc1, being the principal ROS source. Furthermore, blocking the ETC in mammalian cells leads to depletion of NAD⫹, since a major route of NADH oxida-

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tion is removed, resulting in lethal inhibition of aspartate biosynthesis, a critical metabolic process that requires NAD⫹ (9, 10). Hence, inhibition of ETC can be toxic to eukaryotic cells for a variety of reasons. A novel class of experimental antimalarial compounds is that of the endochin-like quinolones (ELQs) (11, 12), compounds that are analogs of ubiquinone that can act as competitive inhibitors of cytochrome bc1 by complexing to the Qi ubiquinone binding site (one of two ubiquinone binding sites on cytochrome bc1 and the one that is oriented toward the luminal side of the inner mitochondrial membrane, as distinct from the Qo site, which is oriented toward the external surface of that membrane) (13, 14). One of these compounds, ELQ-300, is a particularly potent and highly selective inhibitor of Plasmodium falciparum cytochrome bc1 and is an efficacious antimalarial compound both in vitro and in a mouse model of malaria infection (14). ELQ-271 is a highly potent inhibitor of growth of another important apicomplexan parasite, Toxoplasma gondii (13). Hence, these novel compounds show considerable promise as ETC-targeted drugs against several apicomplexan parasites of global importance. Buparvaquone is a hydroxynaphthoquinone that is also related in structure to ubiquinone and is another potential inhibitor of cytochrome bc1 (15). This compound is employed for treatment of the veterinary disease theileriosis, or East Coast cattle fever (15), and has demonstrated efficacy against L. donovani in vitro and to some degree in a BALB/c model of visceral leishmaniasis (16), although its mechanism of action has not been formally established.

Received 20 April 2016 Returned for modification 7 May 2016 Accepted 2 June 2016 Accepted manuscript posted online 13 June 2016 Citation Ortiz D, Forquer I, Boitz J, Soysa R, Elya C, Fulwiler A, Nilsen A, Polley T, Riscoe MK, Ullman B, Landfear SM. 2016. Targeting the cytochrome bc1 complex of Leishmania parasites for discovery of novel drugs. Antimicrob Agents Chemother 60:4972–4982. doi:10.1128/AAC.00850-16. Address correspondence to Scott M. Landfear, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Results of previous studies of several species of Leishmania, including L. major, L. donovani, L. infantum, and L. mexicana, also imply that the ETC is critical for the survival of these pathogens, although those earlier studies employed the promastigote rather than the amastigote, the life cycle stage that causes disease. Application of either antimycin A, an inhibitor of cytochrome bc1, or cyanide, an inhibitor of complex IV, resulted in growth inhibition and death of promastigotes (17–19). Similarly, administration of either antimycin A or 2-heptyl-4-hydroxyquinoline N-oxide, each of which is a specific inhibitor of cytochrome bc1, blocked growth of the cultured insect form epimastigotes of Trypanosoma cruzi (20), the kinetoplastid parasite that is the causative agent of Chagas disease (21). A recent phenotypic screen against T. cruzi identified a novel compound, GNF7686, which selectively targets cytochrome bc1 of that parasite and clears intracellular amastigotes from infected 3T3 cells at a concentration of 1 ␮M (22). Results of those studies imply that the mitochondrial ETC is critical for both Leishmania and T. cruzi and suggest that ELQs or quinolones might also show promise for development of novel antileishmanial or antitrypanosomal drugs, thus potentially broadening the spectrum of the activity of these antiparasitic agents. The high degree of sequence divergence between the Leishmania and human cytochrome b proteins (19% and 15% identity for L. mexicana and L. donovani, respectively, to the human proteins) further suggests that it should be possible to identify potent inhibitors of the parasite enzyme that do not significantly inhibit the human ortholog. The objective of the study described here was to test the potential of ELQs and buparvaquone as cytochrome bc1 inhibitors and antiparasitic agents against two different species of Leishmania, L. mexicana, which causes cutaneous leishmaniasis, and L. donovani, the etiological agent of fatal visceral leishmaniasis. The availability of a library of more than 100 structurally diverse ELQs that had been generated to explore their antimalarial activity (11, 12, 14) provided a rich resource for probing the potential of these compounds as novel antileishmanial agents. Given the sequence divergence of L. mexicana and P. falciparum cytochrome b (23% identity), it is reasonable to expect that the ELQs showing the greatest efficacy against the malaria parasite might not be those most efficacious against Leishmania parasites and that an extensive screening of the ELQ library would be required to determine whether and which ELQs can be effective antileishmanial compounds. More broadly, these studies underscored the potential of the ETC as a target for development of novel antileishmanial therapies, regardless of the particular chemotype that they may represent. MATERIALS AND METHODS Parasite cell cultures and transfection. Wild-type Leishmania mexicana (strain MNYZ/BZ/62/M379) and L. donovani clone Bob (LdBob) (23) promastigotes were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (iFBS) (Thermo Scientific HyClone), 0.1 mM xanthine, and 5 ␮g of hemin/ml. All cultures were maintained at 26°C. Axenic amastigotes of L. mexicana were cultivated at 32°C as described previously (3). Generation of luciferase-expressing reporter lines. The luciferase open reading frame from sea pansy Renilla reniformis (Rluc) was amplified from the pGL4.70[hRluc] vector (Promega). The gene was integrated into the BglII restriction site of plasmid pIR1SAT (24). After insertion, the plasmid was digested with the restriction enzyme SwaI to generate a linearized targeting fragment. Transfection into L. mexicana or L. donovani promastigotes (24, 25) and selection with 50 ␮g/ml nourseothricin

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(ENZO Life) yielded parasites in which the construct was integrated into the small-subunit rRNA gene cluster, leading to a stably maintained transgene and transcription at high levels of luciferase-encoding RNA. This transgenic line was subjected to passage through BALB/c mice as previously described (48) to ensure optimal infectivity for macrophages, and parasites obtained from the mouse lesions were transformed to promastigotes by brief growth in RPMI 1640 medium followed by storage of frozen stabilates in liquid nitrogen. RLuc-expressing clones of L. mexicana or L. donovani were retrieved from the stabilates and maintained by periodic dilution of logarithmic-phase parasites into RPMI medium without antibiotic to a density of 2 ⫻105 cells/ml for L. mexicana or 1 ⫻105 cells/ml for L. donovani. Parasites were maintained for no longer than six passages to ensure optimal maintenance of infectivity. Macrophage infection with transgenic parasites. The J774A.1 mouse macrophage cell line grown in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) and 10% iFBS was infected with stationary-phase parasites selected from 8-day-old parasite cultures at multiplicities of infection of 10:1 and 5:1 with Rluc-L. mexicana and Rluc-L. donovani promastigotes, respectively. Infected macrophages were cultivated overnight at 37°C and 5% CO2 in minimal essential medium (MEM; Invitrogen) containing 10% iFBS. Thereafter, the macrophage monolayer was washed 3 times with Ca2⫹- and Mg2⫹-free phosphate-buffered saline (PBS) to remove the uninternalized promastigotes. Infected macrophages were then scraped off the flask using a rubber policeman, and cell viability was evaluated using Trypan blue exclusion. Infected macrophages (⬎70% viable) were plated into either 96-well Falcon tissue culture plates (for the luminescence assay) or 4-well slides (Thermo Fisher Scientific) (for microscopy), incubated for 3 h before compounds were added, and then incubated for another 96 h. Macrophages were either fixed and stained with Giemsa to assess the level of infection by light microscopy (26) or directly treated as described below for the quantification of luminescence. Determination of luciferase activity and microscopic quantification of parasite loads. Luciferase activity was measured according to the protocol described for the Renilla luciferase assay system (Promega). Briefly, relative light unit (RLU) values from cell extracts were obtained by aspirating medium from each well of a 96-well tissue culture plate, pipetting off the residual buffer, adding 100 ␮l of 1⫻ lysis buffer (Promega) to the infected macrophage monolayer, and incubating at 4°C overnight, conditions that were determined empirically to lead to optimal luciferase signal. After gentle shaking for 15 min at room temperature, 20 ␮l of the cell lysate was transferred to a well of a black flat-bottom 96-well assay plate (Corning Costar Inc.) and 50 ␮l of luciferin substrate in assay buffer (Promega) was added. The luminescence signal was assayed immediately using a Veritas Microplate Luminometer (Turner BioSystems) with integration of signal in a 10-s window. For microscopic quantification of intracellular amastigotes, the number of parasites per macrophage was quantified directly by examining at least 200 macrophages in two independent wells. For comparison of RLU values to microscopically enumerated amastigote numbers, linear regression analysis was performed using GraphPad Prism 6 software. Amastigote growth inhibitory activity. For assessing the activity against amastigotes, test compounds dissolved in 100% dimethyl sulfoxide (DMSO) were diluted serially into MEM–10% iFBS medium at concentrations of 10 ␮M to 1 nM. Compounds were applied to wells containing 1 ⫻ 104 infected macrophages and incubated at 37°C for 96 h in a CO2 incubator. After incubation, the compound-containing medium was aspirated and luciferase assays were performed as described above. Relative amastigote survival was determined by comparing the RLU of the compound-treated cells with the RLU of cells treated with DMSO vehicle only. The percentage of amastigote survival was calculated using the following equation: percent survival ⫽ (mean number of treated cells/mean number of DMSO-treated cells) ⫻ 100. Levels of 50% inhibitory concentrations (IC50s) were calculated by nonlinear regression analysis of the doseresponse curve using GraphPad Prism 6 software.

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Macrophage cytotoxicity assay. For determination of J774A.1 macrophage viability following treatment with compounds, 1 ⫻ 104 cells were incubated with test compounds at 10 concentrations ranging from 1 nM to 10 ␮M. After 96 h of incubation, the compound-containing medium was aspirated and 100 ␮l of 1⫻ lysis buffer for the Renilla luciferase assay system was added to each well followed by incubation at 4°C overnight. Subsequently, SYBR green (Sigma-Aldrich stock at a 10,000⫻ concentration diluted to 1⫻ final concentration)-PBS was added to each well followed by incubation for 60 min with shaking. Fluorescence was measured with a SpectraMax Gemini XPS microplate reader (Molecular Devices GmbH) at wavelengths of 490 nm for excitation and 540 nm for emission. The percentage of cytotoxicity was estimated by comparison to DMSOtreated cells. Cytochrome bc1 assay. An enriched mitochondrial fraction was generated from L. mexicana axenic amastigotes as follows. A total of 2.5 ⫻ 1010 axenic amastigotes were pelleted by centrifugation, resuspended in PBS, and pelleted a second time. The pellet was frozen, thawed, and resuspended in 25 ml lysis buffer consisting of 100 mM Tricine (pH 8.0), 50 mM KCl, and 1 mM phenylmethanesulfonyl fluoride, and the suspension was passaged 3 times through a BioNeb (Glas-Col) cell disruptor at a pressure of 100 lb/in2. The suspension was centrifuged for 20 min at 2,000 ⫻ g, the pellet was discarded, and the supernatant was centrifuged for 40 min at 13,000 ⫻ g. After removal of the supernatant, the pellet was resuspended in 1 ml lysis buffer, brought to a concentration of 15% (vol/ vol) glycerol, and stored at ⫺80°C prior to use. After aliquots of mitochondria were thawed, dodecyl maltoside was added to achieve a final concentration of 10 mg ml⫺1 and the suspension was allowed to equilibrate for 30 min on ice. The dispersed mitochondria were centrifuged at 10,000 ⫻ g for 10 min, and the supernatant containing active cytochrome bc1 was retained. The reduction of cytochrome c levels was measured in buffer containing 50 mM KCl, 100 mM Tricine, 2.5 mM sodium azide, 50 ␮M oxidized horse heart cytochrome c (Fisher), and 50 ␮M decylubiquinol (pH 8.0) freshly prepared from a 10 mM stock by addition of a few crystals of sodium borohydride with subsequent quenching with dilute HCl. The difference between the levels of absorbance at 550 nm and 542 nm was monitored spectrophotometrically at 30°C, and this absorbance difference was recorded as a function of time to generate initial enzymatic reaction rates. To initiate the assay, the quinol was added to the buffer and the background reduction of cytochrome c was allowed to progress for approximately 30 s, after which dispersed mitochondria were added. The background rate of cytochrome c reduction was subtracted from the initial rate of cytochrome c reduction upon addition of mitochondria in the presence and absence of inhibitors. Mitochondria were preincubated with each ELQ for 30 min on ice prior to addition to the assay mixture to ensure complete binding of each inhibitor to cytochrome bc1. ATP determination. For measurement of ATP, an ATPlite luminescence assay luciferin-luciferase assay kit (PerkinElmer Inc.) was employed. Axenic amastigotes were treated with various concentrations of buparvaquone or ELQ-260 for 1 h at 32°C. Subsequently, cells were lysed with the somatic cell ATP-releasing buffer provided in the kit. The luciferin substrate and luciferase enzyme were added, and bioluminescence was assessed using a Veritas Microplate Luminometer. The cellular ATP level determined using control DMSO-treated cells was assigned a value of 1.0, and other values were normalized accordingly to obtain relative ATP levels. Measurement of levels of reactive oxygen species. Intracellular ROS levels were measured in treated and untreated cells with the permeant probe 2=,7=-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes) as described previously (27). Axenic amastigotes (2 ⫻ 106 cells/ ml) were cultured for 1 h in the absence or presence of different concentrations of either buparvaquone or ELQ-260 (0 to 10 ␮M). Amastigotes were then harvested and resuspended in PBS, and the cells were incubated with 20 ␮M H2DCFDA for 1 h at 37°C in the dark. Relative fluorescence levels were monitored in a Spectra Max Gemini XPS Spectrofluorometer

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(Molecular Devices) at 530 nm using an excitation wavelength of 507 nm. For all measurements, the value corresponding to the basal fluorescence produced by cells alone was subtracted from each measurement. The ROS level measured in control DMSO-treated cells was assigned a value of 1.0, and other values were normalized accordingly to obtain the relative ROS levels. Measurement of mitochondrial membrane potential. Mitochondrial membrane potential (⌬␺m) was measured using JC-1 cationic mitochondrial vital lipophilic dye (Molecular Probes) as described previously (28). Axenic amastigotes (2 ⫻ 106 cells/ml) were cultured for 1 h in the presence or absence of buparvaquone or the cytochrome bc1 inhibitor antimycin A (0 to 10 ␮M) as a positive control. Cells were harvested, resuspended in Hanks’ balanced salt solution (HBSS), and incubated with JC-1 (10 ␮g/ml) for 10 min at 37°C. After washing was performed twice with HBSS, fluorescence was measured spectrofluorometrically at both 530 nm and 590 nm using an excitation wavelength of 480 nm. The ratios of the values obtained at 590 nm and 530 nm were normalized to that obtained with DMSO alone and plotted as the relative ⌬␺m values. Determination of NADⴙ and NADH levels. NAD⫹/NADH ratios were determined using an NAD⫹/NADH-Glo Assay detection kit (Promega) according to the manufacturer’s instructions. Axenic amastigotes were cultured for 1 h at 32°C in the presence or absence of different concentrations of either buparvaquone (0 to 10 ␮M) or rotenone (0 to 400 ␮M), lysates were prepared, and luminescence was recorded using Falcon solid opaque plates and a Veritas Microplate Luminometer. The NAD⫹/ NADH ratio observed with control DMSO-treated cells was assigned a value of 1.0, and other values were normalized accordingly to obtain the relative NAD⫹/NADH ratios. Synthesis and purification of ELQs. Anhydrous solvents and reagents were purchased from various fine chemical suppliers and were used without further purification. Inert atmosphere operations were conducted under argon in flame-dried glassware. Compounds were identified by analysis of 1H nuclear magnetic resonance (NMR) spectra collected on a Bruker 400-MHz instrument. Final compounds were judged to be ⬎95% pure by high-performance liquid chromatography (HPLC) analysis using an HP1100 HPLC instrument at 254 nm with a Phenomenex Luna C8(2) reverse-phase column (5 micron particle size, 50 mm by 2 mm inner diameter [i.d.]) at 40°C and were eluted with methanol–water– 0.5% trifluoroacetic acid (TFA) and acetonitrile–water– 0.5% TFA at 0.4 ml/min. ELQ-118, ELQ-120, ELQ-136, and ELQ-390 were synthesized as described previously (11, 12). ELQ-233, ELQ-271, ELQ-300, ELQ-314, ELQ-316, ELQ-317, ELQ-319, ELQ-351, ELQ-372, ELQ-380, ELQ-384, ELQ-385, ELQ-388, and ELQ-404 were synthesized using methods described in references 14 and 29, employing a Suzuki reaction (30) as the key step, while ELQ-245, ELQ-260, and ELQ-274 were synthesized using a Sonogashira reaction (31) as the key step. ELQ-337, ELQ-338, and ELQ370 were synthesized as described in reference 32. Buparvaquone was purchased from Sigma-Aldrich. Statistical analysis. Results were expressed as means ⫾ standard deviations (SD) of the results determined for 2 to 4 samples in at least 2 independent experiments. The results were analyzed using GraphPad Prism 6 software. For dose-response curves, data were fitted by nonlinear regression to sigmoidal fit. Alternatively, a spline curve was employed when data did not converge due to limited or no inhibition (see, e.g., Fig. 3D and E).

RESULTS

Growth-inhibitory activity of ELQs against intracellular amastigotes of L. mexicana and J774.A1 macrophages. To determine whether ELQs are effective in killing disease-causing amastigotes, we employed transgenic strains of parasites expressing a luciferase gene, similarly to the approach described by Roy et al. (33). We integrated the Renilla luciferase gene (34) into the ribosomal DNA (rDNA) locus of L. mexicana and monitored luminescence as a measure of parasite growth in the presence and absence of various

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FIG 1 Validation of luminescence assay for quantifying the effect of ELQs on L. mexicana amastigotes expressing Renilla luciferase and growing inside J774A.1 macrophages. (A) Relative luminescence units (RLU) were measured from increasing numbers (from 0 to 25 ⫻ 105) of intracellular amastigotes that had been enumerated microscopically. The data were fitted to a straight line using linear regression with an r2 value of 0.985, demonstrating linearity of the luminescence signal as a function of amastigote number. Standard deviations are too small to be visible in this graph. (B) A dose-response curve was determined for killing of intracellular amastigotes by ELQ-118 by microscopic enumeration of parasites, generating an IC50 of 0.69 ␮M. (C) A dose-response curve was determined for ELQ-118 using the luminescence assay (open circles), resulting in an IC50 of 1.4 ␮M. The viability of J774A.1 macrophages at various concentrations of ELQ-118 was also determined, using SYBR green to quantify cellular nucleic acid. All values were normalized to those determined without ELQ-118 but with DMSO vehicle.

compounds. To validate the luciferase assay under the experimental conditions used in this study, we compared the number of intracellular amastigotes enumerated microscopically, i.e., using the conventional assay for quantifying Leishmania infections of macrophages, with the luminescence signal generated by luciferase activity (Fig. 1A). The high degree of correlation between these two signals (r2 ⫽ 0.985) demonstrated that luciferase activity accurately reflects the number of intracellular parasites over a wide range of parasite numbers. To further demonstrate the utility of the luciferase assay, we produced initial dose-response curves for ELQ-118 employing microscopic enumeration (Fig. 1B) and luciferase analysis (Fig. 1C, open circles) and obtained IC50s of 0.69 ␮M and 1.4 ␮M, respectively, demonstrating a close correlation between the two methods. Inhibition of amastigote growth could result from toxicity of ELQs toward the amastigotes or their host cells. To provide a facile screen that would initially eliminate ELQs that were toxic to mammalian cells, we monitored growth of J774.A1 macrophages, initially at a 10 ␮M concentration, using the SYBR green-based assay (35) to quantify levels of host cell nucleic acid (Fig. 1C, filled circles). This cell screen identified 30 ELQs that inhibited host cell growth at 10 ␮M by ⬍25% over the course of 4 days (Table 1). In this screen, primarily ELQs with numerical designations larger than ELQ-200 were tested, as the aliphatic side chains at position 3 on the ELQs with lower numbers such as ELQ-118, -120, and -136 were metabolically unstable, whereas the aromatic side chains on ELQs with numerical designations above 200 were typically associated with higher metabolic stability (14). These minimally macrophage-toxic ELQs were subsequently screened for those that inhibited growth of L. mexicana amastigotes by ⬎80% under conditions of application for 4 days at a 10 ␮M concentration. This subset of ELQs was then examined in dose-response experiments performed with both intracellular amastigotes and J774.A1 macrophages using 9 concentrations of each compound (see Fig. 1C as an example) to quantify the potency (IC50) against intracellular amastigotes versus the low level of inhibition of macrophage growth (Table 1). The results demonstrated that 15 ELQs had IC50s for amastigotes that were below 5 ␮M, while inhibition of host cell growth was typically very limited (similarly to that reported in Fig. 1C), precluding determina-

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tion of IC50s. Thus, the best ELQs have potencies in the same concentration range as many clinically employed drugs used to treat leishmaniasis (36). However, this level of potency does not approach that for the best ELQs against P. falciparum and T. gondii, where the IC50s are in the low nanomolar range (11, 13). While carrying out the studies described above, we also investigated the effect of a compound that is structurally related to both ubiquinone and ELQs, the hydroxynaphthoquinone buparvaquone (Table 1). We chose to investigate buparvaquone because it had previously been shown to inhibit growth of intracellular amastigotes of L. donovani with an IC50 of 0.05 ␮M (37) and might thus act as an inhibitor of cytochrome bc1. Examined against intracellular amastigotes of L. mexicana, buparvaquone exhibited an IC50 of 0.15 ␮M (Table 1). Efficacy of ELQs and buparvaquone against L. donovani. Since visceral leishmaniasis is the most lethal of the leishmaniases (1), we also tested the efficacy of several of the most potent ELQs and buparvaquone against L. donovani. For these experiments, a reporter luciferase-expressing line of L. donovani was also generated by integration of the Renilla luciferase gene into the rDNA locus. Dose-response curves and measurement of inhibition of macrophage growth revealed that ELQ-245, -260, -271, -274, and -314 also exhibited low micromolar IC50s (Table 1) against intracellular L. donovani amastigotes, confirming that L. mexicana and L. donovani are similarly susceptible to this class of compounds. For both species, buparvaquone exhibits a lower IC50 (0.060 ␮M for L. donovani) than any of the ELQs and is nontoxic to macrophages (Table 1). ELQs and buparvaquone inhibit cytochrome bc1 activity in isolated mitochondria of Leishmania mexicana. To determine whether ELQs and buparvaquone can inhibit cytochrome bc1 activity in Leishmania parasites, as they do for P. falciparum (14), mitochondria were isolated from L. mexicana axenic amastigotes and cytochrome bc1 activity was measured in the presence and absence of a 10 ␮M concentration of each of the 5 ELQs or buparvaquone. The initial rate of reduction of cytochrome c by cytochrome bc1 was strongly reduced by all 6 compounds compared to the activity of cytochrome bc1 in the absence of this inhibitor (Fig. 2), confirming that these compounds can act as inhibitors of L. mexicana cytochrome bc1 activity, at least in the context of iso-

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TABLE 1 Effects of ELQs on intracellular amastigotes of L. mexicana and L. donovani and on J774A.1 macrophagesa IC50 (␮M) for amastigotes of:

ELQ no. or name

L. mexicana

L. donovani

Macrophage growth (% at 10 ␮M)

118

0.7 ⫾ 0.5

ND

2⫾2

120

3

ND

0

136

4⫾1

ND

0⫾0

233

8

ND

33

245

4⫾3

4⫾2

12 ⫾ 10

260

1.2 ⫾ 0.1

3⫾1

12 ⫾ 8

271

0.8 ⫾ 0.2

4⫾1

21 ⫾ 3

274

3⫾1

7⫾5

13 ⫾ 13

300

5⫾4

ND

0⫾0

314

1.8 ⫾ 0.2

2⫾1

16 ⫾ 13

316

12

ND

1

317

2⫾0

ND

7⫾7

319

2⫾2

ND

15 ⫾ 4

Structure

(Continued on following page)

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TABLE 1 (Continued) IC50 (␮M) for amastigotes of:

ELQ no. or name

L. mexicana

L. donovani

Macrophage growth (% at 10 ␮M)

337

6⫾3

ND

0.5 ⫾ 0.7

338

9

ND

0

351

10

ND

10

370

4⫾1

ND

7 ⫾ 11

372

3⫾1

ND

12 ⫾ 10

380

34

ND

17

384

0.910 ⫾ 0.002

ND

26 ⫾ 19

385

8.4

ND

0.2

388

8

ND

17

390

5⫾3

ND

2⫾2

404

5⫾4

ND

6⫾6

Buparvaquone

0.2 ⫾ 0.1

0.0609 ⫾ 0.0005

12 ⫾ 11

Pentamidine

1.1 ⫾ 0.8

ND

14 ⫾ 10

Structure

a The leftmost column designates each ELQ by number or name, and the rightmost column shows the corresponding chemical structure. Intermediate columns indicate the IC50s (⫾ standard deviations) for intracellular amastigotes of either L. mexicana or L. donovani, determined from dose-response curves, and the percentages of inhibition (⫾ standard deviations) of macrophage growth at a concentration of 10 ␮M. ND, not determined. Data represent means and standard deviations of results of least 2 independent experiments.

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FIG 2 Inhibition of cytochrome bc1 (CytC reduction) activity, in mitochondria isolated from axenic amastigotes of L. mexicana, by ELQs and buparvaquone. Measurements were made in the presence of the control DMSO vehicle and in the presence of 5 ELQs or buparvaquone (BPVQ) applied at a concentration of 10 ␮M. Results were normalized to the activity in the DMSO control. Data points in this and subsequent figures represent the means and standard deviations (error bars) of results from 3 biological replicates.

lated mitochondria. Similar experiments (not shown) also showed strong inhibition of L. donovani cytochrome bc1 activity by ELQ-271. Effect of buparvaquone and ELQs on depletion of cellular ATP. To determine whether inhibition of cytochrome bc1 activity by buparvaquone or ELQs impairs synthesis of ATP by disrupting oxidative phosphorylation, the ATP levels in axenic amastigotes were measured as a function of inhibitor concentration (Fig. 3A). A 1-h treatment with 10 ␮M buparvaquone reduced the level of ATP by ⬃90%, and an IC50 of 0.16 ⫾ 0.06 ␮M (n ⫽ 3) was determined for this effect. The reduction in cellular ATP levels was not simply the result of parasite death, as 97% ⫾ 3% (n ⫽ 3) of the amastigotes were still viable following the 1 h of incubation with 10 ␮M buparvaquone, as determined by luciferase activity. In contrast, treatment of parasites with 10 ␮M ELQ-260 produced a reduction in ATP levels of only 24.5% ⫾ 11.5% after 1 h (Fig. 3B) and reduced ATP levels by only ⬃50% after 96 h of incubation (data not shown). Effect of buparvaquone on other mitochondrial functions. In addition to impairing production of ATP as a cellular energy source, inhibition of the mitochondrial ETC could have other potentially toxic effects, such as generation of ROS (7), depletion of the essential coenzyme NAD⫹ due to the failure of the inhibited ETC to oxidize NADH via NADH dehydrogenase (38), and/or impairment of the mitochondrial membrane potential (⌬⌿m) by inhibition of proton pumping associated with ETC activity (39). To test the effect of buparvaquone on each of these mitochondrial functions, these parameters were measured (see Materials and Methods) in the presence of buparvaquone or a control inhibitor of either NADH dehydrogenase (rotenone) or cytochrome bc1 (antimycin). Application of buparvaquone to axenic amastigotes for 1 h resulted in enhanced levels of ROS, with increases of up to ⬃3-fold seen when buparvaquone was administered at concentrations ranging between 10⫺5 and 10⫺6 M (Fig. 3C). In contrast, ELQ-260 did not induce an increase in ROS levels (Fig. 3D). Bu-

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parvaquone applied at concentrations of up to 10⫺5 M (Fig. 3E), the limit beyond which it begins to inhibit the viability of host macrophages, did not alter ⌬⌿m levels, and application of buparvaquone also did not significantly reduce the NAD⫹/NADH ratio (Fig. 4A). The cytochrome bc1 inhibitor antimycin significantly decreased ⌬⌿m levels (Fig. 3F), and the NADH dehydrogenase inhibitor rotenone exhibited the expected reduction in the NAD⫹/NADH ratio (Fig. 4B), demonstrating that control inhibitors of the ETC did exhibit inhibition of each of the mitochondrial functions that were not inhibited by buparvaquone. Recent studies of mammalian cells confirmed that inhibition of electron transport blocks proliferation but demonstrated the unexpected result that addition of 10 mM aspartate was able to release the replication block (9, 10). This effect was attributed to the reduction in the level of oxidized NAD⫹ in the absence of ETC activity and the resultant impairment in NAD⫹-dependent aspartate biosynthesis, which was overcome by supplementation of the medium with this amino acid. To determine whether the lethal effects of ETC inhibition could be overcome by aspartate in Leishmania parasites, L. mexicana axenic amastigotes were incubated with various concentrations of buparvaquone with and without supplementation with 10 mM aspartate (Fig. 4C). Unlike results reported for mammalian cells, aspartate was not able to restore growth of parasites whose ETC had been inhibited by buparvaquone. This result is consonant with the observation that buparvaquone does not deplete NAD⫹ levels when applied to axenic amastigotes and, therefore, that aspartate biosynthesis should not be inhibited. Buparvaquone is cytocidal rather than cytostatic. To determine whether buparvaquone inhibited growth of intracellular amastigotes by a cytocidal mechanism or a cytostatic mechanism, J774A.1 macrophages that had been infected overnight with luciferase-expressing L. mexicana promastigotes were treated with either DMSO vehicle or 10 ␮M buparvaquone for increasing periods of time from 1 to 96 h (Fig. 5A). For infected macrophages treated with DMSO, the number of intracellular amastigotes decreased by ⬃20% over 96 h, while treatment with 10 ␮M buparvaquone resulted in complete death of the amastigotes by 96 h. This result indicates that buparvaquone is cytocidal, because application of this compound leads to parasite death rather than simply growth arrest (40). DISCUSSION

Previous investigations have suggested that insect stage Leishmania (17–19) and T. cruzi (20) are susceptible to inhibitors of cytochrome bc1, but those experiments did not interrogate the diseasecausing stage of the parasite life cycles or employ compounds that have potential for drug development such as the ELQs or buparvaquone. The principal contribution of the current study was to confirm the potential of cytochrome bc1 as a valid target for development of novel antileishmanial drugs. While several ELQs exhibited selective potency in the low micromolar range against intracellular amastigotes of both L. mexicana and L. donovani, they did not achieve the low-nanomolar efficacy that has made ELQs attractive scaffolds for development of new and extremely potent drugs against P. falciparum and T. gondii. Although it is possible that synthesis of new ELQs could generate more-robust members of this class, the observation that buparvaquone strongly inhibits L. mexicana cytochrome bc1 and is selectively toxic to intracellular amastigotes at nanomolar concentrations suggests that this hy-

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FIG 3 Effects of buparvaquone and ELQ-260 on mitochondrially linked properties, including cellular ATP levels, reactive oxygen species (ROS), and mitochondrial membrane potential (⌬⌿m). L. mexicana axenic amastigotes were treated for 1 h with a range of concentrations of buparvaquone (A, C, and E), ELQ-260 (B and D), or the cytochrome bc1 inhibitor antimycin (F). Measurements were normalized to the values obtained in the presence of DMSO vehicle but the absence of added compound (0 M). (A and B) Relative levels of cellular ATP. (C and D) Relative levels of ROS. (E and F) Relative levels of ⌬⌿m.

droxynaphthoquinone represents a more promising chemotype for drug development. Consistent with the higher potency of buparvaquone against intracellular amastigotes, this compound was able to robustly deplete ATP at submicromolar concentrations following 1 h of incubation, whereas 10 ␮M ELQ-260 did not substantially reduce ATP levels after a 1-h treatment. These results suggest that inhibition of the ETC in amastigotes manifests its lethal effect at least partly via depletion of ATP stores. In contrast, the fact that ELQs reduce synthesis of ATP in axenic amastigotes modestly and slowly (⬃50% reduction after 96 h) suggests either that their kinetics of inhibition of the ETC is much slower than that of buparvaquone or that their toxicity toward intracellular amastigotes does not result from inhibition of the ETC, even though application of ELQs to isolated mitochondria can inhibit cytochrome bc1 activity (Fig. 2). This unanticipated result suggests that ELQs may have another target in amastigotes.

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The ability of buparvaquone to increase the generation of ROS, presumably because it blocks cytochrome bc1 activity, thus causing release of electrons to molecular oxygen (7), is another potential mechanism of toxicity for this compound. The NAD⫹/NADH ratio is not altered by buparvaquone, suggesting that NADH is still reduced by NADH dehydrogenase but that the electrons generate ROS due to downstream blockage of the ETC. Similarly, ⌬⌿m is maintained in buparvaquone-poisoned mitochondria, possibly by the continued action of NADH dehydrogenase. These studies have revealed the mechanism of action of buparvaquone as an inhibitor of cytochrome bc1 activity and elucidate the biochemical consequences of this inhibition, i.e., reductions in ATP levels and increases in ROS levels, that most likely contribute to antileishmanial toxicity. There are two fundamental structural differences between buparvaquone and ELQs (Table 1): (i) buparvaquone has a carbonyl moiety at position 1 rather than in the secondary amino group of

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FIG 4 Effects on buparvaquone and rotenone on the cellular NAD⫹/NADH ratio. (A and B) L. mexicana axenic amastigotes were incubated for 1 h in the indicated concentrations of buparvaquone (A) or the control NADH dehydrogenase inhibitor rotenone (B). (C) Axenic amastigotes were incubated for 96 h in a range of buparvaquone concentrations in the presence (filled circles) or absence (open circles) of 10 mM aspartate, and relative survival rates were determined using the luciferase assay. The indicated IC50s in the presence and absence of 10 mM aspartate were derived by fitting the dose-response curves, as indicated in Materials and Methods.

ELQs; and (ii) buparvaquone has an aliphatic side chain at position 2 instead of the aromatic side chain at position 3 that is present in the pharmacologically active ELQs. In future structure-activity studies of buparvaquone relevant to further exploration of its potential for drug development, it will be interesting to exchange these structural features one at a time to determine whether it is the carbonyl moiety or the side chain at position 2 (or both) that is necessary for the potent activity of buparvaquone and its analogs against the Leishmania ETC. One potential advantage of buparvaquone, or of other compounds that selectively poison the parasite ETC, is that it targets a multisubunit protein, cytochrome bc1. Leishmania parasites have remarkably plastic nuclear genomes and are often able to amplify segments of genomic DNA that encode the targets of cytotoxic compounds, thus attaining resistance to the cytotoxin (41). For example, the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene is amplified when parasites are exposed over extended periods of time to the cytotoxic DHFR inhibitor methotrexate, and these mutants are highly resistant to methotrexate due to the pronounced increase in the level of the DHFR-TS target protein (42). This mechanism of resistance is unlikely to be available to parasites exposed to buparvaquone, because (i) amplification of the cytochrome b open reading frame does not achieve an increase in the level of the multisubunit pro-

tein and (ii) the cytochrome b gene is carried by mitochondrial maxicircle DNA (43) and not within the plastic nuclear genome. Although buparvaquone has been studied previously for its effect on amastigotes in vitro and in a murine model of visceral leishmaniasis (37), its activity against cytochrome bc1 has not been investigated. The relatively modest efficacy of this compound in the murine model of disease, as shown by an ⬃60% reduction in liver parasite burden, was attributed to probable poor distribution of the compound after injection, poor oral absorption, and relatively rapid turnover by liver microsomes. Further studies of water-soluble phosphate derivatives of buparvaquone (44) have demonstrated their ability to permeate skin, and an anhydrous gel formulation (45) of this prodrug was more effective than the parent compound in that study, although it still reduced parasite burdens in the liver by only ⬃34%. Buparvaquone incorporated into phosphatidylserine liposomes demonstrated increased efficacy against L. infantum chagasi in a hamster model of visceral leishmaniasis at a 60-fold-lower dose than free buparvaquone (46) and reduced levels of amastigotes in the liver by ⬃67%. Hence, while some advances in modification of this chemotype or mode of delivery have been made, development of buparvaquone into a useful drug is likely to require a committed medicinal chemistry program in which diverse modifications of the parent scaffold are undertaken to increase in vivo performance. Indeed,

FIG 5 Effect of buparvaquone on intracellular amastigotes as a function of time and concentration. (A) J774A.1 macrophages were infected with L. mexicana promastigotes expressing Renilla luciferase, incubated for 16 h, washed with PBS, and reincubated in the presence of either DMSO vehicle (solid bars) or 10 ␮M buparvaquone (BPQ). Relative luminescence units (RLU) were measured at the indicated times thereafter. (B) Dose response curves were determined for buparvaquone applied to infected macrophages for increasing periods of time. Percent survival rates were quantified as RLU values compared to the value measured in the presence of DMSO vehicle only. The reported IC50s were determined from fitting the dose-response curves obtained at all times of incubation.

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such a medicinal chemistry program was required to convert endochin, a compound with high in vitro potency against Plasmodium parasites but low in vivo efficacy in a murine model of malaria (11), into highly potent antimalarial compounds such as ELQ-300 and ELQ-400 (14, 29, 47). One critical modification to the original scaffold was to incorporate aromatic side chains such as the diarylether group at position 3, thus leading to greatly enhanced metabolic stability (14). Accordingly, generation of dozens of buparvaquone analogs, including those with aromatic side chains at position 2 or 3, as well as metabolizable prodrugs that increase aqueous solubility and oral bioavailability of the series (32), may be required to realize the full potential of the buparvaquone chemotype as an antileishmanial drug. Additionally, a search for alternate chemotypes that selectively inhibit parasite electron transport may produce other promising leads for drug development. ACKNOWLEDGMENTS Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000128. This support was delivered via the Oregon Clinical & Translational Research Institute of the Oregon Health & Sciences University. This work was also supported by R21 AI102874 (to S.M.L.), R21 AI023682 (to B.U.), and R21 AI100569 (to M.K.R.). This project was additionally supported by funds from the Veterans Affairs Merit Review Program, award number i01 BX003312 (to M.K.R.), and by funds from the U.S. Department of Defense Peer Reviewed Medical Research Program (PR130649) (to M.K.R.).

FUNDING INFORMATION This work, including the efforts of Michael K. Riscoe, was funded by Department of Veterans Affairs (i01 BX003312). This work, including the efforts of Michael K. Riscoe, was funded by Department of Defense (PR130649). This work, including the efforts of Michael K. Riscoe, Buddy Ullman, and Scott M. Landfear, was funded by HHS | National Institutes of Health (NIH) (UL1TR000128, AI102874, and AI023682). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funding institutions.

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