Experimental Parasitology 125 (2010) 310–314

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Experimental Parasitology

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Research Brief

Leishmania donovani: Oral therapy with glycosyl 1,4-dihydropyridine analogueshowing apoptosis like phenotypes targeting pteridine reductase 1 in intracellularamastigotes

Jaspreet Kaur a, Nasib Singh b, Biswajit Kumar Singh c, Anuradha Dube b, Rama Pati Tripathi c,Prashant Singh d, Neeloo Singh a,*

a Drug Target Discovery and Development Division, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, CSIR, Lucknow-226001, Indiab Division of Parasitology, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, CSIR, Lucknow-226001, Indiac Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, CSIR, Lucknow-226001, Indiad Department of Chemistry, D.A.V. (P.G.) College, Dehradun-248001, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 August 2009Received in revised form 19 February 2010Accepted 23 February 2010Available online 26 February 2010

Keywords:Leishmania donovaniDihydropyridineAntileishmanial activityFlow cytometryCell cycle arrest

0014-4894/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.exppara.2010.02.011

* Corresponding author. Fax: +91 0522 2623405.E-mail address: neeloo888@yahoo.com (N. Singh).

Glycosyl 1,4-dihydropyridine analogue (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threopentofuranos-4-yl)-1-phenyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid diethyl ester) synthesized inour laboratory, inhibited Leishmania donovani infection in vitro and in hamsters (Mesocricetus auratus)when administered orally. This analogue is nontoxic, cell-permeable and orally effective. This glycosyldihydropyridine analogue functioned through arrest of cells in sub-G0/G1-phase, triggering mitochon-drial membrane depolarization-mediated programmed cell death of the intracellular amastigotes.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

The drugs recommended for the treatment of visceral leishman-iasis (VL), i.e., pentavalent antimonials, pentamidine, amphotericinB and its lipid formulations, paromomycin, allopurinol and miltefo-sine have limitations, including development of resistance, paren-teral administration, long courses of treatment, toxic side effects,and high costs (Murray et al., 2005; Sundar and Chatterjee,2006). In order to generate an adequate armory of effective, safeand cheaper chemotherapeutic agents to treat VL, new and effec-tive drug targets are required to combat this disease. Enzymes,metabolites or proteins present in the parasite but absent fromtheir mammalian host are considered as ideal targets for rationaldrug design. In the search for better therapeutics against leishman-iasis we focused on a validated drug target viz. folate biosyntheticpathway which is unique to the parasite (Bello et al., 1994). Pteri-dine reductase1 (PTR1) enzyme in Leishmania is essential for devel-opment of successful antifolate chemotherapy (Nichol et al., 1985).

ll rights reserved.

In search for specific inhibitors of PTR1 (Genbank Accession No.AY547305) we have previously reported phenyl 1,4-dihydropyri-dine ring as the lead structure showing impressive antileishmanialefficacy with an IC50 of 18 lM in intracellular amastigotes (Kumaret al., 2008). Dihydropyridine analogues have exhibited importanttherapeutic and pharmacological properties as the integral back-bone of several calcium channel blockers (Kappe, 2000), antihyper-tensive agents (Atwal et al., 1991), a1a-antagonists (Kappe et al.,1997), and neuropeptide Y (NPY) antagonists (Poindexter et al.,2002). A broad range of biological effects including antiviral, anti-tumor, antibacterial and anti-inflammatory activities have beendescribed for these compounds (Kappe, 1993). Amlodipine andlacidipine, conventional antihypertensive 1,4-dihydropyridinederivatives have been reported to be active against experimentalVL and triggered programmed cell death (PCD) in promastigotes(Palit and Ali, 2008). The main objective of the present study wasto evaluate the antileishmanial efficacy both in vitro and in vivoand their possible mode of killing mediated by glycosyl 1,4-dihy-dropyridine analogue (GDA) (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threo pentofuranos-4-yl)-1-phenyl-1,4-dihy-dro-pyridine-3,5-dicarboxylic acid diethyl ester) on intracellularamastigotes of Leishmania donovani clinical isolate.

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2. Materials and methods

2.1. Parasite culture

Transgenic parasites overexpressing PTR1-GFP chimera (PTR1tagged at the N-terminal with GFP) were cultured at 25 �C in M-199 medium (Gibco-BRL) supplemented with 10% heat inactivatedFBS (Gibco-BRL), 100 U penicillin, 100 lg/ml streptomycin and inthe presence of 150 lg/ml geneticin sulfate (G418) (Kumar et al.,2007).

2.2. Macrophages

J774A.1 mouse (BALB/c) macrophage cell line was maintainedin RPMI 1640 medium (Gibco-BRL) adjusted to contain 2 g/l so-dium bicarbonate, 6 g/l HEPES, 10% HIFBS and 100 U penicillinand 100 lg/ml streptomycin, at 37 �C in an atmosphere of 5%CO2 and air.

2.3. Infected macrophages

J774A.1 cells (5 � 105 cells/ml) were seeded in 75 cm sq tissueculture flask and incubated overnight using RPMI 1640 mediumcontaining 10% HIFBS. After 24 h, these cells were infected withL. donovani promastigotes overexpressing PTR1-GFP chimera(5 � 106) in the log phase of growth in serum free media for 2–6 h. The infected cells were then maintained in RPMI containing10% FBS from 0–18 h at 37 �C with 5% CO2 and air (Singh et al.,2003). Images were collected using 63�, 1.4 NA (oil) Plan Apochro-mate lens on Leica DM5000 B fluorescence microscope. Greenimages were collected after excitation at 488 nm.

2.4. Isolation of L. donovani amastigotes from infected macrophages

J774A.1 cells grown in 75 cm sq tissue culture flasks infectedwith L. donovani promastigotes were used to purify amastigotes.Briefly, flasks containing infected J774A.1 were flushed, cells har-vested by centrifugation at 201g for 10 min at 4 �C and then resus-pended in PBS (pH 7.2). Cells were disrupted by passage through a27-gauge needle and centrifuged at 201g for 10 min at 4 �C. Thepellet was discarded and the supernatant was transferred in freshtube. This supernatant was centrifuged at 3220g for 5 min at 4 �C.Pellet (amastigotes) was suspended in PBS and viewed under aphase contrast microscope for purity check.

2.5. Antileishmanial activity in vivo

Syrian male golden hamsters (Mesocricetus auratus) were in-fected intracardially with 1 � 107 amastigotes isolated from thespleen of heavily infected donor hamsters. Splenic biopsies werecarried out on day 25 p.i. for assessing the status of infection bymaking spleen dab smears. Animals having 10–15 amastigotesper 100 spleen cell nuclei were selected and used for screeningpurposes. Four to five animals per group were treated orally withthe GDA at 6.25, 12.5, 25, 50 and 100 mg/kg dose for 5 consecutivedays. Splenic biopsies were again performed on day 7 post treat-ment and the parasite burden in treated and untreated groupwas quantitated. Percent inhibition of parasite multiplication wascalculated by the following formula:

PI ¼ AT� 100IT� TI

where PI-percent inhibition; AT-actual number of amastigotes/100spleen cell nuclei in treated animals; IT-initial number of amastig-otes/100 spleen cell nuclei in treated animals; and TI-times increasein untreated control animals. The use of animals for all the experi-

ments was in compliance with the relevant guidelines of the insti-tutional animal ethics committee.

2.6. DNA content analysis by flow cytometry

Amastigotes isolated from infected macrophages treated withGDA (18 lM for 24 h) were harvested by centrifugation at 3220g,for 5 min at 4 �C. Cells were washed once in 1 ml PBS and thenfixed by incubation in 70% ethanol: 30% PBS for 1 h at 4 �C. Priorto analysis, fixed cells were harvested by centrifugation at 3220g,for 10 min at 4 �C, washed in 1 ml PBS and then resuspended in1 ml PBS with RNAse A (100 lg/ml) and propidium-iodide (Molec-ular Probes) at 10 mg/ml. The cells were incubated at 35 �C for45 min and then analysed by using a flow cytometer (Becton Dick-inson FACSAria serial no-P9M500001). Ten thousand cells wereanalysed for each sample. Cell cycle distribution was modeledusing the ModFit LT for Mac V3.0.

2.7. Measurement of mitochondrial membrane potential

Mitochondrial damage upon treatment with compound inamastigotes was assessed by flow cytometry using a cell-perme-able dye, mitotracker deep red (Molecular Probes). Mitotrackersare aldehyde fixable cationic lipophilic fluorochrome that passivelydiffuses through the plasma membrane of viable cells and is selec-tively sequestered in mitochondria with an active membrane po-tential and permits the examination of membrane potential inadherent cells (Haugland, 1996). Amastigotes isolated from in-fected macrophages treated with GDA (18 lM for 24 h), washedin PBS and loaded in dark for 30 min with mitotracker deep red(10 lM) as per the manufacturer’s instructions. Analysis for meanfluorescence intensity (MFI) was done using FACSAria and Cell-Quest Pro software.

2.8. Statistical analysis

The data are presented as mean ± SD. The statistical significanceof differences in percentage between treated and untreated wasanalysed by One-Way ANOVA using GraphPad Prism software.

3. Results

3.1. Determination of the IC50 of GDA mediated death in vivo

The GDA exhibited excellent in vivo antileishmanial efficacy byoral route against established infection of L. donovani. It imparted89.9 ± 2.3% inhibition of parasite at 50 mg/kg dose. At higher doseof 100 mg, it exhibited almost same (90.2 ± 5.0%) growth inhibitoryeffect. An ED50 of 25.0 mg/kg was obtained (Fig. 1). At lower con-centrations such as 12.5 and 6.25 mg, the % inhibition was foundto be 52.0 ± 4.2% and 24.8 ± 2.4%, respectively. GDA was devoidof any toxicity towards animals as no mortality was seen duringthe course of treatment.

3.2. GDA induces sub-G0/G1-phase cell cycle arrest in intracellularamastigotes

We performed a cell cycle analysis by flow cytometry after PIstaining of the parasites incubated for 24 h at 18 lM of the GDA.As can be seen from Fig. 2C and D, the GDA induced a decreasein number of amastigotes after 24 h in S-phase relative to theamastigotes without treatment (S: 26.85% vs. 0% for amastigotesand GDA-treated amastigotes, respectively). In addition, the GDAincreased the percentage of amastigotes in G2/M phase by a factorof 4 relative to amastigotes without treatment (7.76% vs. 31.67%

Fig. 1. Analysis of antileishmanial activity of GDA in vivo: Syrian golden hamsters.(Mesocricetus auratus) were infected intracardially with 1 � 107 amastigotesisolated from the spleen of heavily infected donor hamsters. Animals were treatedorally with the inhibitor at 6.25, 12.5, 25, 50 and 100 mg/kg dose for 5 consecutivedays. Splenic biopsies were performed on day 7 post treatment and the parasiteburden in treated and untreated group was quantitated as detailed in Section 2.Data are presented as mean ± SD for four or five animals per group from triplicatesvalues. Comparison among the experimental groups was done by One-Way ANOVAtest. The upper level of significance was chosen as P < 0.001.

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for amastigotes and GDA-treated amastigotes, respectively). Theeffect of GDA on G2/M phase in amastigotes was largely at the ex-pense of S-phase cells with a non-significant change in G0/G1-phase cell population compared with the amastigotes withouttreatment. The drug-induced cell cycle perturbations such as de-crease in the S-phase have been reported to correlate with a re-sponse to chemotherapy. G2/M is a crucial period for furthercellular proliferation. Drug-induced increase in G2/M phase isassociated with DNA double strand breaks and extensive chromo-

Fig. 2. GDA causes sub-G0/G1 arrest of L. donovani amastigotes. Infected macrophages w(C) isolated amastigotes and (D) isolated amastigotes after 24 of treatment with GDA.experimental groups was done by One-Way ANOVA test. The upper level of significance

some damage (Sorenson and Eastman, 1988). Further, treatmentwith GDA resulted in a 24.27% sub-G1 peak, characteristic of apop-tosis, at same time point (Fig. 2D). This suggests that a check in thecell cycle could simultaneously initiate late events of PCD such asnuclear condensation and DNA nicking. However, in macrophages(Fig. 2A) and in macrophages after 48 h of infection (Fig. 2B), theproportion of cells in different phases of the cell cycle was compa-rable with isolated amastigotes.

3.3. GDA depolarizes the mitochondrial membrane potential ofamastigotes

The loss of mitochondrial membrane potential is a characteris-tic feature of metazoan apoptosis and has been observed to play akey role in drug-induced death in protozoans such as Leishmania(Sen et al., 2004). Following treatment, membrane potential wasdetermined by cytofluorometric measurement of the mitochon-drial dependent uptake and retention of Mitotracker Red (CMXRos)into mitochondria. CMXRos is an aldehyde fixable cationic lipo-philic fluorochrome that passively diffuses through the plasmamembrane of viable cells and is selectively sequestered in mito-chondria with an active membrane potential and permits theexamination of the membrane potential in cells (Haugland, 1996;Shibayama-Imazu et al., 2006). Propidium-iodide staining was uti-lized to gate the necrotic cell population and debris from the viablecell population (Darzynkiewicz et al., 1997). Treatment with 18 lMGDA resulted in reduction of mitotracker positive cells from 83.90%(control) to 41.60% (at 24 h of treatment) or mean fluorescenceintensity (MFI) decreased from 13,108 (control) to 6324 (at 24 hof GDA treatment) (Fig. 3A and B).

ere incubated with 18 lM GDA for 24 h. (A) Macrophages, (B) infected macrophages,Data are presented as mean ± SD from triplicates values. Comparison among thewas chosen as P < 0.001.

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4. Discussion

Our previous study established the antileishmanial property ofGDA in vitro (Kumar et al., 2008). Further to explore the effect ofGDA against L. donovani amastigotes we have investigated thetherapeutic efficacy of GDA in experimental VL and its apoptosislike features in vitro.

GDA showed a high therapeutic index in vivo and can give �90%decreases in parasite burden in L. donovani infected hamsters. Thisdosage which eliminated �90% of parasites (i.e. that was essen-tially curative) and that did not also result in >10% mean weightloss, which we arbitrarily chose as an indication of drug toxicity.The GDA showed leishmanicidal effect on intracellular organismsboth in vitro and in vivo model, suggesting that interaction of thecompound with host cell or with cellular events is important forits inhibitory effect. The efficacy of GDA is due to excellent cell per-meability. Our molecule resembles monastrol, a structurally rathersimple DHPM, which is the only cell-permeable molecule currentlyconsidered as a lead for the development of new anticancer drug(Kapoor et al., 2000). Orally active long lasting antihypertensiveagents of dihydropyridine specificity have been reported (Wanget al., 2006) resulting from the increased chemical stability of theurea functionality. GDA exhibited superior oral antileishmanicidalactivity.

The amastigote stage in Leishmania is of prime importance inperpetuating the disease and is the target for antileishmanialdrugs. Amastigotes are a replicative stage that periodically escapefrom the host cell by a poorly defined mechanism and rapidly rein-fect other phagocytes (i.e. macrophages or dendritic cells) andsome non-phagocytic cell types (i.e. fibroblasts) (Bogdan et al.,2000). We have been able to successfully carry out in vitro infectionof J774A.1 macrophage culture along the same lines of humaninfection by Leishmania parasites. In this way we are confident ofhaving mimicked the intracellular metabolism of amastigote. Pre-vious study has shown that infection of murine macrophages withwild-type L. chagasi, fusion of developing phagosomes with lyso-somes is delayed for 24–48 h (Rodriguez et al., 2006). In our studythe promastigotes from a clinical isolate of L. donovani, were ableto invade the macrophages, differentiate into amastigotes within6 h and also proliferate in the macrophage in vitro for 7 days indi-cating that we can thus confidently extrapolate our results to theclinical scenario.

Our results showed that parasites invade the macrophages(J774A.1), where it survives by modulating the host immune re-sponse via inhibiting macrophage apoptosis (Fig. 2B). L. donovaniwas the first parasite reported to enhance host cell viability by

Fig. 3. GDA causes depolarization of mitochondrial membrane potential in L. donovaniControl and (B) after 24 h of treatment with GDA. Data are presented as mean ± SD from trANOVA test. The upper level of significance was chosen as P < 0.001.

inhibiting growth factor deprivation-induced apoptosis (Mooreand Matlashewski, 1994). Leishmania parasites induce the activa-tion of several pathways (NF-jB, PI3K and p38 MAPK) that inhibitapoptosis in macrophages (Ruhland et al., 2007). Upon the estab-lishment of infection and differentiation from the disseminativeform (promastigote) to the proliferative form (amastigote), Leish-mania spp. may inhibit macrophage apoptosis by suppressing therelease of cytochrome c that is related to the manipulation of theBcl-2 family of proteins (Ruhland et al., 2007).

We have used infected macrophages to investigate the chemo-therapeutic effect of GDA and the consequent DNA damage in-duced in amastigotes. In accordance with previous studies incancer cells (Park et al., 2006), and on Leishmania promastigotes(Sen et al., 2007; Das et al., 2008) a significant G2/M arrest couldbe observed with in 24 h with a simultaneous sub-G0/G1 arrestat same time point after treatment with GDA. The effect of inhibi-tor on G2/M phase arrest in amastigotes was largely at the expenseof the S-phase, with a non-significant change in G0/G1-phase cellpopulation compared with untreated amastigotes. The GDA in-creased the percentage of amastigotes in G2/M phase showed highmitotic index (MI). MI is a measure for the proliferation status of acell population. It is defined as the ratio between the number ofcells in mitosis and the total number of cells. While existing in clo-sely knit groups, unicellular organisms acquire a new level of indi-viduality and can express features of apoptosis under situations ofstress prompted us to explore whether or not, while living in closeassociation inside the macrophages, the parasites have the capabil-ity to undergo apoptosis. Importantly, it lends acceptance to thesuggestion that, in situations of close existence of the parasites,such as within the host cell, the cell cycle pattern is differentmay be due to difference in the levels of gene expression (Kauret al., 2009).

In addition, due to its unique physicochemical property, com-pound can easily pass through the plasma membrane and evokestructural and functional changes in cellular membrane integrity(Kappe, 2000). All these factors might contribute towards the in-creased permeability of the mitochondrial membrane. The induc-tion of mitochondrial dysfunction in the apoptotic process wasapparent in the Mitotracker Red study results. The study of mito-chondrial potential has become a focus of apoptosis regulation asmany investigations demonstrate a major functional impact ofmitochondrial alterations on apoptosis (Gottlieb, 2000). Mitochon-drial membrane permeabilization (MMP) is considered as the‘‘point-of-noreturn” in numerous models of programmed celldeath. MMP affects the inner and outer mitochondrial membranes(IM and OM, respectively) to a variable degree. OM permeabiliza-

amastigotes. Infected macrophages were incubated with 18 lM GDA for 24 h. (A)iplicates values. Comparison among the experimental groups was done by One-Way

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tion culminates in the release of proteins that normally are con-fined in the mitochondrial intermembrane space (IMS), includingcaspase activators (e.g., cytochrome c) (Bhaumik et al., 1999). Par-tial IM permeabilization disrupts mitochondrial ion and volumehomeostasis and dissipates the mitochondrial transmembrane po-tential. Our study on intracellular amastigotes provides significantimplications for drug development.

Based on the phenyl-1,4-dihydropyridine ring as the lead struc-ture, this compound may be used as a main stream antileishmanialdrug for cost-effective oral therapy of the neglected disease VL.

Acknowledgments

We are thankful to Mr. A.L. Vishwakarma, Technical Officer SAIFdivision for his help regarding the flow cytometry work. We aregrateful to Department of Biotechnology, India (Grant No. BT/PR5452/BRB/10/430/2004) for financial assistance for this study.This is CDRI communication no. 7727.

References

Atwal, K.S., Swanson, B.N., Unger, S.E., Floyd, D.M., Moreland, S., Hedberg, A.,O’Reilly, B.C., 1991. Dihydropyrimidine calcium channel blockers. 3-Carbamoyl-4-aryl-1,2,3,4-tetrahydro-6-methyl-5-pyrimidine carboxylic acid esters asorally effective antihypertensive agents. Journal of Medicinal Chemistry 34,806–811.

Bello, A.R., Nare, B., Freedman, D., Hardy, L., Beverley, S.M., 1994. PTR1: a reductasemediating salvage of oxidized pteridines and methotrexate resistance in theprotozoan parasite Leishmania major. Proceedings of the National Academy ofSciences 91, 11442–11446.

Bhaumik, S., Anjum, R., Rangaraj, N., Pardhasaradhi, B.V., Khar, A., 1999. Curcuminmediated apoptosis in AK-5 tumor cells involves the production of reactiveoxygen intermediates. FEBS Letters 456, 311–314.

Bogdan, C., Donhauser, N., Döring, R., Röllinghoff, M., Diefenbach, A., Rittig, M.G.,2000. Fibroblasts as host cells in latent Leishmaniosis. Journal of ExperimentalMedicine 191, 2121–2130.

Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997.Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death(necrosis). Cytometry 27, 1–20.

Das, R., Roy, A., Dutta, N., Majumder, H.K., 2008. Reactive oxygen species andimbalance of calcium homeostasis contributes to curcumin inducedprogrammed cell death in Leishmania donovani. Apoptosis 13, 867–882.

Gottlieb, R.A., 2000. Role of mitochondria in apoptosis. Critical Reviews inEukaryotic Gene Expression 10, 231–239.

Haugland, R.P., 1996. Handbook of Fluorescent Probes and Research Chemicals.Molecular Probes, Eugene, OR.

Kapoor, T.M., Mayer, T.U., Coughlin, M.L., Mitchison, T.J., 2000. Probing spindleassembly mechanisms with monastrol, a small molecule inhibitor of the mitotickinesin, Eg5. Journal of Cell Biology 150, 975–988.

Kappe, C.O., 1993. One hundred years of the biginelli dihydropyridine synthesis.Tetrahedron 49, 6937.

Kappe, C.O., Fabian, W.M.F., Semones, M.A., 1997. Conformational analysis of 4-aryl-dihydropyrimidine calcium channel modulators. A comparison of ab initio, semiempirical and X-ray crystallographic studies. Tetrahedron 53, 2803.

Kappe, C.O., 2000. Biologically active dihydropyrimidones of the Biginelli-type, aliterature survey. European Journal of Medicinal Chemistry 35, 1043–1052.

Kaur, J., Singh, B.K., Tripathi, R.P., Singh, P., Singh, N., 2009. Leishmania donovani: aglycosyl dihydropyridine analogue induces apoptosis like cell death viatargeting pteridine reductase 1 in promastigotes. Experimental Parasitology123, 258–264.

Kumar, P., Sundar, S., Singh, N., 2007. Degradation of pteridine reductase 1 (PTR1)enzyme during growth phase in the protozoan parasite Leishmania donovani.Experimental Parasitology 116, 182–189.

Kumar, P., Kumar, A., Verma, S.S., Dwivedi, N., Singh, N., Siddiqi, M.I., Tripathi, R.P.,Dube, A., Singh, N., 2008. Leishmania donovani pteridine reductase 1:biochemical properties and structure modeling studies. ExperimentalParasitology 120, 73–79.

Moore, K.J., Matlashewski, G., 1994. Intracellular infection by Leishmania donovaniinhibits macrophage apoptosis. Journal of Immunology 152, 2930–2937.

Murray, H.W., Berman, J.D., Davies, C.R., Saravia, N.G., 2005. Advances inleishmaniasis. Lancet 366, 1561–1577.

Nichol, C.A., Smith, G.K., Duch, D.S., 1985. Biosynthesis and metabolism oftetrahydrobiopterin and molybdopterin. Annual Review of Biochemistry 54,729–764.

Palit, P., Ali, N., 2008. Oral therapy with amlodipine and lacidipine, 1,4-dihydropyridine derivatives showing activity against experimental visceralLeishmaniasis. Antimicrobial Agents and Chemotherapy 52, 374–377.

Park, C., Kim, G.Y., Kim, G.D., Choi, B.T., Park, Y.M., Choi, Y.H., 2006. Induction of G2/M arrest and inhibition of cyclooxygenase-2 activity by curcumin in humanbladder cancer T24 cells. Oncology Reports 15, 1225–1231.

Poindexter, G.S., Bruce, M.A., LeBoulluec, K.L., Monkovic, I., Martin, S.W., Parker,E.M., Iben, L.G., McGovern, R.T., Ortiz, A.A., Stanley, J.A., Mattson, G.K.,Kozlowski, M., Arcuri, M., Antal-Zimanyi, I., 2002. Dihydropyridineneuropeptide Y Y1 receptor antagonists. Bioorganic and Medicinal ChemistryLetters 12, 379–382.

Rodriguez, N.E., Gaur, U., Wilson, M.E., 2006. Role of caveolae in Leishmania chagasiphagocytosis and intracellular survival in macrophages. Cellular Microbiology8, 1106–1120.

Ruhland, A., Leal, N., Kima, P.E., 2007. Leishmania promastigotes activate PI3K/Aktsignaling to confer host cell resistance to apoptosis. Cellular Microbiology 9, 84–96.

Sen, N., Das, B.B., Ganguly, A., Mukherjee, T., Tripathi, G., Bandyopadhyay, S.,Rakshit, S., Sen, T., Majumder, H.K., 2004. Camptothecin induced mitochondrialdysfunction leading to programmed cell death in unicellular hemoflagellateLeishmania donovani. Cell Death and Differentiation 11, 924–936.

Sen, R., Bandyopadhyay, S., Dutta, A., Mandal, G., Ganguly, S., Saha, P., Chatterjee, M.,2007. Artemisinin triggers induction of cell-cycle arrest and apoptosis inLeishmania donovani promastigotes. Journal of Medicinal Microbiology 56,1213–1218.

Shibayama-Imazu, T., Sonoda, I., Sakairi, S., Aiuchi, T., Ann, W.W., Nakajo, S., Itabe,H., Nakaya, K., 2006. Production of superoxide and dissipation of mitochondrialtransmembrane potential by vitamin K2 trigger apoptosis in human ovariancancer TYK-nu 520 cells. Apoptosis 11, 1535–1543.

Singh, N., Singh, R.Th., Sundar, S., 2003. Novel mechanism of drug resistance in kalaazar field isolates. The Journal of Infectious Diseases 188, 600–607.

Sorenson, C.M., Eastman, A., 1988. Mechanism of cw-diamminedichloroplatinum(II)-induced cytotoxicity: role of G2 arrest and DNA double-strand breaks.Cancer Research 48, 4484–4488.

Sundar, S., Chatterjee, M., 2006. Visceral leishmaniasis – current therapeuticmodalities. Indian Journal of Medical Research 123, 345–352.

Wang, X., Quan, Z., Wang, J.K., Zhang, Z., Wang, M., 2006. A practical and greenapproach toward synthesis of N3-substituted dihydropyrimidinones: usingAza-Michael addition reaction catalyzed by KF/Al2O3. Bioorganic and MedicinalChemistry Letters 16, 4592–4595.