Journal of Inorganic Biochemistry 129 (2013) 43–51

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry

j ourna l homepage: www.e lsev ie r .com/ locate / j inorgb io

Anti-plasmodial activity of aroylhydrazone and thiosemicarbazone ironchelators: Effect on erythrocyte membrane integrity, parasitedevelopment and the intracellular labile iron pool

Asikiya Walcourt a,⁎, Joseph Kurantsin-Mills a,b,c, John Kwagyan d, Babafemi B. Adenuga e,Danuta S. Kalinowski f, David B. Lovejoy f, Darius J.R. Lane f,⁎⁎, Des R. Richardson f,⁎⁎⁎a Department of Physiology and Biophysics, Howard University College of Medicine, Washington, DC 20059, USAb Department of Medicine, The George Washington University Medical Center, Washington, DC 20037, USAc Department of Pharmacology and Physiology, The George Washington University Medical Center, Washington, DC 20037, USAd Design, Biostatistics & Population Studies, Center for Clinical & Translation Science and Department of Community and Family Medicine, Howard University College of Medicine, Washington,DC 20059, USAe Department of Medicine, Howard University College of Medicine, USAf Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia

⁎ Correspondence to: A. Walcourt, Department of PhyUniversity College of Medicine, 520 W Street NW, Wash806 9700; fax: +1 202 806 4479.⁎⁎ Correspondence to: D.J.R. Lane, Department of PBlackburn Building (D06), University of Sydney, SydnAustralia. Tel.: +61 2 9351 6144; fax: +61 2 9351 3429.⁎⁎⁎ Correspondence to: D.R. Richardson, Department ofBlackburn Building (D06), University of Sydney, SydnAustralia. Tel.: +61 2 9036 6548; fax: +61 2 9351 3429.

E-mail addresses: awalcourt@howard.edu (A. Walcou(D.J.R. Lane), d.richardson@med.usyd.edu.au (D.R. Richard

0162-0134/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.jinorgbio.2013.08.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 July 2013Received in revised form 19 August 2013Accepted 19 August 2013Available online 26 August 2013

Keywords:AroylhydrazoneThiosemicarbazoneIron chelatorPlasmodium falciparumErythrocytes

Iron chelators inhibit the growth of themalaria parasite, Plasmodium falciparum, in culture and in animal andhumanstudies. We previously reported the anti-plasmodial activity of the chelators, 2-hydroxy-1-naphthylaldehydeisonicotinoyl hydrazone (311), 2-hydroxy-1-naphthylaldehyde 4-methyl-3-thiosemicarbazone (N4mT), and2-hydroxy-1-naphthylaldehyde 4-phenyl-3-thiosemicarbazone (N4pT). In fact, these ligands showed greatergrowth inhibition of chloroquine-sensitive (3D7) and chloroquine-resistant (7G8) strains of P. falciparum in culturecompared to desferrioxamine (DFO). Thepresent study examined the effects of 311, N4mTandN4pTon erythrocytemembrane integrity and asexual parasite development. While the characteristic biconcave disk shape of the eryth-rocytes was unaffected, the chelators caused very slight hemolysis at IC50 values that inhibited parasite growth. Thechelators 311, N4mT and N4pT affected all stages of the intra-erythrocytic development cycle (IDC) of P. falciparumin culture. However, while these ligands primarily affected the ring-stage, DFO inhibited primarily trophozoite andschizont-stages. Ring, trophozoite and schizont-stages of the IDC were inhibited by significantly lower concen-trations of 311, N4mT, and N4pT (IC50 = 4.45 ± 1.70, 10.30 ± 4.40, and 3.64 ± 2.00 μM, respectively) than DFO(IC50 = 23.43 ± 3.40 μM). Complexation of 311, N4mT and N4pTwith iron reduced their anti-plasmodial activity.Estimation of the intracellular labile iron pool (LIP) in erythrocytes showed that the chelation efficacy of 311, N4mTandN4pT corresponded to their anti-plasmodial activities, suggesting that the LIPmay be a potential source of non-heme iron for parasite metabolism within the erythrocyte. This study has implications for malaria chemotherapythat specifically disrupts parasite iron utilization.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

The initiation of human malaria begins with the female Anophelesmosquito injecting sporozoites into the blood circulation during a blood

siology and Biophysics, Howardington, DC, USA. Tel.: +1 202

athology and Bosch Institute,ey, New South Wales, 2006

Pathology and Bosch Institute,ey, New South Wales, 2006

rt), darius.lane@sydney.edu.auson).

ghts reserved.

meal [1]. These sporozoites migrate to the liver, pass through Küpffercells and then actively invade hepatocytes. Each invading sporozoite dif-ferentiates and divides mitotically into thousands of liver merozoitesthat, when released, invade erythrocytes, thereby beginning the asexuallifecycle of Plasmodium falciparum [1]. The merozoites then mature asex-ually during the parasite's intra-erythrocytic development cycle (IDC)through the ring, trophozoite, and schizont-stages [2]. The completecycle spans approximately 48 h [1,2].

Maturation of the parasite to the schizont-stage entails: (i) proteolyticdegradation of the host hemoglobin; (ii) detoxification of the liberatedheme to prevent damage to both parasite and host cell; (iii) reductionof the intra-erythrocytic hemoglobin concentration and consequentlyintra-erythrocytic colloid-osmotic pressure; (iv) initiation of biochemicalreactions involving proteases that destabilize the erythrocyte cytoskele-ton; and (v) eventual host-cell lysis. These events lead to the release of

44 A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

15–32 non-motile merozoites into the circulation, which are capable ofinvading new erythrocytes to begin a new IDC [2–5]. The simultaneousappearance of merozoites every 48 h causes the fever seen in patientswith clinical malaria [6,7]. The ever increasing mortality and morbidityrate of P. falciparum malaria due to drug-resistance underscores the ur-gent need to develop effective, less expensive drugs that allow for the ex-ploration of new therapeutic strategies against this disease.

Intra-erythrocyte development and growth of P. falciparum aredependent on iron and are repressed by iron chelators, as demonstratedby the anti-malarial activity of the clinically-used ligand, desferrioxamine(DFO; Fig. 1A) [8–10]. This finding prompted research into theanti-malarial activity of the lipophilic aroylhydrazone class of ironchelators, such as pyridoxal isonicotinoyl hydrazone (PIH; Fig. 1A),2-hydroxy-1-naphthaldehyde m-fluorobenzoyl hydrazone (HNFBH)and salicylaldehyde isonicotinoyl hydrazone (SIH; Fig. 1A) [11,12].Aroylhydrazone chelators have been shown to be orally effective, eco-nomical to synthesize and markedly membrane-permeant, thus over-coming the many clinical disadvantages of DFO [13].

We previously showed that 2-hydroxy-1-naphthylaldehydeisonicotinoyl hydrazone (311; Fig. 1A), 2-hydroxy-1-naphthylaldehyde4-methyl-3-thiosemicarbazone (N4mT; Fig. 1A), and 2-hydroxy-1-naphthylaldehyde 4-phenyl-3-thiosemicarbazone (N4pT; Fig. 1A), areeffective inhibitors of the in vitro growth of chloroquine-sensitive 3D7and chloroquine-resistant 7G8 strains of P. falciparum [14]. The che-lators, 311, N4mT andN4pT, are Schiff base compounds formed betweenhydrazides or thiosemicarbazides and an aldehyde [15]. In compar-ison to the hexadentate iron chelator, DFO, the aroylhydrazone, 311,and thiosemicarbazones, N4mT and N4pT, are tridentate chelatorsthat strongly bind iron and possess high iron-chelation and anti-proliferative efficacies in vitro [13,15–18].

Fig. 1. (A) Line drawings of the chemical structures of the iron chelators studied, including DFO, 31light microscopy after a 48 h incubation with: (i) control medium; (ii) 0.5% DMSO; (iii) 311 (15 μ

The efficacy of iron chelators at inhibiting P. falciparum developmentand growth indicates the important role of iron in its life cycle [8–12,14].Indeed, iron is required for the activity of a number of plasmodial pro-teins, including the rate-limiting enzyme, ribonucleotide reductase,which catalyzes the de novo synthesis of deoxyribonucleotides that arerequired for DNA synthesis in the parasite [19,20].

Since malaria parasites are cultured in human erythrocytes, the ef-fect of anti-malarial drugs on the growth and proliferation of variousstages of the parasite during the IDC could bedue to direct effectswithinthe P. falciparum cell, and/or to indirect effects elicited by drug interac-tions within the host erythrocyte or at the erythrocyte membrane[21–23]. As invasion and survival of P. falciparum depend on the normalfunctioning of the erythrocytemembrane [22], changes in its propertiesare likely to interferewith the IDC of the parasite. Ziegler and colleagues[23,24] have shown that a number of amphiphiles that cause the forma-tion of stomatocytes (i.e., erythrocytes with an elongated area of centralpallor), but not echinocytes (i.e., spiculated erythrocytes), inhibit thegrowth of P. falciparum. In other studies, it was demonstrated thatlicochalcone A, a potent membrane-active agent, is incorporated intothe erythrocyte membrane, transforming normal discoid parasitizedand non-parasitized erythrocytes into echinocytes [24]. The erythrocyteshape changes caused by licochalcone A correlated with its apparenthalf-maximal inhibitory concentration (IC50) value in inhibiting growthof P. falciparum, although no data were reported on cellular hemolysis[24].

In the present study, we designed experiments to determine theeffects of 311, N4mT and N4pT on uninfected human erythrocyte mor-phology and membrane integrity (estimated by hemolysis) by incubat-ing erythrocytes at concentrations similar to those used in the in vitroinhibition of parasite growth. We also examined the effect of these

1, N4mT, N4pT, PIH, SIH and BIP. (B) Giemsa-stained normal human erythrocytes viewed byM); (iv) N4mT (15 μM); or (v) N4pT (15 μM). Magnification: 1000×, oil immersion.

45A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

chelators on specific stages of P. falciparum development and growthduring the IDC. The mechanism by which the chelators inhibit parasitedevelopment and growth was assessed after their complexation withiron and also by analyzing the labile iron pool (LIP) in erythrocytes.These studies indicate that the ring, trophozoite and schizont-stages ofthe parasite IDC are inhibited by 311, N4pT, andN4mT, at lower concen-trations thanDFO. The chelators did not alter red cell shape, but they didcause very slight hemolysis. Furthermore, results obtained from ironcomplexation experiments, and the use of the iron-sensing fluorescentprobe, calcein, indicate that the ligands readily permeate erythrocytesto chelate intracellular labile iron.

2. Materials and methods

2.1. Chemicals

Synthesis and characterization of PIH, 311, N4mT and N4pT wereperformed using standard techniques [15,16]. The chelator, 2,2′-bipyridine (BIP, N99% pure; Fig. 1A), was purchased from Acros Or-ganics, Morris Plains, NJ. Anti-calcein antibody, which quenches calceinfluorescence, was a gift from Professor Z.I Cabantchik, Department ofBiological Sciences, Institute of Life Sciences, Hebrew University ofJerusalem, Jerusalem, Israel. Salicylaldehyde isonicotinoyl hydrazone(SIH) was a gift from Professor Prem Ponka (Lady Davis Institute forMedical Research,Montréal, Canada). DFOwas purchased fromNovartisPharma AG, Basel, Switzerland and DMSO was from Fisher Scientific,Norcross, GA. Calcein-acetoxymethyl ester (calcein-AM) was obtainedfrom Molecular Probes, Eugene, OR. Other reagents used include:RPMI-1640 (Mediatech Cellgro, Alexandria, VA), human A+ serum(pooled from different A+ serum from non-immune donors; InterstateBlood Bank Inc., Memphis, TN), 2,8-[3H]-hypoxanthine (Amersham,Pharmacia Biotech, Little Chalfont, England), D-sorbitol and scintillationfluid (Fluosafe; Fisher Scientific, Norcross, GA).

2.2. Cell culture

The chloroquine-sensitive strain of P. falciparum (3D7) used inthese studies was provided by O. Muratova and D. Keister (NationalInstitutes of Health, Bethesda, MD). Parasites were maintained ascontinuously growing cultures in washed human erythrocytes (A+)using culture medium containing RPMI-1640 supplemented with25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),23 mM NaHCO3, 10 mM D-glucose and 10% (v/v) heat-inactivatedhuman serum (A+), in accordancewith themethods of Trager & Jensen[25]. Cultures were maintained at 3–5% parasitemia and 2% hematocritin an atmosphere containing 90%N2, 5% CO2 and 5%O2. Synchronizationto the ring-stage (≥90% rings) was achieved by using 5% D-sorbitoltreatment [26]. The gross morphological characteristics of the parasiteswere assessed by light microscopic examination of thin blood smearsstained with Giemsa. The degree of parasitemia was examined bycounting a representativefield of 1000 cells and determining the percent-age of cells parasitized. For the determination of IC50 values, parasiteswere cultured at 2% parasitemia and 2% hematocrit. When required fordrug assays, synchronized ring-stage cultures were used after suspensionin RPMI-1640 supplemented with 10% heat-inactivated human serum(A+) and buffered to pH 7.4 with 25 mM HEPES, 23 mM NaHCO3 and10 mM D-glucose.

2.3. Preparation of iron chelators

Aroylhydrazone and thiosemicarbazone chelators were dissolved inDMSO as 10 mM stock solutions [18] and diluted in culture medium, sothat the final concentration of DMSO was 0.5% (v/v). In contrast, DFOwas prepared in culture medium alone due to its high solubility. Stocksolutions were then filtered through a Swinnex Millipore filter (poresize 0.2 μm) for sterilization.We demonstrated in previous experiments

that DMSO at this low concentration had no effect on parasite growth[14]. All drug solutions were prepared fresh before an experiment andused immediately.

2.4. Effects of 311, N4mT and N4pT on erythrocyte shape

Non-parasitized human erythrocytes were incubated with 311,N4mT, or N4pT at the same molar concentrations used for P. falciparumculture following the methods of Ziegler et al. [24]. Control wellscontained non-parasitized erythrocytes without the chelators in culturemedium. Non-parasitized erythrocytes in wells containing 0.5% DMSOin culture medium served as negative controls. Briefly, various concen-trations of the test compound were added to 600 μL of non-parasitizedwashed human erythrocytes (blood group A) at 3% hematocrit in para-site growth medium (see above) in Eppendorf tubes, then 200 μL ofeach suspension were pipetted into Falcon 96-well microtiter plates.Each chelator concentrationwas tested in duplicatewells. Cells were in-cubated in a Thermo FormaModel 370 Steri-Cycle Incubator at 37 °C ina humidified atmosphere of 90% N2, 5% CO2 and 5% O2. After a 48 h in-cubation period, the plates were placed on a shaking table for 1 minand 100 μL of each sample were then transferred into Eppendorf tubesand treated with 50 μL of 3% glutaraldehyde in phosphate buffer(pH 7.4) for 1 h. The cells were washed three times with sterilephosphate-buffered saline (PBS, pH 7.4; Biofluids, Biosource Interna-tional, Inc., Camarillo, CA). A fraction (20 μL) of this sample was spreadonmicroscope slides, allowed to dry, treatedwithmethanol and stainedwith Giemsa for light-microscopic examination of erythrocyte shape[24]. Representative views of the cells under the microscope werephotographed using different light filters to generate clear images.

2.5. Assessment of potential chelator-induced erythrocyte hemolysis

Assayswere conducted according to themethod of Pradines and col-leagues [27] with modifications. Human erythrocytes were washedthree times and resuspended in RPMI-1640 medium (without phenolred) supplemented with 10% human serum and buffered to pH 7.4with 25 mM HEPES and 25 mM NaHCO3 to a hematocrit of 2–2.5%.The erythrocyte suspensions were aliquoted at 200 μL/well into Falcon96-well microtiter plates in triplicate. Final concentrations of 311,N4pT, and N4mT ranged from 0.5 to 3.0 μM. Erythrocytes incubated inRPMI-1640 medium (without phenol red) and without any of the che-lators, but containing 0.5% DMSO, served as a negative control. For com-parison, erythrocytes were also incubatedwith DFO or PIH (10–60 μM).Standard hemoglobin solutions (Fisher Scientific, Norcross, GA) weredilutedwith RPMI-1640medium (without phenol red) and used to pre-pare the standard curve for the hemolysis assay.

The optical density of the standard hemoglobin solutions and the su-pernatants of the cell suspensions were measured using an automaticplate reader (PerkinElmer Victor2™ MultiLabel Counter, PerkinElmer,Inc., Shelton, CT) at awavelength of 415 nm. These datawere expressedas a percentage of the mean corpuscular hemoglobin concentration(MCHC) obtained after complete cellular lysis. As 10% human serum(A+) was used in the standard culture media for parasite growth[12,14,25,28], we also assessed the hemoglobin concentration in theautologous plasma. Therefore, aliquots of the autologous plasma of thedonor blood were also diluted with RPMI-1640 medium (without phe-nol red) to either 10% or 50% plasma, or used without dilution (100%).The rationale for using these plasma concentrations was to spectro-photometrically compare their heme-containing proteins. This wasrequired, since 10% plasma is the concentration used in the culturemedium for parasite growth. Additionally, 50% plasma and 100%(i.e., undiluted) plasma were also included to assess the effect of an in-crease in heme-containing proteins at higher % plasma concentrations.The optical density of the diluted and undiluted plasma (100%) wasmeasured at 415 nm for hemoglobin (i.e., the absorption maximum ofthe hemoglobin Soret band). The results of the diluted and whole

46 A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

autologous plasma without any of the chelators, but containing 0.5%DMSO, served as an additional negative control. These results werecompared to the supernatant optical density of the RPMI-DMSO controland the supernatants of cell suspensions incubated with chelators.

2.6. Assessment of chelator effect on stage-specific parasite growth

To evaluate the effects of the chelators on specific stages of parasitegrowth, synchronized (≥90% rings) parasite cultures [26]were incubatedwith 311, N4mT, N4pT (at 2.5–15 μM) or DFO (10–60 μM) in duplicatewells in 24-well plates. Plates were transferred to a candle jar and incu-bated at 37 °C for 48 h [25]. Control cultures without the chelators wereincubated concurrently under the same conditions. Giemsa-stained slideswere prepared from the parasite cultures from the different chelator con-centrations at 0, 24 and 48 h after drug incubation and examined using alight microscope. The sensitivity of parasite development to the chelatorswas determined by counting the number of rings, trophozoites and schiz-onts per 1000 erythrocytes at the different time intervals. Representativeviews of the parasite IDC under themicroscopewere photographed usingdifferent light filters for clear images.

2.7. Effect of chelators and their FeIII complexes on parasite growth

To determine the role of iron chelation in the anti-plasmodial effectsobserved, we assessed the growth of parasites cultured with chelatorspre-complexedwith iron. The FeIII complexes of the chelators were pre-pared by adding FeCl3 to the ligands in a 1:1 metal:ligand equimolarratio for the hexadentate chelator (DFO), and a 2:1 metal:ligand ratiofor the tridentate chelators (311, N4mT and N4pT), using previousmethods [29]. The solutionswere thenmixed thoroughly and incubatedfor 1 h at 37 °C before being added to parasite cultures. Complexation ofthe chelators with Fe3+ ionswasmonitored spectrophotometrically be-tween 200 nm and 800 nm [30]. The effects of the chelators and theirFeIII complexes on parasite growth were examined by adding variousconcentrations to 600 μL of ring-stage synchronized parasitized (3D7)red cells (2.0% parasitemia and 2.0% hematocrit) in a 24-well plate.In all these studies, DFO served as a positive control, while negative-control wells contained only the vehicle (0.5% DMSO). After a24 h/37 °C incubation of the parasitized erythrocytes with the chelatorsor their FeIII complexes, [3H]-hypoxanthine was added to the wells(1 μCi/well) and incubated for a further 24 h (48 h culture). The parasiteswere then harvested to determine [3H]-hypoxanthine incorporation. Thehalf-maximal inhibitory concentration (IC50) values were determined byanalyzing these data implementing non-linear regression analysis oflog-dose–response curves using GraphPad Prism® Analysis software(GraphPad Prism® Software, Inc., San Diego, CA).

2.8. Intracellular iron chelation properties of 311, N4mT and N4pT

To evaluate the intracellular chelating activities of 311, N4mT, andN4pT in intact red cells, we used the iron-sensing fluorescent probe,calcein, as described by Epsztejn and colleagues [31] and Darbari andcolleagues [32]. The ligands, BIP and SIH, were used as FeII and FeII/FeIII

control chelators, respectively. Erythrocyte suspensions (2 × 109 cells/mL) were briefly incubated for 30 min/37 °C in medium containingNaCl (125 mM), HEPES (20 mM; pH 7.35), D-glucose (5 mM) andcalcein-AM (250 nM). Following this incubation, the cells were washedfour times with HEPES-saline-glucose buffer to remove extracellularcalcein-AM and the cell suspension was adjusted to 107 cells/mL. Onemilliliter of the calcein-loaded erythrocytes (107 cells/mL)was pipettedinto a spectrofluorometer cuvette, followedby the addition of an aliquotof anti-calcein antibody (1:1000 final titer). The intracellular calceinfluorescencewas then recorded on a PerkinElmer LS-50B LuminescenceSpectrometer (PerkinElmer, Inc. Shelton, CT). The addition of anti-calcein antibody to the erythrocyte suspensions quenched any extra-cellular fluorescence in the suspension (which was less than 1% of the

initial signal) and assured that all recorded fluorescence signals origi-nated from within the erythrocytes [32].

The fluorescence of calcein is quenched by the binding of iron[31,32], and in the current study, calcein fluorescence was measuredat an excitation wavelength of 488 nm and an emission wavelength of512 nm. Following a stable basal fluorescence signal (F1), aliquots of311, N4mT, N4pT, BIP or SIH (100 μM final) were added to the suspen-sion,which caused an increase in the fluorescence signal (F2). Consider-ing this, the addition of the chelator presumably led to competitivebinding of intracellular labile iron, subsequent release of calcein-bound iron, and an increase in fluorescence intensity. Therefore, thefractional increase in fluorescence (dF) reflects the labile iron concen-tration. This measurement together with the values of the dissociationconstant (Kd) and the total intracellular calcein concentration [CA]t,which were determined as described by Epsztejn and colleagues [31]and Darbari and colleagues [32], was used to calculate and comparethe chelation of labile iron in erythrocytes. The erythrocyte labile ironconcentration, [LIP], was calculated using the equation:

LIP½ � ¼ dF� CA½ �t� �þ Kd � dF 1−dFð Þ½ �

where dF represents the fractional increase in fluorescence intensityafter addition of BIP, SIH, 311, N4mT, orN4pT to the erythrocyte suspen-sions, Kd is the dissociation constant of calcein and iron within humanerythrocytes [28,32], and [CA]t is the total intracellular concentrationof calcein [31,32].

2.9. Statistical analysis

Data are expressed as mean ± standard deviation. Data were com-pared against the respective control in experiments using the Mann–Whitney test which is appropriate for small sample sizes. Results wereconsidered statistically significant at p b 0.05.

3. Results

3.1. Effects of 311, N4mT and N4pT on erythrocyte shape

Incubation of human erythrocytes in RPMI-1640 culture mediumcontaining 0.5% DMSO (solvent control), or 2.5–15 μM of 311, N4mTor N4pT had no apparent effect on erythrocyte shape, as shown byGiemsa-staining (Fig. 1B). Indeed, as determined by light microscopy,the cells used in this study demonstrated the characteristic biconcave,discoid shape of normal, healthy erythrocytes. The peripheral zone ofsuch erythrocytes is thicker (resulting in a longer optical path length)and so appears darker, whereas the central zone is thinner (due to thebiconcave shape) and consequently appears pale (Fig. 1B). The size ofthis zone of central pallor and the overall staining intensity of the eryth-rocyte is related to the content of hemoglobin. The control and thechelator-treated cells both showed the normalminor characteristic var-iations in shape and size seen in Giemsa-stained preparations (Fig. 1B)[33]. After 48 h of incubationwith the chelators (2.5–15 μM), the eryth-rocytes remained as biconcave disks (i.e., discocytes), with the sametypical features as control cells. Thus, under the present incubation con-ditions, the chelators did not affect erythrocyte morphology.

3.2. Effects of 311, N4mT and N4pT on erythrocyte membrane integrity

The hemolytic effect of 311, N4mT, N4pT, DFO, or PIH on eryth-rocytes was assessed by comparing the supernatant hemoglobinconcentrations of the cells incubated in vehicle-containing controlmedium [RPMI 1640 (without phenol red), 0.5% DMSO and 10%human plasma] to the supernatant hemoglobin concentration ofcells incubated with various chelator concentrations (Table 1). Asshown in Table 1, the supernatant hemoglobin in the controls,expressed as a percentage of MCHC, was 0.0175 ± 0.0005%. As

47A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

10% serum was used in the standard culture media for P. falciparum,and was used in the present hemolysis experiments, we also mea-sured the hemoglobin concentration in the autologous plasma todetermine the extent to which it contributed to the apparent degreeof hemolysis. The concentrations of hemoglobin (determined spectro-photometrically at 415 nm) in 10% plasma, 50% plasma, and whole(100%) autologous plasma, expressed as percentage of MCHC, were0.0158 ± 0.0013%, 0.0564 ± 0.0029% and 0.0892 ± 0.0039%, respec-tively. The level of hemoglobin measured in the 50% and 100% autolo-gous plasmas was significantly higher (p b 0.01) than the levelsdetected in the supernatant of the RPMI-1640-DMSO control and 10%plasma (Table 1).

When erythrocytes were incubated in the RPMI-1640 control mediacontaining increasing concentrations of the chelators reflecting thoseused to inhibit P. falciparum growth (see below), therewas a very slight,but significant increase in hemolysis for all concentrations tested com-pared to the control (Table 1). The supernatant hemoglobin valuesfrom cells incubated with the chelators were significantly (p b 0.05)less than the apparent hemoglobin concentration in the 50% and 100%autologous plasmas. Importantly, all cell supernatant hemoglobinvalues (except for 20 μM DFO; p N 0.05) were significantly greater(p b 0.05) than the apparent hemoglobin values of the 10% autologousplasma. Thus, the incubation of the erythrocytes with the chelatorscaused a minor, albeit significant, increase in hemolysis. It is alsoworth noting that the incubation of erythrocytes in control mediumalone was sufficient to cause a small, but significant (p b 0.05) increasein hemolysis when compared to the 10% autologous plasma.

3.3. Effect of chelators on stage-specific parasite growth

As previously reported, the chelators, 311, N4mT and N4pT, aresignificantly more effective at inhibiting the growth of chloroquine-

Table 1Comparative hemolytic effect of 311, N4mT, N4pT, DFO or PIH. Supernatant hemoglobinwasmeasured at 415 nm and expressed as a percentage of the mean corpuscular hemoglobinconcentration (MCHC). Heme-containing proteins in the autologous plasma (i.e., 10% plasma,50% plasma, and whole (100%) plasma) are shown for comparison to the supernatant he-moglobin concentrations in control and chelator-treated samples. Results are mean ± SD(3 experiments). The percent MCHC in the supernatants after incubation under the variousconditions was compared to the control using the Mann–Whitney test.

Sample Concentration (μM) % MCHC p value

Control – 0.0175 ± 0.0005 –

10% plasma – 0.0158 ± 0.0013 p b 0.0550% plasma – 0.0564 ± 0.0029 p b 0.01100% plasma – 0.0892 ± 0.0039 p b 0.01311 0.5 0.0193 ± 0.0005 p b 0.05

1 0.0195 ± 0.0010 p b 0.051.5 0.0200 ± 0.0006 p b 0.052 0.0203 ± 0.0005 p b 0.053 0.0197 ± 0.0005 p b 0.05

N4mT 0.5 0.0211 ± 0.0004 p b 0.051 0.0208 ± 0.0007 p b 0.051.5 0.0213 ± 0.0002 p b 0.052 0.0208 ± 0.0007 p b 0.053 0.0214 ± 0.0004 p b 0.05

N4pT 0.5 0.0215 ± 0.0006 p b 0.051 0.0218 ± 0.0002 p b 0.051.5 0.0222 ± 0.0004 p b 0.052 0.0227 ± 0.0006 p b 0.053 0.0225 ± 0.0006 p b 0.05

DFO 10 0.0193 ± 0.0005 p b 0.0520 0.0182 ± 0.0005 p N 0.0530 0.0186 ± 0.0008 p N 0.0545 0.0192 ± 0.0001 p b 0.0560 0.0182 ± 0.0002 p b 0.05

PIH 10 0.0195 ± 0.0004 p b 0.0515 0.0188 ± 0.0003 p b 0.0520 0.0190 ± 0.0006 p b 0.0525 0.0193 ± 0.0004 p b 0.0530 0.0192 ± 0.0008 p b 0.05

resistant (7G8) and chloroquine-sensitive (3D7) strains of P. falciparumin vitro than DFO [14]. The IC50 values for these aroylhydrazone andthiosemicarbazone chelators were significantly (p b 0.001) lower thanthose determined for DFO (Table 2). The chelators, 311 and N4pT,were the most effective among these compounds, with mean IC50values more than 4-times lower than the IC50 for DFO in the 7G8 and3D7 strains of the parasite (IC50 = 18.5 μM and 23.5 μM), respectively.Furthermore, the IC50 values of 311 and N4pT were 11- to 14-timeslower than the IC50 of their parent compound, PIH (IC50 = 50 μM), inthe 3D7 malaria strain [14].

The asexual IDC of P. falciparum involves specific molecular and bio-chemicalmechanisms that result in themanifestation of themajormor-phological stages (detectable by Giemsa stains of thin blood films)throughout the IDC represented as ring-stage, trophozoite-stage, orschizont-stage parasites. In the present study, the effects of various con-centrations of 311, N4mT, and N4pT (2.5–15 μM) and DFO (10–60 μM)on the developmental stages of the synchronized chloroquine-sensitivestrain of P. falciparum (3D7) cultures were investigated by determiningthe numbers of rings, trophozoites and schizonts at 0, 24 and 48 h of in-cubation with each of the chelators. Each chelator caused inhibition ofall developmental forms of the parasite (Table 3). However, markedlyhigher concentrations of DFOwere required to inhibit the developmentof rings to trophozoites and schizonts compared with 311, N4mT andN4pT. Indeed, relative to DFO, these novel chelators significantly(p b 0.01) inhibited the developmental progression of the parasite atits ring-stage at concentrations as low as 2.5 μM (Fig. 2A). Additionally,during a 48 h incubation, 311, N4pT and N4mT caused significantlygreater stage-specific parasite growth inhibition than DFO (Table 3).

Fig. 2B demonstrates the effect of the chelator N4pT (2 μM) onthe stage-specific growth of chloroquine-sensitive P. falciparum(3D7) cultured within human erythrocytes. Fig. 2Bi–Bii show con-trol (i.e., pre-treatment, 0 h) Giemsa-stained erythrocytes harboringthe ring (Fig. 2Bi) and schizont (Fig. 2Bii) stages of chloroquine-sensitiveP. falciparum (3D7). Notably, during the ring and schizont-stages of theIDC, the infected erythrocyte membrane surface showed the characteris-tic protruding cyto-adherent knobs or “blebs” (see arrows, Fig. 2Bi & Bii)that are indicative of increased host cell permeability [34].

Fig. 2Biii–Biv show P. falciparum (3D7)-containing erythrocytes orig-inally containing ring- and schizont-stage parasites that were incubatedwith vehicle-control medium (i.e., culture medium + 0.5% DMSO) for24 h (2Biii; ring-stage) or 48 h (2Biv; schizont-stage). As Giemsa specif-ically stains phosphate groupswithin DNA, the parasite stainsmarkedlymore intensely with Giemsa than the enucleated host cell. It should benoted that Maurer's dots or clefts, which appear as pink-stained stip-pling or dots in the erythrocyte cytoplasm or attached to the plasmamembrane, are also apparent (see arrows, Fig. 2Bv and Bvi). These aresingle-membrane organelles localized beneath the host plasma mem-brane. The parasite generates this membranous network connected toboth the parasitophorous vacuoles and the erythrocyte plasma mem-brane for nutrient uptake, immune evasion, cell signaling, merozoiteegress, phospholipid biosynthesis and possibly other biochemical path-ways [34].

Notably, 24 h after incubation with 2 μM of N4pT, all parasite stagesceased to develop (Fig. 2Bv). Also, incubation of the parasite with 2 μMof N4pT for 48 h revealed significant inhibition and no schizonts,

Table 2Growth inhibition efficacy (IC50; μM) of chelators in cultures of chloroquine-sensitive(3D7) and chloroquine-resistant (7G8) Plasmodium falciparum. Results are mean ± SD(n = 6).

Chelator 3D7 P. falciparum 7G8 P. falciparum Statistical significancea

DFO 23.43 ± 3.40 18.54 ± 4.70 N/A311 4.45 ± 1.70 4.46 ± 0.30 311 vs. DFO, p ≤ 0.001N4mT 10.30 ± 4.40 10.81 ± 2.00 N4mT vs. DFO, p ≤ 0.001N4pT 3.64 ± 2.00 2.68 ± 1.90 N4pT vs. DFO, p ≤ 0.001

a Significance for both 3D7 and 7G8 P. falciparum; N/A, not applicable.

Table 3Inhibition of P. falciparum stage development after 24 h and 48 h incubation with chelators.

24 hour culture 48 hour culture

P. falciparum IDC stage P. falciparum IDC stage

IDC Rings Trophozoites Schizonts Rings Trophozoites Schizonts

Control (%) 0 20 ± 2.0 20 ± 2.0 0 20 ± 2.0 20 ± 2.0

Chelator % inhibition % inhibition % inhibition % inhibition % inhibition % inhibition

DFO (30 μM) 80 ± 6.0 74 ± 7.0 100 ± 9.5 100 ± 9.5 100 ± 9.5 95 ± 4.8311 (7.5 μM) 99 ± 8.4 90 ± 8.6 100 ± 9.5 100 ± 9.5 100 ± 9.5 100 ± 9.5N4mT (7.5 μM) 80 ± 4.0 60 ± 5.7 80 ± 6.0 80 ± 6.0 80 ± 6.0 80 ± 6.0N4pT (7.5 μM) 100 ± 9.5 100 ± 9.5 100 ± 9.5 100 ± 9.5 100 ± 9.5 100 ± 9.5

Parasites were synchronized at the ring-stage. Giemsa-stained slides of parasite culture were examined microscopically for ring-, trophozoite-, or schizont-stage parasites after a 24 and48 h incubation at 37 °C. The results are expressed as percent of observed inhibition relative to the control cells without chelators during-stages of parasite development. One thousandcells were counted for each compound. Each data point is themean of triplicates. The range of results did not exceed 10% of themean parasitemia in the ring-stage. IDC: intra-erythrocyticdevelopmental cycle.

48 A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

compared to the vehicle-control medium, but showed putative non-viable parasites that did not develop further (Fig. 2Bvi). Confirmationof parasite non-viability was achieved by washing the parasites thatwere previously incubated in 311, N4mT, or N4pT for 24 or 48 h exper-iments several times with culture medium, and then re-culturing theseparasites according to standard methods. Re-examination of Giemsa-stained slides prepared from these cultures showed no parasite growtheven after more than 48 h (data not shown). This observation stronglysuggests that the normal molecular and biochemical mechanismsrequired for P. falciparum growth during the IDC had been irreversiblyrepressed by the chelators.

It is alsoworth noting that after a 24 h or 48 h incubationwithN4pT(2 μM) the erythrocyte morphology was altered to pre-lytic swollencells (i.e., spherocytes; Fig. 2Bv and Bvi). The spherocytemorphology re-flects the adaptation of the parasite to the host environment, whereby itproteolytically degrades the host cell hemoglobin and detoxifies theliberated heme to prevent damage to both parasite and host cell. Impor-tantly, this process progressively reduces the intraerythrocytic hemo-globin concentration, and consequently reduces the colloid-osmoticpressure within the erythrocyte that leads to the flow of water intothe cell. The net effect is that the parasite delays the onset of the criticalhemolytic volume of the host cell that triggers hemolysis [35–37].

3.4. Effect of chelators and their FeIII complexes on parasite growth

To examine the role of iron in the mechanism of parasite growth in-hibition, the iron complexes of 311, N4pT and N4mT were prepared(which are saturated with iron, and thus, cannot deplete cellular iron)and added to synchronized ring-stage 3D7 parasites. Iron-free chelatorsincubated with synchronized ring-stage parasites served as a control.Fig. 3A shows the IC50 values of the free ligands and their FeIII com-plexes. Complexation of DFO, 311, N4mT andN4pTwith FeIII significant-ly (p b 0.01 or p b 0.05) inhibited their anti-plasmodial activity in termsof parasite development and growth when compared to the ligandalone. However, the effect of FeIII complexation varied with the individ-ual chelators examined. For instance, the IC50 of DFO and N4mT in-creased by 53.6% and 47.0%, respectively, whereas the IC50 of 311 andN4pT increased by 102.1% and 191.8%, respectively. These results sug-gest that the inhibitory effect of the chelators on 3D7 parasite develop-ment and growth during the IDC is mediated, at least in part, throughiron-binding.

3.5. Estimation of labile iron by calcein dequenching studies

To further verify that the chelator-mediated effect on parasite devel-opment and growth is due to host iron-binding, we employed the iron-sensing fluorescent probe, calcein [31,32]. Erythrocyte cytosolic labileiron is thought to be in dynamic equilibrium between the FeII and FeIII

oxidation states. Therefore, in the calcein-based assay for the

determination of intracellular labile iron, SIH (an FeII/FeIII chelator)[38] served as a well characterized control. Further, the effect of SIHwas compared to 311, N4mT andN4pT that are also ligandswith affinityfor FeII/FeIII [15] whereas BIP served as a control for exclusive FeII chela-tion [39,40]. Hence, we performed parallel measurements of the eryth-rocyte labile iron concentration in cell suspensions using the FeII or FeIII

specific chelators.After a stable baseline fluorescence signal (F1) was obtained, each of

the following chelators, BIP, SIH, 311, N4mT, or N4pT (100 μM final con-centration), was added to erythrocyte suspensions. This caused an in-crease in the fluorescence signal (F2) due to the competitive bindingto intracellular labile iron and the subsequent release of iron bound tocalcein. Hence, the resulting fractional increase in fluorescence (dF)was reflective of the labile iron concentration. The total intracellularcalcein concentration [Cat] was calculated and compared upon the che-lation of labile iron in the cells by BIP, SIH, 311, N4mT, and N4pT, as de-scribed in the Materials and methods section.

Using the calcein-BIP (FeII) chelator system, the labile FeII concentra-tion estimated in three separate experiments was 1.55 ± 0.23 μM. Onthe other hand, the average labile iron concentration estimated usingcalcein and the FeII/FeIII chelators, SIH and 311, was 2.93 ± 0.44 and3.07 ± 0.46 μM, respectively. In contrast, calcein-N4mT and calcein-N4pT chelator systems gave labile FeII/FeIII estimates of 7.77 ±1.17 μM and 10.93 ± 1.56 μM, respectively. Thus, the measurementof erythrocyte labile FeIII using calcein-N4mT or calcein-N4pT chelatorsystems was 2.6- and 3.5-times greater than those of SIH and 311, re-spectively (Fig. 3B), and this difference was statistically significant(p b 0.001). The measured variables (dF, [calcein], and [LIP]) using theFeII chelator, BIP were also significantly (p b 0.001) lower than thevalues obtained with the FeII/FeIII chelators, SIH, 311, N4mT and N4pT.The lower free calcein concentration in cells incubated with BIP proba-bly reflects the balance between FeII and FeIII in calcein-loaded cells.Considering this, in its mono-protonated form, calcein chelates FeIII

with a low reduction potential, large stability constant (log β111 =33.9) and high pFeIII value [41]. However, calcein also binds FeII [31]and the dequenching of calcein fluorescence obtained after the additionof a ferrous iron-specific chelator such as BIP provides a measure of la-bile FeII levels within cells. The latter process depends on BIP's abilityto rapidly permeate the cell membrane and competitively bind FeII

with high affinity [42,43].

4. Discussion

We previously reported the inhibitory activity of aroylhydrazoneand thiosemicarbazone Fe chelators, including 311, N4mT and N4pT,against the growth of chloroquine-resistant and chloroquine-sensitiveP. falciparum during the IDC [14]. These compounds are tridentateligands that strongly bind ferric iron (FeIII) and have lower affinity forferrous iron (FeII) [13,30]. In the present study, we demonstrated the

Fig. 2. (A) The inhibitory effects of DFO, 311, N4mT and N4pT on the ring-stage of chloroquine-sensitive (3D7) P. falciparum. Parasites were evaluated by examining Giemsa-stainedmicroscope slides prepared from synchronized cultures at 24 h after incubationwith the chelators to determine the sensitivities of different stages of parasite development. One thousandcells were counted for each compound. DFO served as the standard. The results are expressed as percent inhibition relative to that determined in the control cultures without chelators atthe ring-stage of parasite development. Each data point is mean ± SD (3 experiments). **p b 0.01 comparing the respective chelator concentration with the untreated control cultures.(B) Effect of N4pT incubation on parasitized (ring- and schizont-stages) of chloroquine-sensitive P. falciparum (3D7). (i) Control ring-stage at 0 h (i.e., pre-treatment); (ii) control schizont-stageat 0 h (note: arrows in (i) and (ii) indicate the presence of protruding cyto-adherent knobs or “blebs” indicative of increased host cell permeability [34]); (iii) control ring-stage at 24 h afterincubation with vehicle-control; (iv) control schizont-stage at 48 h after incubation with vehicle-control. (v) P. falciparum-infected erythrocytes were incubated for 24 h with 2 μM N4pT;only dead parasites and Maurer's dots or clefts (see arrows) are seen at ring-stage. (vi) P. falciparum-infected erythrocytes were incubated for 48 h with 2 μMN4pT (note: arrows in (v) and(vi) indicate non-viable parasites and Maurer's dots or clefts (see arrows) are seen at the schizont-stage. Non-viable parasites did not recover after re-culturing). Magnification: ×1000, oilimmersion.

49A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

effectiveness of the chelators, 311, N4pT, and N4mT, to inhibit the ring,trophozoite and schizont-stages of P. falciparum development at 3- to4-times lower concentrations compared to DFO. Among the chelators,311 and N4pT were most effective at inhibiting the trophozoite andschizont-stages of parasite growth.

In response to chemical treatments, erythrocytes may alter theirshape [21,44]. Hence, we also investigated the effects of 311, N4mT and

N4pT on erythrocyte shape using non-parasitized erythrocytes treatedwith anti-plasmodial concentrations of the chelators. Echinocytes areinduced by agents that cause red cell membrane evagination, whereasstomatocytes are induced by chemicals that cause invagination of thecell membrane [21,44]. Some amphiphilic agents induce echinocytes orstomatocytes depending on the distribution and incorporation of the am-phiphile into the outer or the inner membrane leaflet [21,45,46]. This

Fig. 3. (A) Effect of DFO, 311, N4mT and N4pT and their iron complexes on P. falciparumgrowth in culture. The FeIII complexes of the chelators were prepared by adding Fe (asFeCl3) to the ligands in a 1:1 ligand:metal ratio for the hexadentate chelator (DFO), anda 2:1 ligand:metal ratio for the tridentate chelators (311, N4mT and N4pT), as describedin the Materials and methods section. Inhibitory effects of the chelators with and withoutFeCl3 were assessed. Parasite growth was assessed after 48 h in the presence of thecompounds by using [3H]-hypoxanthine incorporation. The results are mean ± SD(6 experiments). **p b 0.01 or *p b 0.05 in comparison to the free ligand. (B) Intracellulariron chelation properties of 311, N4mT and N4pT compared to BIP and SIH. The fractionalincrease in fluorescence intensity (dF)was used together with themeasured values of thedissociation constant (Kd) and the total intracellular calcein concentration [Cat], to calculateand compare the chelation properties of the chelators in the erythrocytes. Results arefrom typical experiments with the chelators and the numerical indices are expressed asmeans ± SD(3experiments). Calculation of: [calcein] (μM); [LIP] (μM); anddFare describedin theMaterials andmethods section. ***p b 0.001 in comparison to the [LIP]measuredwithSIH, 311 or BIP.

50 A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

leads to the expansion of one leaflet relative to the other. Furthermore,some compounds have been shown to modify the shape of erythrocytesby becoming incorporated into the erythrocyte membrane, making thecells unsuitable as P. falciparum hosts. Such chemically-altered cellswere shown to inhibit parasite growth not directly related to the drug ef-fect, but via an indirect mechanism on the host cell [23,24].

In the present studies, microscopic examination of Giemsa-stainederythrocytes incubated with the chelators, 311, N4mT, N4pT, showedcharacteristic, biconcave disks, with an area of central pallor. This sug-gests that, unlike other reported amphiphiles that cause erythrocytesto transform into stomatocytes [23] or echinocytes [24], 311, N4mT,N4pT do not cause such effects. In evaluating the effect of the chelatorson erythrocyte integrity, we demonstrated that all three chelatorscaused a very slight, yet statistically significant, increase in hemolysisof uninfected erythrocytes after correction for the apparent hemoglobinlevels in 10% autologous plasma.

As an appropriate approach to elucidate aspects of themechanismofFe chelator-mediated anti-plasmodial activity, we used the iron-sensingfluorescent probe, calcein, to determine the intra-erythrocytic iron

chelation efficacy of host labile iron by BIP (FeII), SIH, 311, N4mT, andN4pT (FeII/FeIII). With calcein and the FeII/FeIII chelators (SIH, 311,N4mT, N4pT; [13,30]), we found significant differences in the erythro-cyte total cytosolic labile iron concentrations. As shown in Fig. 3B, thechelation efficacy is as follows: N4pT N N4mT N 311 ≥ SIH N BIP. Thegreater efficacy of N4pT and N4mT relative to 311 and SIH is probablyrelated to their higher lipophilicity [15]. Additionally, it is well knownthat aroylhydrazone chelators have a low affinity for FeII [13,15,30]and that strong FeIII chelators such as PIH can “push” the redox equilib-rium between FeII and FeIII to the right in solutions containing oxygen[47]. Therefore, the estimates of cytosolic labile iron concentrationusing calcein in the presence of N4mT, N4pT, 311, and SIH can beregarded as a composite measure of both FeII and FeIII pools, dependingon the FeII ⇌ FeIII redox equilibrium in the cell.

The lower free calcein concentration observed in the cells treatedwith BIP (an FeII chelator) probably reflects the shift of the FeII andFeIII redox balance in calcein-loaded cells. Calcein chelates both FeIII

and FeII [31,41] and when present in intracellular compartments, itwill bind labile iron that is complexed to other endogenous chelatorswith relatively low affinities for iron ions, (e.g., citrate) [48] and/or puta-tive iron-transporting chaperones (e.g., poly-r(C)-binding proteins 1–4)[49]. Hence, the level of fluorescence of free calcein indicates the varia-tions in the availability of iron complexed to these low affinity ligands.The affinity of calcein for FeIII is evident from the large stability constant(log β111) and the large pFeIII values reported by Thomas et al. [41]. Thedequenching obtained after addition of BIP provides a measure of thesize of the cytosolic labile FeII pool. The higher concentrations of calceinestimated in cells treated with N4mT and N4pT probably reflect themarked ability of these ligands to rapidly permeate theplasmamembraneand mobilize intracellular iron, which has been previously shown usingSK-N-MC neuroepithelioma cells in culture [15].

Erythrocytes contain up to 20 mMof ferrous iron (FeII) coordinatelybound to the protoporphyrin moiety of heme, but the concentration of“free” cytosolic ferrous (FeII) iron in the erythrocytes is reportedlyvery low [28,32]. This labile iron may constitute metabolically activeand catalytically reactive iron and is thought to be complexed to endog-enous ligands [50,51]. Non-heme iron is essential for the asexual growthof the P. falciparum inmature erythrocytes. During intra-erythrocytic in-fection, P. falciparum generates large millimolar levels of free hemewithin its acidic food vacuole via hemoglobin digestion and limited denovo biosynthesis [52]. Although it has been proposed that some por-tion of heme iron may be liberated from hemoglobin and mobilizedby the parasites for their utilization [52–54], there is no direct evidencefor mobilization of host iron from heme for parasite metabolism. Nota-bly, Goldberg and colleagues recently demonstrated that the parasitelacks the ubiquitous heme oxygenase (HO) pathway that enzymaticallydegrades someheme to biliverdin, or its downstreammetabolite, biliru-bin [55]. Thus, the parasite presumably must rely on an alternativemechanism for heme detoxification and iron acquisition during theIDC. Loyevsky and colleagues [28] demonstrated that labile iron poolsare present within normal and P. falciparum-infected erythrocytes andthat such iron pools are also a potential source of iron for the parasite.More recent studies have also indicated the existence of a chelatablepool of iron in normal erythrocytes that can be bound by lipophilicthiosemicarbazone ligands [56]. Our results demonstrate that the ironchelation efficacies of 311, N4mT and N4pT correspond to their anti-plasmodial activities, suggesting that the intracellular LIP may be a po-tential source of non-heme iron for the parasite within the matureerythrocyte.

Our data also suggest that the inhibition of the intra-erythrocytic de-velopment of the P. falciparum is probably via chelation of iron in theparasite and/or the host cell. Complexation of iron by chelators hasbeen used in several different experimental models to elucidate cellularmechanisms of action. To examine the effect of the complexation on theanti-plasmodial activity of each chelator, the chelators and their FeIII

complexes were added at various concentrations to P. falciparum

51A. Walcourt et al. / Journal of Inorganic Biochemistry 129 (2013) 43–51

cultures. Complexation of 311, N4mT and N4pT with FeIII decreasedtheir anti-plasmodial activity, strongly suggesting that Fe chelation isnecessary for their efficacy.

In summary, we have demonstrated that 311, N4mT, and N4pT havesignificantly greater anti-plasmodial activities compared to DFO.We also showed that the ring, trophozoite and schizont-stages ofP. falciparum are more vulnerable to 311, N4pT, and N4mT comparedto DFO. Iron complexation experiments revealed that the inhibitory ef-fect of the chelators on parasite growth is mediated, in part, throughiron-binding and iron-deprivation. Direct estimation of the labile ironconcentration shows that the chelation efficacies of 311, N4mT andN4pT correspond to their anti-plasmodial activities. These results sug-gest that the labile iron pool may be a potential source of non-hemeiron for parasite metabolism within the mature erythrocyte. Further-more, the promising in vitro data presented in this study should nowbe followed up by using in vivo models of P. falciparum malaria to con-firm the therapeutic efficacy of the novel thiosemicarbazone chelators,N4pT and N4mT.

Acknowledgments

This projectwas supported in part by theNational Institute of Gener-al Medical Sciences (NIGMS) research grant No. 5 SO6 GM008016-39and by the National Institute on Minority Health and Health Disparities(NIMHD) of the National Institutes of Health (NIH) under Award No.G12MD007597 to A. Walcourt; NIH research Grant No. UL1RR031975to J. Kwagyan; NIH research Grant No. UH1HL03679, funded by theNational Heart, Lung, and Blood Institute to J. Kurantsin-Mills, andHoward University General Clinical Research Center Grant No.2MO1RR10284 from the NIH; and a National Health and MedicalResearch Council (NHMRC) Senior Principal Research Fellowship(#571123) and Project Grants (#1021607 and #1021601) to D.R. Rich-ardson. The Cancer Institute New South Wales is thanked for the EarlyCareer Fellowships to D.J.R.L, D.B.L., D.S.K. and Y.S.R. Additionally,D.J.R.L and D.S.K thank the NHMRC for an Early Career Postdoctoral Fel-lowship [1013810] and Project Grant Support [1048972], respectively.

References

[1] A.S.I. Aly, A.M. Vaughan, S.H.I. Kappe, Annu. Rev. Microbiol. 63 (2009) 195–221.[2] R.W. Snow, C.A. Guerra, A.M. Noor, H.Y. Myint, S.I. Hay, Nature 433 (2005) 214–217.[3] A.F. Cowan, B.S. Crabb, Cell 124 (2006) 755–766.[4] T. Bousema, C. Drakeley, Clin. Microbiol. Rev. 24 (2011) 377–410.[5] P.R. Gilson, B.S. Crabb, Int. J. Parasitol. 39 (2009) 91–96.[6] T. Smith, B. Genton, K. Baea, N. Gibson, J. Taime, A. Narara, F. Al-Yaman, H.P. Beck, J.

Hii, M.P. Alpers, Parasitology 109 (1994) 539–549.[7] C.J. Uneke, Parasitol. Res. 101 (2007) 835–842.[8] G. Fritsch, J. Treumer, D.T. Spira, A. Jung, Exp. Parasitol. 60 (1985) 171–174.[9] G.F.Mabeza,M. Loyevsky, V.R. Gordeuk, G.Weiss, Pharmacol. Ther. 81 (1999) 53–75.

[10] A. Traore, P. Carnevale, L. Kaptue-Noche, J. M'Bede, M. Desfontaine, J. Elion, D. Labie,R.L. Nagel, Am. J. Hematol. 37 (1991) 206–208.

[11] M. Loyevsky, V.R. Gordeuk, in: P.J. Rosenthal (Ed.), Antimalarial Chemotherapy:Mechanisms of Action, Resistance, and New Directions in Drug Discovery, HumanaPress, Totowa, NJ, 2001, pp. 307–324.

[12] A. Tsafack, M. Loyevsky, P. Ponka, Z.I. Cabantchik, J. Lab. Clin. Med. 127 (1996)574–582.

[13] D.S. Kalinowski, D.R. Richardson, Pharmacol. Rev. 57 (2005) 547–583.

[14] A. Walcourt, M. Loyevsky, D.B. Lovejoy, V.R. Gordeuk, D.R. Richardson, Int. J.Biochem. Cell Biol. 36 (2004) 401–407.

[15] D.B. Lovejoy, D.R. Richardson, Blood 100 (2002) 666–676.[16] E.M. Becker, D.B. Lovejoy, J.M. Greer, R. Watts, D.R. Richardson, Br. J. Pharmacol. 138

(2003) 819–830.[17] D.R. Richardson, P. Ponka, J. Lab. Clin. Med. 132 (1998) 351–352.[18] D.R. Richardson, E.H. Tran, P. Ponka, Blood 86 (1995) 4295–4306.[19] A. Jordan, P. Reichard, Annu. Rev. Biochem. 67 (1998) 71–98.[20] H. Rubin, J.S. Salem, L.S. Li, F.D. Yang, S. Mama, Z.M. Wang, A. Fisher, C.S. Hamann,

B.S. Cooperman, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 9280–9284.[21] M.P. Sheetz, S.J. Singer, J. Cell Biol. 70 (1976) 247–251.[22] S.A. Desai, S.M. Bezrukov, J. Zimmerberg, Nature 406 (2000) 1001–1005.[23] H.L. Ziegler, D. Staerk, J. Christensen, L. Hviid, H. Hagerstrand, J.W. Jaroszewski,

Antimicrob. Agents Chemother. 46 (2002) 1441–1446.[24] H.L. Ziegler, H.S. Hansen, D. Staerk, S.B. Christensen, H. Hagerstrand, J.W.

Jaroszewski, Antimicrob. Agents Chemother. 48 (2004) 4067–4071.[25] W. Trager, J.B. Jensen, Science 193 (1976) 673–675.[26] C. Lambros, J.P. Vanderberg, J. Parasitol. 65 (1979) 418–420.[27] B. Pradines, F. Ramiandrasoa, J.M. Rolain, C. Rogier, J. Mosnier, W. Daries, T. Fusai, G.

Kunesch, J. Le Bras, D. Parzy, Antimicrob. Agents Chemother. 46 (2002) 225–228.[28] M. Loyevsky, C. John, B. Dickens, V. Hu, J.H. Miller, V.R. Gordeuk, Mol. Biochem.

Parasitol. 101 (1999) 43–59.[29] T.B. Chaston, D.B. Lovejoy, R.N. Watts, D.R. Richardson, Clin. Cancer Res. 9 (2003)

402–414.[30] D.R. Richardson, Antimicrob. Agents Chemother. 41 (1997) 2061–2063.[31] S. Epsztejn, O. Kakhlon, H. Glickstein, W. Breuer, I. Cabantchik, Anal. Biochem. 248

(1997) 31–40.[32] D. Darbari, M. Loyevsky, V. Gordeuk, J.A. Kark, O. Castro, S. Rana, V. Apprey, J.

Kurantsin-Mills, Blood 102 (2003) 357–364.[33] M. Bessis, in: M. Bessis, R.I. Weed, P.F. Leblond (Eds.), Red Cell Shape: Physiology, Pa-

thology, and Ultrastructure, Springer-Verlag, Heidelberg, Germany, 1973, pp. 1–24.[34] H. Wickert, G. Krohne, Trends Parasitol. 23 (2007) 502–509.[35] J.M.A. Mauritz, A. Esposito, H. Ginsburg, C.F. Kaminski, T. Tiffert, V.L. Lew, PLoS Comput.

Biol. 5 (4) (2009) e1000339, http://dx.doi.org/10.1371/journal.pcbi.1000339.[36] I. Safeukui, P.A. Buffet, S. Perrot, A. Sauvanet, B. Aussilhou, S. Dokmak, A. Couvelard,

D.C. Hatem, N. Mohandas, P.H. David, O. Mercereau-Puijalon, G. Milon, PLoS One 8(3) (2013) e60150, http://dx.doi.org/10.1371/journal.pone.0060150.

[37] S.B. Anyona, S.L. Schrier, C.W. Gichuki, J.N. Waitumbi, Malar. J. 5 (64) (2006),http://dx.doi.org/10.1186/1475-2875-5-64.

[38] L.M. Wis Vitolo, G.T. Hefter, B.W. Clare, J. Webb, Inorg. Chim. Acta 170 (2) (1990)171–176.

[39] D.S. Auld, Methods Enzymol. 158 (1988) 110–114.[40] Z.D. Liu, R.C. Hider, Coord. Chem. Rev. 232 (2002) 151–171.[41] F. Thomas, G. Serratrice, C. Béguin, E. Saint Aman, J. Louis Pierre, M. Fontecave, J.

Pierre Laulhère, J. Biol. Chem. 274 (1999) 13375–13383.[42] W. Breuer, S. Epsztejn, Z.I. Cabantchik, J. Biol. Chem. 270 (1995) 24209–24215.[43] A. Staubli, U.A. Boelsterli, Am. J. Physiol. Gastrointest. Liver Physiol. 274 (1998)

G1031–G1037.[44] A. Iglic, J. Biomech. 30 (1997) 35–40.[45] T. Fujii, T. Sato, A. Tamura, M. Wakatsuki, Y. Kanaho, Biochem. Pharmacol. 28 (1979)

613–620.[46] B. Isomaa, H. Hagerstrand, G. Paatero, Biochim. Biophys. Acta 899 (1987) 93–103.[47] M. Hermes-Lima, N.C. Santos, J. Yan, M. Andrews, H.M. Schulman, P. Ponka, Biochim.

Biophys. Acta 1426 (1999) 475–482.[48] A. Jacobs, Blood 50 (1977) 433–439.[49] S. Leidgens, K.Z. Bullough, H. Shi, F. Li, M. Shakoury-Elizeh, T. Yabe, P. Subramanian,

E. Hsu, N. Natarajan, A. Nandal, T.L. Stemmler, C.C. Philpott, J. Biol. Chem. 288 (2013)17791–17802.

[50] R.R. Crichton, Inorganic Biochemistry of IronMetabolism: FromMolecularMechanismsto Clinical Consequences, 2nd edition Ellis Horwood, New York, 2001.

[51] F. Petrat, S. Paluch, E. Dogruoz, P. Dorfler, M. Kirsch, H.G. Korth, R. Sustmann, H. deGroot, J. Biol. Chem. 278 (2003) 46403–46413.

[52] S.E. Francis, D.J. Sullivan Jr., D.E. Goldberg, Annu. Rev. Microbiol. 51 (1997) 97–123.[53] T. Gabay, H. Ginsburg, Exp. Parasitol. 77 (1993) 261–272.[54] T.J. Egan, J.M. Combrinck, J. Egan, G.R. Hearne, H.M. Marques, S. Ntenteni, B.T. Sewell,

P.J. Smith, D. Taylor, D.A. van Schalkwyk, J.C. Walden, Biochem. J. 365 (2002)343–347.

[55] P.A. Sigala, J.R. Crowley, S. Hsieh, J.P. Henderson, D.E. Goldberg, J. Biol. Chem. 287(2012) 37793–37807.

[56] P. Quach, E. Gutierrez, M. Talal Basha, D.S. Kalinowski, P.C. Sharpe, D.B. Lovejoy, P.V.Bernhardt, P.J. Jansson, D.R. Richardson, Mol. Pharmacol. 82 (2012) 105–114.