Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur Campus, West Bengal, India
Dithiocarbamates have emerged as potent carbonic anhydrase (CA) inhibitors in recent years. Given that CAs are importantplayers in cellular metabolism, the objective of this work was to exploit the CA-inhibitory property of dithiocarbamates as a che-motherapeutic weapon against the Leishmania parasite. We report here strong antileishmanial activity of three hitherto unex-plored metal dithiocarbamates, maneb, zineb, and propineb. They inhibited CA activity in Leishmania major promastigotes atsubmicromolar concentrations and resulted in a dose-dependent inhibition of parasite growth. Treatment with maneb, zineb,and propineb caused morphological deformities of the parasite and Leishmania cell death with 50% lethal dose (LD50) values of0.56 �M, 0.61 �M, and 0.27 �M, respectively. These compounds were even more effective against parasites growing in acidicmedium, in which their LD50 values were severalfold lower. Intracellular acidosis leading to apoptotic and necrotic death of L.major promastigotes was found to be the basis of their leishmanicidal activity. Maneb, zineb, and propineb also efficiently re-duced the intracellular parasite burden, suggesting that amastigote forms of the parasite are also susceptible to these metal di-thiocarbamates. Interestingly, mammalian cells were unaffected by these compounds even at concentrations which are several-fold higher than their antileishmanial LD50s). Our data thus establish maneb, zineb, and propineb as a new class ofantileishmanial compounds having broad therapeutic indices.
Leishmaniasis is a vector-borne disease caused by the protozoanparasite of the genus Leishmania. The disease is manifested in
various clinical forms, ranging from self-healing skin ulcers tofatal infection of the visceral organs. With an estimated 1.3 millionnew cases and more than 20,000 deaths every year, leishmaniasiscontinues to be a threat to a huge population living in tropical andsubtropical countries (1).
To date, there is no antileishmanial vaccine for clinical use (2).Treatment options are also few. After several decades of successfuluse against visceral leishmaniasis, the pentavalent antimonialshave become almost obsolete because of resistance developedagainst these drugs (3). This has led to the emergence of a secondline of defense, including amphotericin B, paromomycin, andmiltefosine (4). However, severe side effects, cases of disease re-lapse after an initial cure, and increasing signs of resistance havelimited their efficacy (5–7). The liposomal formulation of ampho-tericin B (AmBisome) is by far the most effective treatment forleishmaniasis, having minimal side effects (8). Despite theeffectiveness of AmBisome, its cost is a major point of concern,especially since this disease is prevalent in poorer sections of com-munities. Novel therapies against all forms of leishmaniasis aretherefore urgently needed.
Carbonic anhydrases (CAs) are a family of metalloenzymesthat catalyze reversible hydration of CO2. By catalyzing this simplereaction, they play vital roles in a wide variety of physiologicalprocesses ranging from ion transport and intracellular pH main-tenance to the metabolic pathways of fatty acid biosynthesis, glu-coneogenesis, and ureagenesis (9). Although mammalian CAshave been extensively studied, much less is known about the func-tion of CAs in microorganisms. There are several recent studiesconfirming the presence of CAs in bacteria (e.g., Mycobacteriumtuberculosis and Helicobacter pylori), fungi (e.g., Cryptococcus neo-formans and Saccharomyces cerevisiae), and protozoa (e.g., Plas-modium falciparum and Trypanosoma cruzi) (10–15). Since mostof these microbes are dreaded pathogens, the idea of using CA
inhibitors to restrict their growth has gained clinical significance(16). Indeed, CA inhibitors were shown to inhibit in vitro growthof bacterial pathogens like H. pylori and Brucella suis (11, 17).These promising results suggested that CAs can be exploited asantibacterial drug targets to circumvent the problem of resistanceagainst classical antibiotics (18).
Analysis of the Leishmania major genome sequence (as well asthe genomes of other species of Leishmania) predicted the pres-ence of two putative CAs (referred to here as LmCA1 and LmCA2,or LmCAs). Our results validated the constitutive expression ofthese two CA transcripts in L. major promastigotes. We also de-tected considerable CA activity in Leishmania cell lysates, therebyconfirming the presence of functional CA in L. major. One of theseLmCAs is a �-CA, which has recently been cloned from Leishma-nia chagasi and was shown to be inhibited by sulfonamides andthiol CA inhibitors. In fact, some of the heterocyclic thiols alsoinhibited in vitro growth of L. chagasi and Leishmania amazonensispromastigotes, albeit at a high concentration (MIC of �256 �M)(19). These findings suggested that LmCAs may be exploited asantileishmanial drug targets.
Received 26 December 2014 Returned for modification 16 January 2015Accepted 22 January 2015
Accepted manuscript posted online 26 January 2015
Citation Pal DS, Mondal DK, Datta R. 2015. Identification of metaldithiocarbamates as a novel class of antileishmanial agents.Antimicrob Agents Chemother 59:2144 –2152. doi:10.1128/AAC.05146-14.
Dithiocarbamates and their metal complexes have long beenused as agricultural fungicides (20). However, their molecular tar-gets remained elusive until recently. The latest reports have estab-lished dithiocarbamates as a general class of CA inhibitors. Theyform coordinate with the active-site zinc ion of CA and inhibit theenzyme at submicromolar concentrations (21, 22). Dithiocar-bamates were shown to inhibit CAs from a number of pathogenicmicroorganisms such as M. tuberculosis, Legionella pneumophila,and C. neoformans (23–25). Although dithiocarbamates inhibitboth �- and �-CAs, they were found to be better inhibitors for�-CAs than other well-known CA inhibitors, such as sulfon-amides and thiols (19, 25). These in vitro CA inhibition studiesencouraged us to explore the possibility of exploiting dithiocar-bamates as a chemotherapeutic weapon against Leishmania para-sites.
Three metal dithiocarbamate complexes, maneb, zineb, andpropineb, were selected for this study after confirmation that theyare efficient inhibitors of CA activity in Leishmania cells. In thisreport, we provide the first evidence of the antileishmanial activityof these metal dithiocarbamates. The ability of these compoundsto target Leishmania promastigotes and amastigotes along withtheir broad therapeutic indices makes them promising candidatesfor drug development against leishmaniasis.
MATERIALS AND METHODSUnless otherwise mentioned, all reagents, including the metal dithiocar-bamates, were purchased from Sigma-Aldrich (St. Louis, MO).
Parasite and mammalian cell culture. Promastigotes of L. major(strain 5ASKH, kindly provided by Subrata Adak of IICB, Kolkata, India)were grown at 26°C in M199 medium (Gibco) supplemented with 15%fetal bovine serum (Gibco), 23.5 mM HEPES, 0.2 mM adenine, 150 �g/mlfolic acid, 10 �g/ml hemin, 120 U/ml penicillin, 120 �g/ml streptomycin,and 60 �g/ml gentamicin. Unless otherwise mentioned, the pH of themedium was adjusted to 7.2. J774A.1 (murine macrophage cell line fromthe National Centre for Cell Science, Pune, India) and NIH 3T3 (murinefibroblast cell line from American Type Culture Collection) cells weregrown in Dulbecco’s modified Eagle’s medium (Gibco) supplementedwith 2 mM L-glutamine, 100 U/ml penicillin, 100 �g/ml streptomycin,and 10% heat-inactivated fetal bovine albumin (Gibco) at 37°C in a hu-midified atmosphere containing 5% CO2.
RNA isolation and RT-PCR. Total RNA was isolated from L. majorpromastigotes using TRIzol reagent (Invitrogen) followed by DNase I(Invitrogen) digestion to remove DNA contaminants. cDNA was synthe-sized from 2 �g of total RNA using an oligo(dT) primer and Moloneymurine leukemia virus (MMLV) reverse transcriptase (RT) (Epicentre).The CA transcripts of L. major were amplified using gene-specific primers:LmCA1F, 5=-GCGCGAATTCATGTCGCTGTGCAGCTG-3=; LmCA1R,5=-GCGCGAATTCCTACAGCTGCCCGTAGC-3=; LmCA2F, 5=-GCGCGAATTCATGAAGACACTTCCTTTCTGTGCCAC-3=; and LmCA2R,5=-GCGCGAATTCTTACCGCACAGCCACGGTAC-3=.
CA activity assay. L. major promastigotes (4 � 107 cells) were resus-pended in 200 �l of lysis buffer (25 mM Tris-sulfate [pH 8.2], 150 mMNaH2PO4, and 1 mM phenylmethylsulfonyl fluoride) and lysed by soni-cation. As described previously, 50 �l of cell lysate was used for measuringCA activity by Maren’s endpoint titration method (26). The average CAactivity from 3 different promastigote cultures was expressed in enzymeunits (EU)/mg, where 1 unit of enzymatic activity is defined as (T0 � T)/Tand T and T0 are, respectively, catalyzed and uncatalyzed times (seconds)required for the drop in pH from 8.5 to 7.0. The total protein concentra-tion in the cell lysate was measured by the method of Lowry et al. (27). Forinhibition studies, the inhibitors (at desired concentrations) were incu-bated with the L. major cell lysate for 15 min at 4°C prior to the assay. The50% enzyme activity inhibitory concentrations (IC50s) were calculated
using OriginPro 8 software. For CA activity assay and inhibition studies inmammalian cells (J774A.1 and NIH 3T3), a similar assay protocol wasfollowed.
Treatment of cells with maneb, zineb, and propineb. The metal di-thiocarbamate complexes maneb (manganese ethylene-bis-dithiocar-bamate), zineb (zinc ethylene-bis-dithiocarbamate), and propineb (zincpropylene-bis-dithiocarbamate) (structures in Fig. 1) were freshly dis-solved in dimethyl sulfoxide (DMSO) to prepare 5 mM stock solutions.According to the experimental requirements, further dilutions were madein DMSO before addition to the culture medium. L. major promastigotesand mammalian cells (J774A.1 or NIH 3T3 cells) were grown in mediumcontaining the metal dithiocarbamates at desired concentrations for 96 hand 72 h, respectively, following which the cells were analyzed. Cells in-cubated with an equivalent concentration of DMSO (0.2%) always actedas untreated controls.
Analysis of cell growth and viability. Following addition of metaldithiocarbamates, the parasite growth was monitored every 24 h by mi-croscopically counting the cells using a hemocytometer. Cell counting wasdone up to 96 h when the control cells reached the late-log phase ofgrowth. The viabilities of Leishmania and mammalian cells were quanti-fied by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] (Invitrogen) assay as described by Gantt et al. with minor mod-ifications (28). Briefly, equal numbers of cells from control and dithiocar-bamate-treated samples were incubated with 0.5 mg/ml MTT for 3 h toallow formation of purple formazan. Then the cells were washed withphosphate-buffered saline (PBS) and harvested by centrifugation, and thecell pellet was dissolved in 100 �l of 0.04 N HCl in isopropanol. Theoptical density (OD) of the solution was measured on a microplate reader(SpectraMax M2e; Molecular Devices) at a wavelength of 595 nm. Sincethe OD of formazan produced by the action of mitochondrial dehydro-genases of metabolically active cells correlates with the number of viablecells, the percentage of viability upon drug treatment was calculated usingthe formula (ODtreated/ODcontrol � 100). The 50% lethal doses (LD50s) forthe parasite populations were calculated using OriginPro 8 software.
Morphological analysis of Leishmania. Untreated and drug-treatedL. major promastigotes were washed in PBS, fixed with 2.5% glutaralde-hyde, postfixed with 1% OsO4, and then dehydrated with increasing con-centrations of ethanol (30 to 100%). After dehydration, the cells wereplaced on silicon wafers for overnight desiccation, coated with gold-pal-ladium, and finally analyzed in a Zeiss Supra 55VP scanning electronmicroscope (SEM) with accelerating voltage of 5 kV. The lengths of thecontrol and dithiocarbamate-treated L. major cells were measured usingMacBiophotonics ImageJ software. At least 40 cells were analyzed for eachexperimental condition.
Intracellular pH measurement. The intracellular pH (pHi) of L. ma-jor promastigotes was measured using the fluorescent probe, BCECF-AM[2=,7=-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, acetoxymethylester] (Invitrogen) as described earlier (29). Briefly, L. major promasti-gotes (1 � 107 cells) were washed in PBS and resuspended in 500 �l ofbuffer A (136 mM NaCl, 2.68 mM KCl, 0.8 mM MgSO4, 11.1 mM glucose,1.47 mM KH2PO4, 8.46 mM Na2HPO4, 1 mM CaCl2, 20 mM HEPES [pH7.0]). The cell suspension was incubated with 10 �M BCECF-AM for 30min at 26°C following which the cells were washed twice in buffer A andfinally resuspended in 100 �l of the same buffer. The ratio of fluorescenceintensities of the sample excited at 490 nm (proton-sensitive wavelength)
FIG 1 Chemical structures of the metal dithiocarbamate complexes: maneb,zineb, and propineb.
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to that excited at 440 nm (isosbestic point) was recorded in a microplatereader (SpectraMax M2e). The emission wavelength was 535 nm for bothcases. The fluorescence ratios were converted into respective pH valueswith the help of an ionophore-based calibration technique (30). For gen-eration of the calibration curve, the BCECF fluorescence intensity ratiowas recorded as a function of pH by incubating the BCECF-loaded para-sites in potassium phosphate buffer of various pH values (pH 5 to 8)containing 5 �g/ml of the ionophore nigericin (Invitrogen).
Annexin V and propidium iodide staining. Double staining of L.major promastigotes with annexin V and propidium iodide (PI) was per-formed using an Alexa Fluor 488 annexin V/dead cell apoptosis kit (Mo-lecular Probes). Briefly, untreated and drug-treated L. major cells (2 �106) were harvested, washed twice in ice-cold PBS and resuspended in 200�l of binding buffer containing 5 �l of Alexa Fluor 488-annexin V conju-gate. The suspension was incubated at room temperature for 30 min in thedark, following which the cells were washed twice with the binding bufferto remove unbound dyes. The cells were then resuspended in PBS con-taining 1 �g/ml PI and incubated for 10 min in the dark. Finally, the cellswere washed in PBS and mounted on poly L-lysine-coated slides, whichwere then embedded in antiquencher solution (1,4-diazabicyclo[2.2.2]octane [DABCO]) containing 4=,6-diamidino-2-phenylindole(DAPI). The fluorescent images were acquired with an Olympus IX81epifluorescence microscope. The percentages of annexin V- and PI-stained cells were quantified with respect to the total number of DAPI-positive cells in a field. All experiments were done in triplicate, and foreach condition, at least 200 cells were analyzed.
Quantitation of intracellular parasite burden. J774A.1 macrophageswere activated with 100 ng/ml lipopolysaccharide for 6 h. L. major pro-mastigotes were added to the activated macrophages at a ratio of 30:1(parasite/macrophage), and the cells were kept for another 6 h. The non-phagocytosed parasites were washed with PBS, and the infected macro-phages were incubated for 18 h in the absence or presence of differentconcentrations of dithiocarbamates. The cells were then fixed with ace-tone-methanol (1:1) and embedded in antiquencher solution (DABCO)containing DAPI, and the parasite load (number of amastigotes per 100macrophages) was quantified by imaging the DAPI-stained cells in anOlympus IX81 epifluorescence microscope. All experiments were done intriplicate, and for each condition, at least 100 macrophages (and the cor-responding number of amastigotes) were analyzed to quantitate the par-asite load.
Statistical analysis. All statistical analyses were done by Student’spaired t test, and the results are expressed as the means � standard devi-ations (SD). P values of 0.05 were considered statistically significant.
RESULTSL. major promastigotes express functional CA that can be inhib-ited by metal dithiocarbamates. The analysis of the genome da-tabase of L. major, the causative agent of cutaneous leishmaniasis,suggested that it encodes two putative CAs (LmCA1 and LmCA2).RT-PCR using gene-specific primers showed constitutive expres-sion of both transcripts in L. major promastigotes (Fig. 2A). TheCA activity in the Leishmania whole-cell lysate was found to be 1.1EU/mg, confirming the presence of at least one functional CA inLeishmania. This activity was inhibited by all 3 metal dithiocar-bamates (i.e., maneb, zineb, and propineb) in a dose-dependentmanner with IC50s of 0.32 �M, 0.28 �M, and 0.14 �M, respec-tively (Fig. 2B).
Stunted growth and abnormal morphology of the metal di-thiocarbamate-treated parasites. To study the effects of thesemetal dithiocarbamates on L. major promastigotes, the parasiteswere grown in the absence or presence of increasing concentra-tions of maneb, zineb, or propineb, and the total numbers of cellswere counted after 96 h of growth. Data presented in Fig. 3Aclearly show that submicromolar concentrations of all three metal
dithiocarbamates inhibited the parasite growth to a significantextent. The treated L. major promastigotes exhibited roundedmorphology with membrane blebs as revealed by SEM images(Fig. 3B). The average lengths of the maneb-, zineb-, andpropineb-treated parasites were 6.3 �m, 5 �m, and 6.8 �m, re-spectively, compared to 11.7 �m for the untreated cells, indicatingthat the metal dithiocarbamate-treated Leishmania cells were un-der stress (Fig. 3C).
Cytotoxicity of metal dithiocarbamates on Leishmania cells.The viability of metal dithiocarbamate-treated L. major promas-tigotes was analyzed by MTT assay. We found that treatment withall 3 metal dithiocarbamates resulted in a concentration-depen-dent reduction in the viability of Leishmania cells, with LD50 val-
FIG 2 Expression of CA transcripts in L. major and inhibition of LmCA ac-tivity by metal dithiocarbamates (DTCs). (A) Total RNA isolated from L.major promastigotes was subjected to RT-PCR using primers specific for theLmCA1 (lane 2) or LmCA2 (lane 5) gene. The respective product sizes (921and 1,887 bp) are indicated. Lanes 1 and 4 represent the respective negative-control reactions without RT. A DNA ladder was loaded in lane 3. (B) Con-centration-dependent inhibition of CA activity (EU/mg) in whole-cell lysatesof L. major promastigotes by maneb (�), zineb (o), and propineb (�). Errorbars represent the means � SD of values from 3 independent experiments. Therespective IC50s are given in the index.
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ues of 0.56 �M, 0.61 �M, and 0.27 �M for maneb, zineb, andpropineb, respectively (Fig. 4A). Since Leishmania parasites residewithin acidic phagolysosomes of the infected host cells, we testedthe effect of these compounds on parasites growing in acidic me-dium (pH 5.5). In this acidic environment, the parasites exhibitedmarkedly increased susceptibility to the metal dithiocarbamates asreflected by their severalfold-lower LD50 values: 0.048 �M, 0.049�M, and 0.041 �M for maneb, zineb, and propineb, respectively(Fig. 4B). This result suggests that these metal dithiocarbamateswould effectively kill the parasites residing inside the acidic
phagolysosomal compartment of host cells. It was encouraging tofind that maneb, zineb, and propineb were also effective againstLeishmania donovani, the causative agent of visceral leishmaniasis,in which they caused dose-dependent growth inhibition as well ascytotoxicity (see Fig. S1 in the supplemental material).
Intracellular acidosis of Leishmania cells upon treatmentwith metal dithiocarbamates. Since in higher organisms, CA ac-tivity is known to be important for intracellular pH maintenance,we next tested whether metal dithiocarbamate-mediated inhibi-tion of LmCAs resulted in intracellular acidosis of Leishmaniaparasites (31). For this, L. major promastigotes were grown in theabsence or presence of maneb, zineb, or propineb, and the intra-cellular pH (pHi) of the cells was measured by loading them withthe fluorescent pH indicator BCECF-AM. The steady-state pHi ofthe untreated cells was found to be almost neutral (i.e., 6.93),whereas the pHi values of the metal dithiocarbamate-treated cellswere �0.2 unit lower in each case, i.e., 6.68, 6.72, and 6.71, respec-tively, for the maneb-, zineb-, and propineb-treated parasites (Ta-ble 1). The extent of intracellular acidification upon metal dithio-carbamate treatment was more pronounced when the parasiteswere grown in acidic medium (pH 5.5). At this acidic external pH,the difference in pHi between the untreated and metal dithiocar-bamate-treated L. major cells was �0.3 pH unit. The untreated
FIG 3 Effect of metal dithiocarbamates on growth and morphology of L.major promastigotes. (A) L. major cells were grown in the absence or presenceof increasing concentrations of maneb (�), zineb (o), or propineb (�), andcells were counted after 96 h of growth. Each data point represents the meanresult � SD from 3 independent experiments. (B) Representative SEM images(at ��6,000 magnification) of untreated L. major promastigotes and thosetreated with 0.625 �M maneb, zineb, or propineb. (C) Bar graph comparingcell length (in �m) of the parasites grown in the absence (untreated) or pres-ence of 0.625 �M maneb, zineb, or propineb. Error bars represent average celllengths � SD of values from at least 40 independent measurements. *, Signif-icant difference (P 0.05) with respect to the untreated control.
FIG 4 Effect of metal dithiocarbamates (DTCs) on viability of L. major cells. L.major promastigotes were grown in normal medium (pH 7.2) (A) or in acidicmedium (pH 5.5) (B) in the absence or presence of the indicated concentra-tions of maneb (�), zineb (o), or propineb (�), and the viabilities of the cellswere measured by the MTT assay after 96 h of growth. The LD50 values ofmaneb, zineb, and propineb for Leishmania cells grown in normal or acidicmedium are given in the indexes.
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cells maintained a pHi of 6.81, whereas the cells treated withmaneb, zineb, and propineb had significantly lower pHis of 6.46,6.53, and 6.49, respectively (Table 1). These findings demon-strated that metal dithiocarbamate treatment caused intracellularacidosis which may be responsible, at least in part, for Leishmaniacell death.
Apoptotic and necrotic cell death of metal dithiocarbamate-treated parasites. The mechanism of Leishmania cell death upontreatment with metal dithiocarbamates was analyzed by annexinV and PI double staining. Annexin V specifically binds to thephosphatidylserine exposed on the outer leaflet of the apoptoticcell membrane, whereas PI is a membrane-impermeant fluores-cent dye that interacts with the DNA and RNA of those cells whosemembrane integrity is lost (e.g., in late apoptotic or necrotic cells)(32). Data presented in Fig. 5A show that, compared to the un-treated cells, there was a significant increase in the number of lateapoptotic (annexin V-positive/PI-positive [annexin V/PI])and necrotic (annexin V-negative [annexin V�]/PI]) cells upontreatment with 0.625 �M maneb, zineb, or propineb. Analysis ofseveral such images revealed that the percentages of late apoptoticand necrotic cells were 2% and 3% for untreated cells, 21% and33% for maneb-treated cells, 19% and 38% for zineb-treated cells,and 26% and 48% for propineb-treated cells, respectively. Thenumbers of early apoptotic cells (annexin V/PI�) were negligiblein all the cases, suggesting that a major proportion of the metaldithiocarbamate-treated cells were at advanced stages of cell death(Fig. 5B).
Reduced intracellular parasite burden upon treatment withmetal dithiocarbamates. Having confirmed the antileishmanialproperties of the metal dithiocarbamates in vitro, we wanted todetermine the effect of these compounds on parasites residinginside the host macrophages. For this, J774A.1 macrophages wereinfected with L. major promastigotes, and the infected cells wereincubated in the absence or presence of maneb, zineb, orpropineb. Data presented in Fig. 6 show that all 3 metal dithiocar-bamates caused a dose-dependent reduction in the number ofintracellular amastigotes. Treatment with 0.625 �M zineb orpropineb resulted in �50% reduction in the parasite load (num-ber of amastigotes/100 macrophages), whereas for maneb-treatedcells the drop in the intracellular parasite load was 43%.
Metal dithiocarbamates are fairly nontoxic to mammaliancells. MTT assay was employed to test the cytotoxicity of maneb,zineb, and propineb on two mammalian cell lines, J774A.1 (mac-rophage) and NIH 3T3 (fibroblast). The cells were treated withvarious concentrations of the metal dithiocarbamates up to amaximum concentration of 5 �M. We found that none of thesecompounds exhibited any cytotoxic effect on mammalian cellseven at a 5 �M concentration (Fig. 7A). Taken together, our datasuggest that these metal dithiocarbamates are fairly nontoxic formammalian cells and are highly selective in targeting the Leishma-nia cells. The fact that the CA activity in J774A.1 and NIH 3T3 cellswas not inhibited even in the presence of 5 �M maneb, zineb, orpropineb is plausibly the reason for this selective cytotoxicity(Fig. 7B).
Variable efficacy and development of resistance against existingdrugs pose serious challenges in controlling leishmaniasis in dif-ferent parts of the world (5–7). A steady pipeline of novel therapiesis therefore critical for our continued effort to combat this com-plex disease. As a significant step toward this, the potent antileish-manial properties of maneb, zineb, and propineb reported herepromise to strengthen the chemotherapeutic repertoire againstleishmaniasis. Interestingly, mammalian cells were not affected bythese metal dithiocarbamates even at a concentration which isseveralfold higher than their antileishmanial LD50s. The efficacyand broad therapeutic indices of this new class of antileishmanialcompounds are thus very striking and warrant further preclinicalstudies for drug development.
Dithiocarbamates and their metal complexes have been used asantifungal agents in agriculture for decades without much knowl-edge about their mode of action (33). A mechanistic insight intotheir fungicidal property was provided only recently when a widerange of dithiocarbamates were found to be strong inhibitors of�-CAs from three fungal pathogens, C. neoformans, Candida albi-cans, and Candida glabrata (25). In subsequent reports, �-CAsfrom M. tuberculosis, �-CA from T. cruzi, and the physiologicallyimportant human �-CAs, e.g., CAI, -II, -IX, and -XII, were alsoshown to be inhibited by submicromolar concentrations of di-thiocarbamates, thereby establishing them as a general class of CAinhibitors (22, 23, 34, 35).
The notion that metal dithiocarbamates may have antileish-manial activity originated from our experiment in which maneb,zineb, and propineb were found to be strong inhibitors of CAactivity in L. major promastigotes. Given that CAs play importantphysiological roles in a variety of organisms, it was necessary totest whether dithiocarbamate-mediated CA inhibition is detri-mental for the parasite growth and/or survival (9). Indeed, 3 metaldithiocarbamate complexes chosen for this study all caused signif-icant growth inhibition and morphological abnormalities of L.major promastigotes. These compounds were also highly cyto-toxic for the parasite as evident from the MTT assay. The submi-cromolar LD50 values of these metal dithiocarbamates (0.56 �Mfor maneb, 0.61 �M for zineb, and 0.27 �M for propineb) indicatethat their in vitro leishmanicidal activity is comparable to that ofamphotericin B, which is thus far the most potent antileishmanialdrug (36). The heterocyclic thiols are the only known CA inhibi-tors reported to exhibit antileishmanial activity. However, theyare effective against L. chagasi and L. amazonensis at a much higher
TABLE 1 Intracellular pH of metal dithiocarbamate-treated L. majorpromastigotes grown in normal (pH 7.2) or acidic (pH 5.5) media
pHea Treatment pHi
7.2 Untreated 6.93 � 0.02Manebc 6.68 � 0.07d
Zinebc 6.72 � 0.03d
Propinebc 6.71 � 0.06d
5.5 Untreated 6.81 � 0.03Manebe 6.46 � 0.05d
Zinebe 6.53 � 0.04d
Propinebe 6.49 � 0.06d
a pHe indicates the pH of the growth medium.b pHi values are experimentally determined intracellular pH values of L. majorpromastigotes grown in the absence (untreated) or presence of metal dithiocarbamates.Means � SD of values from triplicate experiments are shown.c Treatment with 0.625 �M of the respective metal dithiocarbamate.d Significant difference (P 0.05) with respect to the untreated control.e Treatment with 0.078 �M of the respective metal dithiocarbamates.
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concentration (MIC of �250 �M) and hence appear to be lesspotent than maneb, zineb, or propineb (19).
These metal dithiocarbamates were even more effective againstparasites growing in acidic medium (pH 5.5 as opposed to pH 7.2)for which their LD50 values were severalfold lower (0.048 �M formaneb, 0.049 �M for zineb, and 0.041 �M for propineb). Thisresult prompted us to ask whether treatment with metal dithio-carbamates, which are potent CA inhibitors, led to intracellularacidification of Leishmania cells. This was a relevant question be-
cause CAs, by accelerating the rate of CO2/HCO3� buffering in the
cytosol, are known to play important roles in intracellular pHmaintenance (31). This function of CAs is even more critical foractively metabolizing tumor cells, where the interplay betweencytosolic CAII and membrane-bound CAIX helps in regulatingintracellular pH (37). Our results confirmed that indeed the pHi ofmetal dithiocarbamate-treated L. major promastigotes was �0.2unit lower than that of the untreated cells (pHi � 6.93). For Leish-mania cells growing in acidic media (pH 5.5), the pHi dropped by
FIG 5 Extent of apoptosis and necrosis in L. major promastigotes treated with metal dithiocarbamates. (A) L. major promastigotes were grown in the absence(untreated) or presence of 0.625 �M maneb, zineb, or propineb for 96 h, and thereafter the cells were stained with Alexa Fluor 488-annexin V conjugate (green),PI (red), and DAPI (blue). Images were captured by an epifluorescence microscope (at �60 magnification) with appropriate filter sets. (B) Bar graphs representquantitation of early apoptotic (annexin V/PI�), late apoptotic (annexin V/PI), and necrotic (annexin V�/PI) cells expressed as a percentage of the totalnumber of DAPI-positive cells for parasites grown in the absence (gray bars) or presence of 0.625 �M maneb (black bars), zineb (dotted bars), or propineb (whitebars). At least 200 cells were analyzed for each condition, and the error bars represent the means � SD of values from triplicate experiments. *, Significantdifference (P 0.05) with respect to the untreated control.
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almost 0.3 unit upon treatment with maneb, zineb, or propineb,suggesting that the intracellular buffering system of Leishmania isthe target of these metal dithiocarbamates. However, it is alsopossible that other CA-dependent metabolic processes like fattyacid biosynthesis and gluconeogenesis are inhibited by these metaldithiocarbamates (9). Furthermore, the off-target effects of thesecompounds cannot be completely ruled out, and it is to be notedthat pyrrolidine dithiocarbamate at concentrations of 10 �Mand above was shown to inhibit proteasomal activity and NF- B(38, 39).
The mechanism of Leishmania cell death was next investigated.Annexin V/PI staining confirmed that apoptosis and necrosis arethe causes of metal dithiocarbamate-mediated killing of L. majorpromastigotes. The Leishmania cell death is plausibly a conse-quence of intracellular acidosis, as was observed earlier in differentcell types like neuronal cells, tumor cells, and phagocytic cells, inwhich deregulation of intracellular pH acted as a trigger for apop-tosis and/or necrosis (40–42). Several studies suggested that celldeath induced by intracellular acidosis can also be exploited fortherapeutic purposes. For example, lowering of pHi by pharma-cological inhibition of CAIX has been shown to activate ceramide-mediated apoptosis in human cancer cells (43). This finding wascorroborated by in vivo experiments where CAIX gene silencingled to a 40% reduction in xenograft tumor volume in mice (44). H.pylori, when treated with acetazolamide under acidic conditions,lost its cytoplasmic/periplasmic buffering capacity with a simulta-neous decrease in its ability to survive in low pH (45). This obser-vation might explain how gastric ulcer patients were successfullytreated with CA inhibitors with �90% healing efficiency (46). Thepotential clinical benefits of this pHi modulatory property of CAinhibitors are quite appealing and further highlight the possibletherapeutic application of these metal dithiocarbamates.
Can these metal dithiocarbamates be used as antileishmanialdrugs? The reduced parasite burdens of metal dithiocarbamate-treated macrophages suggest that in addition to their in vitro an-tileishmanial activity, these compounds also have the potential totarget intracellular amastigotes and hence can be considered lead
compounds for antileishmanial drug development. The toxicity ofthese metal dithiocarbamates and their cellular metabolites (car-bon disulfide and ethylene/propylene thiourea) might be a matterof concern (47). But it is to be noted that maneb, zineb, andpropineb are reported to be cytotoxic for mammalian cells only ata concentration which is at least 50 times higher than their antil-eishmanial LD50s (48, 49). Similarly, the neuropathic effects ofcarbon disulfide- or ethylene thiourea-induced thyroid tumorswere observed in rats after chronic exposure to a very high dose ofthese compounds (e.g., when exposed to 700 ppm or 9.19 mMcarbon disulfide for 2 h/day, 5 days/week for 12 weeks or fed with�250 ppm or 2.44 mM ethylene thiourea in the diet for 2 years)(50, 51). So, the submicromolar doses at which these metal dithio-carbamates are effective against the parasites should be safe for themammalian cells. Our results also confirmed that maneb, zineb,or propineb, up to a concentration of 5 �M, did not have anycytotoxic effects on 2 mammalian cell lines, i.e., J774A.1 (macro-phage) and NIH 3T3 (fibroblast). From our data, it appears that
FIG 6 Effect of metal dithiocarbamates (DTCs) on intracellular amastigotes.L. major-infected J774A.1 macrophages were incubated for 18 h in the absence(0 �M, gray bar) or in the presence of 0.312 �M or 0.625 �M maneb (blackbars), zineb (dotted bars), or propineb (white bars), following which the cellswere fixed and stained with DAPI. Based on the total number of DAPI-stainednuclei of macrophages and amastigotes in a field, the number of intracellularamastigotes/100 macrophages (parasite load) was calculated from the imagescaptured in an epifluorescence microscope. For each condition, at least 100macrophages (and the corresponding number of amastigotes) were analyzed.Error bars represent the means � SD of values from triplicate experiments. *,Significant difference (P 0.05) with respect to the untreated control.
FIG 7 Effect of metal dithiocarbamates on viability and CA activity of mam-malian cells. (A) J774A.1 (black bars) and NIH 3T3 (white bars) cells weregrown in the absence (untreated) or presence of 5 �M maneb, zineb, orpropineb, and the viabilities of the cells were measured by the MTT assay after72 h of growth. (B) J774A.1 (black bars) and NIH 3T3 (white bars) whole-celllysates were incubated for 15 min in the absence (no inhibitor) or presence of5 �M maneb, zineb, or propineb, following which the CA specific activities(EU/mg) were measured. Data represent the relative specific activities, i.e., thespecific activity in the presence of inhibitor/specific activity in the absence ofinhibitor. The error bars in both experiments represent the means � SD ofvalues from 3 independent experiments.
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these metal dithiocarbamates are more superior inhibitors forLmCAs than their mammalian counterparts, which might be thebasis for their differential cytotoxicity. The encapsulation of thesecompounds within liposomes or biocompatible nanocarriersmight be a way to further reduce their toxic effects and improvetheir bioavailability as well as macrophage targeting.
In conclusion, potent antileishmanial activity coupled withbroad therapeutic indices of maneb, zineb, and propineb is verypromising and provides an important lead toward the develop-ment of a new class of safe and effective antileishmanial drugs. Itwill be tempting to evaluate the efficacy of these metal dithiocar-bamates against the drug-resistant strains of Leishmania.
This work was supported by a Ramalingaswami fellowship (BT/RLF/Re-entry/34/2010) and CSIR grant [37(1622)/14/EMR-11] awarded to R.D.by Department of Biotechnology and Council of Scientific and IndustrialResearch, Government of India, and by generous start-up funds fromIISER Kolkata. D.S.P. and D.K.M. are supported by IISER Kolkata fellow-ships.
We thank Kashinath Sahu for his assistance with the SEM imaging.
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