Eosin B as a Novel Antimalarial Agent for Drug-Resistant Plasmodium falciparum

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2006, p. 3132–3141 0066-4804/06/$08.00⫹0 doi:10.1128/AAC.00621-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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Eosin B as a Novel Antimalarial Agent for Drug-Resistant Plasmodium falciparum† Kristen M. Massimine,1,2 Michael T. McIntosh,2 Lanxuan T. Doan,1 Chloe´ E. Atreya,1 Stephan Gromer,3 Worachart Sirawaraporn,4 David A. Elliott,2‡ Keith A. Joiner,2‡ R. Heiner Schirmer,3 and Karen S. Anderson1* Department of Pharmacology1 and Infectious Diseases Section, Department of Internal Medicine,2 Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520; Biochemistry Center, Im Neuenheimer Feld 504, Heidelberg University, D-69120, Heidelberg, Germany3; and Department of Biochemistry, Mahidol University, Bangkok, Thailand4 Received 19 May 2006/Returned for modification 27 May 2006/Accepted 10 June 2006

Plasmodium falciparum, the causative agent of malaria, is a protozoan parasite that has a significant impact on health worldwide. Due to the emergence and spread of resistance to antimalarial drugs, novel chemotherapeutics for the treatment of malaria are urgently needed. To achieve this goal, antiparasitic research needs not only to identify new, chemically diverse molecules for the circumvention of cross-resistance but also to discover novel targets (44). Ideal putative targets will have a variety of characteristics, including involvement in an essential feature of the parasite life cycle, parasitic selectivity over the host, and low potential for resistance development (44). Along these lines, we recently identified a compound that acts as an inhibitor for a novel chemotherapeutic target in the related apicomplexan organism Toxoplasma gondii. This compound, eosin B (4⬘,5⬘-dibromo-2⬘,7⬘-dinitrofluorescein), targets a unique non-active-site region on the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme (2). Inhibitors specific to this area have the potential to be powerful antiparasitics since they target folate metabolism, an essential feature of the parasitic life cycle, through a different mode of action than the currently utilized antifolate therapies. Furthermore, the nonactive target site is unique to the bifunc-

tional parasitic enzyme—it is not contained within mammalian TS and DHFR counterparts—and therefore provides for a high level of selectivity. Cell culture and biochemical assays agree that the average 50% inhibitory concentration (IC50) of eosin B for T. gondii is 180 ␮M (2). Importantly, the relative safety of eosin has been established previously; the FDA has shown that eosin is not carcinogenic to mice after a dietary exposure as high as 2.0% and therefore has approved it for use in drugs and cosmetics (16). Specifically, in terms of eosin B, single or intermittent exposure of rat embryos to either 120 ␮M or 600 ␮M does not affect in vitro development of blastocysts (13). Furthermore, eosin B-treated blastocysts can be transferred to either naturally mated or pseudopregnant recipients with no effect on embryo survival to term (13). With this in mind, it can be suggested that eosin B treatment is safe, does not promote genotoxicity, and, based on our findings with T. gondii, may serve as a useful lead compound for drug discovery against parasitic infection. We therefore wanted to evaluate eosin B as a potential lead compound for antimalarial drug development by testing its effects on P. falciparum. Since this organism also contains a bifunctional DHFR-TS protein, eosin B may exert selective antimalarial effects either through a mechanism similar to that observed in T. gondii, by an alternative mode of action, or via a combination of the two. Our data reveal that eosin B is a potent inhibitor of a variety of drug-resistant malarial strains in cell culture with a low average IC50 of 124 nM. There is no indication of eosin B cross-resistance with other drugs, suggesting that this compound is acting via a novel mechanism within the parasites.

* Corresponding author. Mailing address: Yale University School of Medicine, SHM B350-B, 333 Cedar St., New Haven, CT 06520. Phone: (203) 785-4526. Fax: (203) 785-7670. E-mail: karen.anderson@yale .edu. † Supplemental material for this article may be found at http://aac .asm.org/. ‡ Present address: Department of Cell Biology and Anatomy, University of Arizona Medical Center, Tucson, AZ 85724. 3132

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4ⴕ,5ⴕ-Dibromo-2ⴕ,7ⴕ-dinitrofluorescein, a red dye commonly referred to as eosin B, inhibits Toxoplasma gondii in both enzymatic and cell culture studies with a 50% inhibitory concentration (IC50) of 180 ␮M. As a non-active-site inhibitor of the bifunctional T. gondii dihydrofolate reductase-thymidylate synthase (DHFRTS), eosin B offers a novel mechanism for inhibition of the parasitic folate biosynthesis pathway. In the present study, eosin B was further evaluated as a potential antiparasitic compound through in vitro and cell culture testing of its effects on Plasmodium falciparum. Our data revealed that eosin B is a highly selective, potent inhibitor of a variety of drug-resistant malarial strains, with an average IC50 of 124 nM. Furthermore, there is no indication of cross-resistance with other clinically utilized compounds, suggesting that eosin B is acting via a novel mechanism. The antimalarial mode of action appears to be multifaceted and includes extensive damage to membranes, the alteration of intracellular organelles, and enzymatic inhibition not only of DHFR-TS but also of glutathione reductase and thioredoxin reductase. In addition, preliminary studies suggest that eosin B is also acting as a redox cycling compound. Overall, our data suggest that eosin B is an effective lead compound for the development of new, more effective antimalarial drugs.

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Furthermore, several lines of evidence indicate that the mode of action of eosin B is multifaceted, containing a variety of mechanisms including non-active-site inhibition of DHFR-TS as well as the glutathione reductase (GR) and thioredoxin reductase (TrxR) enzymes. Taken together, our data suggest that eosin B may be utilized as a lead compound for the development of new, more effective antimalarial drugs.

MATERIALS AND METHODS

content was determined by the bicinchoninic acid assay (53) using serum albumin as a standard. Selectivity was calculated as (IC50 of eosin on mammalian cells)/ (IC50 of eosin on parasites). Eosin B stage specificity. To determine the point within the Plasmodium life cycle where eosin B exerts its effects, FCR3 parasites synchronized to the ring stage were split into two populations and allowed to grow either in the presence or in the absence of 1 ␮M eosin B. Ten hours after the commencement of the initial ring stage, drug treatment was initiated and was allowed to continue for the duration of one complete 48-h erythrocytic life cycle. Parasite morphology was observed via Giemsa-stained blood smear analysis at several time points post-drug treatment: 0, 18, 30, and 42 h. Additionally, eosin B reversibility was tested by removing drug pressure from a subpopulation of eosin B-treated parasites at the 18-h time point. Monitoring continued for the remainder of the time points. Light and electron microscopy on Plasmodium. Epifluorescence microscopy was performed on infected cells using a Microphot FXA microscope (Nikon Inc., Melville, NY). Images were captured on a Photometrics SenSys charge-coupled device (CCD) camera, processed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Giemsa-stained images were captured on a Zeiss Axioplan microscope with a Diagnostic Instruments camera and processed with SPOT Camera software. For thin-section transmission electron microscopy, cells were fixed in 2.5% glutaraldehyde (EM Science), 2% paraformaldehyde, 2 mg/ml tannic acid, 0.3 mg/ml saponin, 50 mM KCl, 5 mM MgCl, and 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at room temperature. They were then washed three times in 0.1 M cacodylate buffer and then postfixed for 1 h in 1% osmium tetroxide (EM Science) in the same buffer at 4°C. After three washes in water, the samples were stained for 1 h at room temperature in 2% uranyl acetate (EM Science), washed again in water, and dehydrated in a graded series of ethanol. Samples were then incubated for 1 h in a lead acetate solution, washed with acetone, and embedded in resin. Ultrathin (50- to 60-nm-thick) sections were cut using a Reichert Ultracut ultramicrotome, collected on Formvar- and carboncoated nickel grids, and stained with 2% uranyl acetate and lead citrate before examination with either a Philips (Eindhoven, The Netherlands) 410 or a Hitachi electron microscope under 80 kV. Intracellular accumulation of eosin B. To visualize how eosin B associated with P. falciparum, infected red blood cells were enriched for trophozoite-stage parasites. A total of 2 ⫻ 107 cells were incubated in culture medium containing 1 ␮g/ml Hoechst 33342 for 20 min at 37°C. After a subsequent wash, a set of parasites was killed (60°C for 10 min). Both live and dead parasites were then incubated with 100 ␮M of eosin B for 30 min at either 37°C or 4°C. After a wash, cells were visualized on a Microphot FXA microscope (Nikon Inc., Melville, NY). Images were captured on a Photometrics SenSys CCD camera and processed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Evaluation of DHFR-TS enzyme inhibition by eosin B. The Plasmodium falciparum DHFR-TS plasmid was from Worachart Sirawaraporn (Mahidol University). DHFR-TS was purified according to a previous protocol (46). The DHFR-TS protein concentration was estimated using the Bio-Rad system with serum albumin as a standard. Steady-state experiments were carried out by mixing 2⫻ enzyme solution (1 ␮M enzyme, 1 mM EDTA, 50 mM MgCl2, 50 mM Tris, pH 7.8, 1 mM dUMP, 1 mM NADPH, and eosin B [0.075 to 0.5 mM] in DMSO) with an equal volume of 400 ␮M radiolabeled CH2H4 folate. Control reactions were conducted in which the vehicle, DMSO, was used in place of eosin B. Reactions were monitored from 0 to 50 s and were terminated by quenching with 67 ␮l of KOH solution (10% sodium ascorbate, 200 mM 2-mercaptoethanol, 0.78 M KOH, pH 12.6). The amount of DMSO was kept constant in all experiments at 4%. The resulting tritiated folates were quantified by high-performance liquid chromatography in combination with a radioactivity flow detector. Separation was accomplished using a BDS Hypersil C18 reverse-phase column (250 by 4.6 mm; Keystone Scientific, Bellefonte, PA) with 10% methanol–180 mM triethylammonium bicarbonate (pH 8) at 1 ml/min. The high-performance liquid chromatography effluent from the column was mixed with scintillation cocktail (Mono-Flow V; National Diagnostics) at a flow rate of 5 ml/min. Radioactivity was monitored continuously with a Flo-One radioactivity flow detector (Packard Instruments, Downers Grove, IL). Sample injection was automated using a Waters (Milford, MA) 712B WISP autosampler. Evaluation of GR and TrxR enzyme inhibition by eosin B. Human GR (hGR) (34, 42), P. falciparum GR (PfGR) (15), human TrxR (hTrxR) (20), and P. falciparum TrxR (PfTrxR) (28) were prepared and assayed according to published procedures. All enzymes are homodimeric proteins containing 1 flavin adenine dinucleotide per subunit of approximately 55 kDa. The Mrs per subunit are 52,400 for hGR, 57,200 for PfGR, 55,200 for hTrxR, and 60,300 for PfTrxR.

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Chemicals. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Boehringer Mannheim Biochemicals (Indianapolis, IN) unless otherwise indicated. Eosin B was purchased from ABCR (A Better Choice for Research Chemicals; Karlsruhe, Germany). Radiolabeled reagents included [methyl-3H]thymidine (specific activity, 6.7 Ci/mmol) and [3H]hypoxanthine (specific activity, 10 to 30 Ci/mmol), purchased from Amersham Corp. (Arlington Heights, IL). Radiolabeled dihydrofolate was synthesized by reduction of [3⬘,5⬘,7,9-3H]folic acid (Moravek Biochemicals, Brea, CA) with sodium hydrosulfite. Radiolabeled methylenetetrahydrofolate (CH2H4 folate) was synthesized by enzymatic conversion of radiolabeled dihydrofolate to form tetrahydrofolate (H4 folate), which was subsequently condensed with formaldehyde to yield CH2H4 folate (39). The (6R)-L-methylenetetrahydrofolate enantiomer was purified by DE-52 anion-exchange chromatography (Whatman) (10). Hoechst 33342 was from Molecular Probes (Eugene, OR). Cell lines and culture conditions. The nucleated mammalian cell lines used in this study were primary human foreskin fibroblasts (HFF) (ATCC CRL-1635) grown as monolayers as described previously (7) and HeLa cells grown as monolayers at 37°C under an atmosphere of 5% CO2 in Dulbecco’s modified Eagle medium (Gibco), supplemented with penicillin-streptomycin. Erythrocytes of blood group A⫹ were obtained from the Red Cross and were stored at 4°C until needed for Plasmodium culture. In vitro culture of P. falciparum. The four strains of P. falciparum used in this study were FCR3/Gambia subline F-86 (ATCC 50005), obtained from the ATCC (Manassas, VA); a knobless FCR3, which was a generous gift from Kasturi Haldar (Northwestern University); and Dd2 (MRA-150) and HB3 (MRA-155), obtained from MR4, ATCC (Manassas, Va.). The strains were maintained in erythrocytes of blood group A⫹ at a hematocrit between 2 and 5% and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA), pH 7.4, supplemented with 25 mM HEPES, sodium bicarbonate (2 g/liter), 11 mM glucose, gentamicin (25 to 40 ␮g/ml), and 92 ␮M hypoxanthine and containing human plasma (for FCR3, Dd2, and HB3) or serum (for knobless FCR3) at 10% as described previously (54). For maintenance of cultures, the medium was changed daily. Parasite synchronization was achieved by weekly treatment of ring-stage infected red blood cells (iRBC) with either 0.3 M L-alanine or 5% sorbitol (35). Trophozoites were enriched to a parasitemia of ⬎50% with 1% gelatin (19, 25) or 65% Percoll (30) depending on the strain. Evaluation of in vitro antimalarial activity. Effects of eosin B on P. falciparum were assessed by measuring the incorporation of the radiolabeled nucleic acid precursor [3H]hypoxanthine (6, 12). The test compounds (eosin B, fluorescein, phenolphthalein, methotrexate [MTX], pyrimethamine [PYR], and chloroquine [CQ]) were initially dissolved in either dimethyl sulfoxide (DMSO) or ethanol and subsequently serially diluted in a 96-well flat-bottom microtiter plate (Falcon) with culture medium (RPMI-1640, 10% A⫹ human plasma, 40 ␮g/ml gentamicin). Parasitized erythrocytes (5% parasitemia, 5% hematocrit) synchronized to the ring stage were added to these various inhibitor concentrations. Plates were then placed in a gas-tight box that was flushed with a gas mixture of 5% O2 and 5% CO2 balanced with 90% N2, sealed, and incubated at 37°C. After 24 h, [3H]hypoxanthine (0.5 ␮Ci/well) diluted in culture medium was added to each well, and following a further 18-h incubation, the cells were harvested onto glass fiber filters (Wallac, Finland) with a semiautomatic cell harvester (Skatron, Leir, Norway) and incorporation of [3H]hypoxanthine into nucleic acids was measured by liquid scintillation counting. All data points were collected in triplicate for each experiment. Dose-response curves and IC50s of the averaged data were generated by using GraphPad Prism software, where the IC50 was defined as the amount of test compound required to inhibit [3H]hypoxanthine uptake by 50% compared to the control. Controls consisted of complete medium as a substitute for the test compound. Evaluation of eosin B toxicity. To determine the toxicity of eosin B as well as the selectivity of the compound for the parasites, eosin B was tested on mammalian cell lines (HFF and HeLa). Effects were assessed by measuring the incorporation of the radiolabeled nucleic acid precursor [3H]thymidine. Protein

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RESULTS Eosin B selectively inhibits the growth of drug-resistant P. falciparum. The effect of eosin B on P. falciparum growth was assayed by measuring the incorporation of [3H]hypoxanthine in growing ring-stage parasites. Initially, tests were performed on FCR3, a West African strain from Gambia that is resistant to chloroquine (26). It was observed that the amount of [3H]hypoxanthine taken up decreased with increasing eosin B concentration, indicating a clear decrease in malarial parasite survival; eosin B was found to inhibit FCR3 growth with an average extracellular IC50 of 111.5 nM (Table 1). It was determined that the DMSO concentrations used to dissolve the eosin B did not have an inhibitory effect on parasite growth (data not shown). Furthermore, the ability of eosin B to inhibit the growth of dividing mammalian cells was tested. Assays performed in triplicate determined the IC50s of eosin B to be 151.6 ␮M (n ⫽ 4) and 320 ␮M (n ⫽ 1) for actively dividing HFF and HeLa cells, respectively. This indicates that at the concentrations needed for 50% parasitic growth inhibition (IC50 for FCR3, 111.5 nM; n ⫽ 7), eosin B is nontoxic to mammalian host cells. Specifically, eosin B demonstrated a ⬎1,000-fold difference in inhibitory concentration between parasite and mammalian cells, thus further indicating a considerable amount of selectivity for the parasite. Promising initial results with eosin B indicated that further testing of this compound on other drug-resistant Plasmodium strains was warranted. Two strains were selected on the basis of their origin and drug susceptibility properties: HB3, a PYRresistant strain from Honduras (4), and Dd2W, a West African (Gambian) strain that is resistant not only to PYR but to CQ as well (58). Notably, eosin B had pronounced effects on both HB3 and Dd2W, with IC50s of 141.3 and 21.9 nM, respectively.

TABLE 1. Activity of antimalarial compounds on various P. falciparum strains Mean IC50 (nM) of the indicated compound for the following strain: Compound

EosinB PYR MTX CQ

FCR3a

Knobless FCR3b,c

HB3d

Dd2c,e

111.5 f 2.5 13.7 ND g

219.4 41.5 124.2 5806.0

141.3 9174.0 21.2 ND

21.9 5,580.0 50.5 7250.0

a From Gambia. Values are means of three to seven experiments, each performed in triplicate. b From K. Haldar, Northwestern University. c Values represent either one experiment or the means of two experiments, each performed in triplicate. d From Honduras. Values are means of two or four experiments, each performed in triplicate. e From Southeast Asia. f Selectivity indices for HFF and HeLa cells were 1,360 and 2,870, respectively. g ND, not determined.

These effects indicate a lack of cross-resistance with other known antimalarial compounds (Table 1). Eosin B enters P. falciparum via an active uptake process. To determine the localization of eosin B in iRBC, the autofluorescence properties of this molecule were exploited in fluorescence microscopy studies. After a 30-min exposure to the drug, RBC infected with live parasites selectively took up the compound at 37°C. Eosin B was excluded from uninfected RBC as well as from iRBC that contained heat-killed parasites (Fig. 1). This finding indicates that eosin B does not easily diffuse through the host cell membrane and requires active parasitic modification of the RBC to gain intracellular entry. This, in turn, accounts for the selective uptake of eosin B into parasitized red blood cells. Eosin B causes severe cytopathic effects on Plasmodium. The long-term effects of eosin B on Plasmodium were examined, and long-term-treated parasites were compared to non-drugtreated parasites at the ultrastructural level (Fig. 2). Electron microscopy studies revealed that trophozoites treated with eosin B exhibited several pronounced cytopathic effects. Compared to untreated cells (Fig. 2a), the eosin B-treated parasites (Fig. 2b and c) had obviously distended mitochondria. In addition, proper formation of the digestive vacuole appeared to be substantially altered. Multiple vacuoles containing single hemozoin crystals were evident in several cases. In situations where the hemozoin was not separated, the food vacuole appeared condensed, with no evident digestive vacuolar membranes. Furthermore, compared to normal parasites, there appeared to be a decrease in the amount of rough endoplasmic reticulum and a substantial number of ruptures between what should be very closely associated membranes. Despite these alterations, other organelles such as the apicoplast and nucleus appeared unaffected. This morphology was distinct from that observed with other clinically utilized antimalarial compounds such as MTX and CQ (see the supplemental material) (43, 61). For example, MTX treatment did not appear to affect the morphology of the trophozite, while eosin B caused damage at this stage. This observation was in agreement with literature reports which state that inhibitors of nuclear division, such as MTX, act chiefly on schizont development (1).

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Disulfide reductase concentrations were measured by using a value of 11.3 mM⫺1 cm⫺1 at ␭vis,max (463 nm ⫾ 2 nm) for bound flavin adenine dinucleotide. Prior to experimentation, eosin B was freshly prepared at a stock concentration of 500 ␮M to 5 mM in no more than 2% DMSO. As shown by control assays, the concentrations of DMSO used have no inhibitory effect on the activity and stability of the enzymes studied. All solutions were kept away from light, stored at 4°C, and replaced once the absorbance spectrum began to change. Concentrations of eosin B were determined by measuring the absorbance of approximately 10 ␮M solutions in GR assay buffer and using an ε of 61.2 mM⫺1 cm⫺1 at 519 nm. Eosin B was tested as an enzyme inhibitor by monitoring the oxidation of NADPH (ε, 6.22 mM⫺1 cm⫺1) in an optical test at 340 nm. The contribution of eosin B to the absorbance at this wavelength appeared to be constant throughout the assays. IC50s were determined in assays where the substrate concentrations were equivalent to 5 times the Km. Ki values and Ki⬘ values for GR inhibition were determined according to Cornish-Bowden (8). In each series of experiments, three substrate concentrations were used (either 10, 40, and 100 ␮M NADPH or 52.5, 175, and 875 ␮M glutathione oxidized disodium salt [GSSG]). The eosin B concentrations in the assay ranged from 260 nM to 5.3 ␮M. For determination of Ki or Ki⬘ values, the data of a given experiment were plotted according to Dixon (1/v against [I]) and according to Cornish-Bowden ([S]/v against [I]) (8); as detailed in reference 8, comparison of the two plots yields the type of inhibition. Evaluation of eosin B as a substrate of the disulfide reductases. Eosin B was tested as a substrate of the disulfide reductases. In these assays, the natural substrate (GSSG or thioredoxin) was replaced by eosin B up to a concentration of 50 ␮M. The enzyme concentration in the assay was at least 500 nM and therefore more than 200-fold higher than that in assay mixtures containing natural substrates; values around 500 nM represent physiologically occurring enzyme concentrations. Km and kcat were determined assuming Michaelis-Menten kinetics.

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Downloaded from http://aac.asm.org/ on January 7, 2016 by guest FIG. 1. Intraparasitic uptake of eosin B. iRBC were preincubated for 20 min at 37°C in culture medium containing 1 ␮g/ml Hoechst 33342. A proportion of parasites were heat killed (HK) by exposing them to 60°C for 10 min. Subsequently, samples were incubated with 100 ␮M eosin B for 30 min at 37°C. Additionally, RBC with live parasites were also incubated with 100 ␮M eosin B at 4°C for over 2 h. iRBC containing live parasites selectively acquired eosin B at 37°C, as evident by the exclusion of the compound from parasite-free RBC and iRBC containing heat-killed parasites. Exclusion of eosin uptake at 4°C indicates that an active process is required for eosin B uptake into the parasites. BF, bright field.

Eosin B reversibly inhibits parasite maturation. To determine at which erythrocytic stage eosin B exerts its effects, Plasmodium parasites in mid-ring stage were treated with eosin B for the duration of one complete 48-h erythrocytic life cycle. Parasite morphology, observed via Giemsa-stained blood smears, indicated that parasites grew normally in the presence of eosin B until the onset of schizogony (Fig. 3). At this point, disorganized segmenters were apparent, but the

release of merozoites or the formation of rings was not evident. This finding provides evidence that eosin B inhibits maturation of the parasites during the late-trophozoite stage of development. The reversibility of eosin B was tested by removing drug pressure from a subpopulation of treated parasites once they reached the trophozoite stage. Without continuous contact with the drug, parasites were able to mature throughout the

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entire 48-h life cycle and subsequently invade erythrocytes (data not shown). This suggests that the detrimental effects of eosin B are reversible. Eosin B has unique features that account for its inhibitory properties. The identification of eosin B as a nanomolar inhib-

itor of P. falciparum led to the screening of other compounds with similar scaffolds. Two compounds were selected on the basis of structural similarity: fluorescein, the unliganded precursor of eosin B which lacks the halogen and nitro groups, and phenolphthalein, a compound resembling the cyclic form of

FIG. 3. Stage selectivity of eosin B. FCR3 parasites synchronized to the ring stage were grown either in the presence or in the absence of 1 ␮M eosin B. Drug treatment commenced 10 h into the initial ring stage and continued for the duration of one complete 48-h erythrocytic life cycle. Giemsa analysis was performed after drug treatment at 0, 18, 30, and 42 h. Eosin B-treated parasites progressed until the late trophozoite/early schizont stage.

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FIG. 2. Cytopathic effects of eosin B. The long-term effects of eosin B on Plasmodium were examined, and eosin-treated parasites were compared to non-drug-treated trophozoites at the ultrastructural level. Eosin-treated parasites had obviously distended mitochondria (M), improper formation of the digestive vacuole (D) and the hemozoin (Z) within, and a distinct lack of membrane integrity, as evident by the separation of the parasite plasma membrane (PPM) from the parasitophorous vacuolar membrane (PVM). Hemoglobin (H) content appeared normal. (a) No drug; (b) 0.6 ␮M eosin B; (c) 1 ␮M eosin B.

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FIG. 4. Structure of eosin B and derivatives. (A) Eosin B; (B) fluorescein; (C) phenolphthalein.

pounds to levels higher than that supplied in the external medium. Alternatively, inhibition of other enzymatic targets could also have contributed to this difference. Based on the stage specificity, cytopathic effects, and structure of eosin B (52), it was believed that eosin B was inhibiting one or more thiol reductases. Initial observations on PfGR, hGR, PfTrxR, and hTrxR revealed that eosin B is an inhibitor of each of these enzymes, with IC50s of 1.5 ␮M, 0.6 ␮M, 4.2 ␮M, and 13 ␮M, respectively (Table 2). Because TrxR does not exist in red blood cells (21), further studies of hTrxR were not pursued. Examination of the glutathione reductases revealed Ki⬘ values of 0.5 ␮M and 1.3 ␮M for hGR and PfGR, respectively (Table 2), although the type of inhibition exerted by eosin B on these enzymes could not be unambiguously assessed. When GSSG was treated as the substrate for the glutathione reductases, and its concentration was varied, eosin B appeared to act as an uncompetitive inhibitor. However, when the NADPH concentration was varied instead, the type of inhibition was not clear: mixed noncompetitive and uncompetitive inhibition revealed itself as a possibility. Eosin B acts as a substrate of hGR and PfTrxR. Eosin B was tested as an alternative substrate for the disulfide reductases by replacing the natural substrates (GSSG or thioredoxin, respectively) with the compound and measuring the enzymes’ ability to oxidize NADPH. No detectable activity was evident for either PfGR or hTrxR; however, hGR revealed apparent kinetic constants of 16.5 ␮M and 0.016 s⫺1 for Km and kcat, respectively. This resulted in a kcat/Km value of approximately 1,000 M⫺1 s⫺1 (Table 3). Additionally, eosin B appeared to be utilized as a substrate for PfTrxR, with a kcat of 0.108 s⫺1, a Km of 10.6 ␮M, and thus a kcat/Km of 10,200 M⫺1 s⫺1 (Table 3). These findings indicate that a metabolic modification of eosin B is occurring via the dithol enzymes.

TABLE 2. Eosin B inhibition of disulfide reductasesa

FIG. 5. Steady-state kinetics of eosin B on DHFR-TS. Steady-state time courses were carried out with 1 ␮M P. falciparum DHFR-TS, 400 ␮M CH2H4 folate, and varying concentrations of eosin B (0.075 to 0.5 mM). An IC50 of 320 ␮M was obtained by plotting the rates of the steady-state reactions versus the eosin B concentration.

Enzyme

IC50 (␮M)

Ki⬘ (␮M)

hGR PfGR PfTrxR

0.6 1.5 4.2

0.5 1.3 NDb

a The type of inhibition could not be unambiguously assessed. For the glutathione reductases, inhibition was uncompetitive with respect to GSSG and mixed type (noncompetitive or uncompetitive) with respect to NADPH. b ND, not determined.

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eosin B in that it contains phenolic and benzoic acid moieties (Fig. 4). Despite their structural similarity, neither of these compounds had any inhibitory effects on parasite growth when tested in the range of 0.153 nM to 20 ␮M or 40 nM to 40 ␮M for phenolphthalein and fluorescein, respectively. This finding indicates that eosin B has unique structural components that allow for its inhibitory properties on P. falciparum. Eosin B inhibits several enzymes, including DHFR-TS, GR, and TrxR. Since inhibition of T. gondii DHFR-TS appears to be the primary mode of action for the antitoxoplasmosis effect of eosin B (2), the effectiveness of this compound on the Plasmodium enzyme was tested. Preliminary steady-state screening demonstrated that eosin B inhibited the bifunctional P. falciparum DHFR-TS reaction in which CH2H4 folate is converted to H4 folate. Specifically, steady-state time courses for the reaction of 1 ␮M DHFR-TS with 400 ␮M CH2H4 folate in the presence of eosin B revealed an IC50 of 320 ␮M (Fig. 5). Steady-state inhibition of DHFR-TS by eosin B is not saturable, indicating that eosin B is a noncompetitive inhibitor of this enzyme. The ca. 3,000-fold difference in the IC50 for DHFR-TS enzymatic inhibition compared to the cell culture IC50 could be due to the parasite’s ability to concentrate com-

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TABLE 3. Eosin B as a substrate of disulfide reductases Enzyme

kcat (s⫺1)

Km (␮M)

kcat/Km (M⫺1 s⫺1)

hGR PfGR PfTrxR

0.016 ⬍0.003 0.108

16.5 NDa 10.6

970 ND 10,200

a

ND, not determinable.

DISCUSSION

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The primary affordable drugs available for malaria treatment in tropical countries are chloroquine, other 4-aminoquinolines, quinine, pyrimethamine, and sulfadoxine. Although these drugs remain the standards of antimalarial chemotherapy, their usefulness is threatened by the emergence and spread of multidrugresistant strains of Plasmodium falciparum (48). The work presented here demonstrates that eosin B has potent selective antimalarial activity against strains that are resistant to one or more of these standard drugs (Table 1). This lack of crossresistance with the currently utilized therapies suggests that eosin B could serve as a lead compound for development of novel antimalarial drugs. We have identified eosin B as an inhibitor of drug-resistant P. falciparum with an average IC50 of 124 nM (Table 1). This IC50 is approximately 1,500-fold less than that observed to block T. gondii replication (2). There are three possible explanations for the IC50 discrepancy between organisms. One explanation is the variability of drug access to the different parasites; P. falciparum invades mature erythrocytes, while T. gondii replicates within nucleated cells replete with full biosynthetic machinery. It is therefore reasonable to assume that relatively direct access of eosin B to the Plasmodium parasite occurs, whereas drug trapping within the various compartments of the Toxoplasma host cell slows down and reduces the amount of eosin B available for interaction with the parasites. Another concurrent explanation for the differential toxicity is that eosin B concentrates inside the malarial parasite, which, in turn, allows for higher intraparasitic concentrations of the compound. Such concentration mechanisms could occur because the compound gets protonated and trapped inside the parasite. An additional explanation for the differential toxicity of eosin B observed between T. gondii and P. falciparum is that the compound exerts alternative modes of action in the two parasites; evidence to support this point is discussed below. It is noteworthy that eosin B selectively concentrates inside iRBC versus nonparasitized erythrocytes (Fig. 1). The trace amount of eosin B within the uninfected RBC cytosol suggests that the anionic compound does not easily diffuse through the host cell membrane and, since erythrocytes cannot undergo endocytosis, instead directly accesses the parasite from the external medium through a pathway that is not present in the uninfected RBC. As such, active parasitic modification of the RBC must account for the selective uptake of eosin B into parasitized red blood cells. Two proposed pathways that could be responsible for such an occurrence are the parasitophorous duct (45) and the tubovesicular network pathway (TVN) (36). Once the drug is adjacent to the parasite’s plasma membrane, the parasite itself is believed to be responsible for the uptake of the drug, since only live organisms are capable of accumu-

lating the compound (Fig. 1). Furthermore, since the uptake process appears to be temperature dependent, it can be concluded that an active process is required for the parasites to internalize the drug. We have demonstrated that, once inside the parasite, eosin B exerts extensive cytopathic damage, with the most significant morphological changes observed in the parasitic membranes, digestive vacuole, and mitochondria (Fig. 2b and c). This morphology is distinct from that observed with either MTX or CQ treatment. Notably, CQ and MTX treatment did not result in any damage to the parasite plasma membrane or the parasitophorous vacuolar membrane (43, 61) (see also the figure in the supplemental material). Furthermore, since MTX treatment did not appear to affect the morphology of the trophozite (supplemental material) (1) while eosin B treatment caused damage at this stage, it was concluded that inhibition of DNA synthesis is not the primary mechanism of action of eosin B in P. falciparum. The almost 3,000-fold difference between the Plasmodium parasitic and DHFR-TS enzyme IC50s corroborates this hypothesis. Further insight into eosin B’s primary mechanism of action can be gathered from the membrane structure of drug-treated parasites as observed by transmission electron microscopy. The large number of ruptures between what should be very closely associated membranes suggests that eosin B is causing a disruption in membrane integrity. The enlargement of the mitochondrial organelles suggests that eosin B causes a loss of mitochondrial osmoregulatory properties and/or affects the mitochondrial respiration rate; the parent structure of xanthene dyes is known to be intrinsically responsible for exerting such effects (41). Overall, each of the morphological effects exerted by eosin B is similar to that observed with artemisinin treatment (14, 27, 38). Since artemisinin is an oxidant drug, it seems logical to think that eosin B may be operating by this mode of action as well. This could occur by either enhancing oxidative stress or restraining the parasite defense mechanisms for dealing with such stress, or both. Consistent with the idea of oxidative stress, our data demonstrate that eosin B inhibits both PfGR and PfTrxR, with IC50s of 3.3 ␮M and 8.0 ␮M, respectively (Table 2). These enzymes are parts of two major NADPH-dependent redox systems in the Plasmodium parasite—the glutathione system, comprising glutathione reductase and glutathione, and the thioredoxin system, which is based on thioredoxin reductase and thioredoxin. These systems are considered to be of great importance for parasite survival, since they are functional in the removal of reactive oxygen species (ROS) (40, 47). Antioxidant defense is an essential function for the parasite, considering that it lives in an extremely prooxidant environment due to the endogenous toxic metabolites arising not only from parasite metabolism and catabolism of hemoglobin but also from the host’s immune system (3). The importance of these systems is highlighted by the work of Krnajski and colleagues, who demonstrated that PfTrxR is essential for the survival of P. falciparum erythrocytic stages (33). Additionally, inhibition of the thioredoxin system is known to contribute to mitochondrial swelling (49, 50). Thus, eosin B inhibition of PfTrxR may partially account for the mitochondrial distortion seen in Fig. 2. Overall, we believe that eosin B inhibition of both PfGR and

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cell types (63). Another argument in favor of choosing a host cell enzyme as a target is the a priori prevention of drug resistance (32). Thus, compounds inactivating both PfGR and erythrocyte GR may be equally important for the chemotherapy of malaria. Similarly, since oxidized glutathione can be reduced by the thioredoxin system, it would be of interest to develop a pharmacological agent, such as eosin B, that inhibits both thioredoxin reductase and glutathione reductase (31). Notably, the ability of eosin B to inhibit multiple enzymatic targets is most likely specific and not due to indiscriminate binding and off-target, nonmechanism toxicities. As addressed in our earlier work (2), several studies were performed to rule out the possibility of eosin B aggregate formation and nonspecific enzymatic inhibition. Based on these studies, we believe that the nonspecific activity of eosin B is an unlikely explanation for this compound’s mechanism of action. Furthermore, our previous studies indicate that the enzymatic binding and inhibition of eosin B are not time dependent. Thus, covalent binding is not indicated. This result is supported by Waheed et al., who suggest that eosin B binding occurs mostly through electrostatic interactions (57). In this light, eosin B is a prime candidate for antimalarial drug development, since it is an effective inhibitor of multiple dithiol targets (31). There is a substantial amount of precedent for the development of dyes for utilization as antimalarials (56). The bestknown examples are the currently utilized 4- and 8-aminoquinolines, which are derivatives of methylene blue, a phenothiazine dye that was discovered in the late 19th century to have antimalarial activity. In addition, xanthenes (24, 56) and xanthones (22, 23, 29, 59, 60) have been demonstrated to have antimalarial properties. These findings suggest that there are some inherent properties of the tricyclic pharmacophore that warrant further investigation. In comparing the structures of eosin B and analogs such as fluorescein and phenolphthalein, it is apparent that eosin’s aromatic ring substitutions, which uniquely contain a free carboxyl group as well as nitro and bromine functionalities (Fig. 4), are important for the antimalarial effects of eosin B. One contributing factor for this may be the bromine atoms, which allow eosin to resist destruction by its self-generated reactive oxygen species, thereby prolonging its ability to produce singlet oxygen (11). Another important factor may be one or both of the nitro groups; the antimalarial activities of several compounds, such as 4,4⬘-dinitro-2,2⬘-stilbenedisulfonic acid and p-(aminoacyl)diphenyl thioethers, are abolished upon the removal of their nitro functional group, presumably because a part of their activity derives from the nitro-catalyzed production of superoxide (55). Studies to affirm the importance of the nitro and bromo constituents will be performed in our laboratory. In addition, eosin B offers several other unique advantages which warrant its future development: (i) selectivity (a large differential between host and parasitic cytotoxicity is observed with eosin B), (ii) target multiplicity (the concurrent specific inhibition of multiple parasitic enzymes, as well as the redox cycling properties of eosin B, most likely results in either an additive or a synergistic effect, which in turn accounts for the low IC50 observed in cell culture), (iii) lack of cross-resistance and low potential for resistance formation (eosin B circumvents cross-resistance issues with other currently utilized antimalarial drugs; furthermore, by hindering more than one en-

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PfTrxR effectively interrupts the parasite’s main mechanisms for cellular control of redox processes and contributes to the eventual arrest of parasite growth in the late-trophozite to early-schizont stage (Fig. 3). Growth inhibition is observed at this point in the parasite life cycle due to the route of entry of eosin B into the parasite. As mentioned above, the compound appears to directly access the parasite from the external medium through a pathway, such as the TVN, that evades the erythrocyte cytosol. Since the TVN is not induced until 33 h of infection (18), eosin B cannot enter the parasite until the later stages of development. Hence, the effects of eosin B are not apparent until the late stages of parasite growth. Our initial studies suggested that eosin B was an uncompetitive inhibitor when the concentration of GSSG, used as a substrate, was varied. However, when NADPH concentrations were varied, a mixed noncompetitive mode of inhibition was also observed. The ambiguity of this assessment could be due to the fact that PfGR in dilute solutions is very unstable in the presence of NADPH and loses activity even in the absence of an inhibitor (31). The complexity of eosin B inhibition kinetics is further highlighted by the fact that eosin B acts not only as an inhibitor but also as a substrate of both hGR and PfTrxR (Table 3). This finding points to a metabolic modification of eosin B and suggests that this compound is capable of undergoing redox cycling, a process by which pharmacological compounds exert their effects by producing ROS. As such, eosin B-generated ROS could be a contributing factor to the antimalarial effects found in cell culture. Since a multitude of radicals and oxidants are usually formed due to ROS, it is considered inconclusive to assess the redox cycling capabilities of eosin B via antioxidant reversal. Rather, ROS generation via redox cycling is considered likely, since eosin B has the ability to generate oxygen free radicals under other circumstances as well (11). It is noteworthy that eosin B inhibits not only PfGR and PfTrxR but hGR and hTrxR as well. Despite the importance of hGR, there is good evidence that the enzyme is not essential for normal erythrocyte function and that the reduced life span of hGR-deficient red blood cells is tolerable (17). Furthermore, there are compelling arguments suggesting that we should not neglect human erythrocyte GR as a potential antimalarial target (32, 63). These arguments include clinical and epidemiological observations as well as pharmacological studies showing that a low level of hGR activity—which is usually restricted to erythrocytes—does not significantly interfere with erythrocyte function but offers protection from malaria. The reasoning behind this protection is twofold. First, hGR is most likely also involved in maintaining the intracellular redox environment and thus supports the intraerythrocytic growth of the parasite (15); the parasite generates glutathione at least 100 times faster than the host cell, and about 10% of this glutathione is actively pumped as GSSG into the host cell compartment (18). Second, inhibition of hGR would mimic the naturally occurring glucose-6-phosphate deficiencies which are known to be selective in mitigating parasitic infection (5, 37, 51, 62). Furthermore, since erythrocytes lack the capability for de novo protein synthesis, they are uniquely sensitive to enzyme inhibitors, while cells which are able to synthesize enzymes de novo are not. Thus, a single dose of the inhibitor is expected to have a longer-lasting effect in RBC than in other

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zymatic target, resistance formation would be slowed), (iv) target importance (each of the enzymatic targets is vital for parasitic growth), and (v) non-active-site inhibition (eosin B exerts either noncompetitive or uncompetitive inhibition on all targeted enzymes; uncompetitive inhibitors are particularly toxic to the cellular compartment where they operate, and thus they are often more-promising drug candidates than other reversibly acting inhibitors) (9). Taken together, our data indicate that eosin B selectively inhibits the growth of drugresistant Plasmodium strains and therefore qualifies as a good candidate for lead optimization and future drug development. ACKNOWLEDGMENTS

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Several people aided in the improvement of the manuscript. We thank Kasturi Haldar for the generous gift of the knobless FCR3 Plasmodium strain and Isabelle Coppens for her guidance. This work was supported by an NIH grant to K.S.A. (AI 44630), a RO1 grant entitled Mechanism Based Drug Selection and Design (AI46416) to K.A.J., and a DFG grant to S.G. (Gr 2028/1-2). K.M.M. was supported by a T32 AI07404 Interdisciplinary Parasitology Training Grant and a New Initiatives in Malaria Research award (475890) from the Burroughs Wellcome Fund.

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