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JAC Advance Access originally published online on August 11, 2008
Journal of Antimicrobial Chemotherapy 2008 62(5):998-1002; doi:10.1093/jac/dkn319
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Antileishmanial activity mediated by apoptosis and structure-based target study of peganine hydrochloride dihydrate: an approach for rational drug design

Pragya Misra1, Tanvir Khaliq2, Anshuman Dixit2, Souvik SenGupta3, Mukesh Samant1, Shraddha Kumari1, Awanish Kumar1, Pramod K. Kushawaha1, H. K. Majumder3, Anil K. Saxena2, T. Narender2 and Anuradha Dube1,*

1 Division of Parasitology, Central Drug Research Institute, Lucknow 226001, India 2 Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226001, India 3 Department of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata 700032, India

* Corresponding author. Tel: +91-522-2612411; Fax: +91-522-2623405; E-mail: anuradha_dube{at}rediffmail.com

Received 4 April 2008; returned 9 May 2008; revised 27 June 2008; accepted 30 June 2008


   Abstract
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Objectives: The aim of this study was to resolve the putative pathway responsible for death induced by peganine hydrochloride dihydrate isolated from Peganum harmala seeds at cellular, structural and molecular level in Leishmania donovani, a causative agent of fatal visceral leishmaniasis.

Methods: The mode of action was assessed using various biochemical approaches including phosphatidylserine exposure, estimation of mitochondrial transmembrane potential and in situ dUTP nick end labelling staining of nicked DNA in the parasite. Molecular modelling and molecular dynamics studies were conducted with DNA topoisomerase I to identify the target of peganine hydrochloride dihydrate mediating apoptosis. Further, DNA topoisomerase I inhibition by peganine hydrochloride dihydrate was also assessed using an L. donovani topoisomerase I relaxation assay.

Results: Peganine hydrochloride dihydrate, besides being safe, was found to induce apoptosis in both the stages of L. donovani via loss of mitochondrial transmembrane potential. Molecular docking studies suggest that a binding interaction with DNA topoisomerase I of L. donovani (binding energy of –79 kcal/mol) forms a stable complex, indicating a possible role in apoptosis. The compound also inhibits L. donovani topoisomerase I.

Conclusions: The compound induces apoptosis in L. donovani and inhibits DNA topoisomerase I.

Keywords: apoptosis , mitochondrial transmembrane potential , TUNEL assay , molecular docking


   Introduction
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Most antileishmanial drugs are expensive, toxic and have unacceptable side effects; dosing is also complicated by the fact that they are given parenterally.1 Moreover, cases of drug resistance are on the rise.2 This has caused a renewed interest in the study of medicinal plants as a source for new antiparasitic leads. Further, understanding the mode of action and binding modes of these natural products to specific target sites may be used to design potent, novel, selective and less toxic antileishmanial analogues of these compounds on a structural basis.

In our earlier studies, activity-guided fractionation and isolation from Peganum harmala seeds led to the identification of oral antileishmanial activity in peganine hydrochloride dihydrate (1), a small molecule with simple structural features [Figure S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)], easily synthesizable and non-toxic to hosts. In this paper, we show that the leishmanicidal effect of 1 seems to be the consequence of induction of programmed cell death (PCD) both in the intracellular amastigote and extracellular promastigote forms of Leishmania cells targeting DNA topoisomerase I.


   Materials and methods
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Enzyme and chemicals

Recombinant type I DNA topoisomerase was prepared as described previously.3 Camptothecin (Sigma, St Louis, MO, USA) was dissolved in DMSO at 20 mM concentrations and kept frozen at –20°C.

Parasite culture

The promastigotes of Leishmania donovani were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 100 U/mL penicillin (Sigma) and 100 mg/L streptomycin (Sigma) at 26°C. Transgenic parasites expressing green fluorescent protein were cultured in the presence of geneticin sulfate (G418) (100 mg/L).

Double staining with annexin V and propidium iodide (PI)

Externalization of phosphatidylserine on the outer membrane of untreated and 1-treated promastigotes was measured by the binding of annexin V-FITC and PI as previously described.4

Mitochondrial membrane potential determination

The dissipation of mitochondrial membrane potential ({Delta}{Psi}m) is a characteristic feature of apoptosis.5 To determine the changes in {Delta}{Psi}m, we used the JC-1 dye as a probe according to the method of Dey and Moraes.6 Briefly, cells were collected after treatment with 1 for various time periods and incubated for 7 min with 10 µM JC-1 at 37°C, washed, resuspended in medium and measured for fluorescence. The ratio of the reading at 590 nm to the reading at 530 nm (590:530) was considered as the relative {Delta}{Psi}m value.

In situ labelling of DNA fragments by TUNEL assay

In situ detection of DNA fragments by terminal deoxyribonucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) was performed using the DeadEndTM Fluorometric TUNEL System (Promega). Briefly, promastigotes and intracellular amastigotes (106) treated with or without 1 were fixed in 4% formaldehyde and permeabilized with 0.2% (v/v) Triton X-100 followed by incubation with buffer containing a nucleotide mix as per the manufacturer’s protocol. The samples were counterstained with PI (10 mg/L) and visualized under a high resolution fluorescence camera (Leica DFC320) mounted on a Leica DMS000B microscope. Images were captured and processed as described earlier. At least 20 microscopic fields were observed for each sample.

Molecular modelling and molecular dynamics studies

The binding site in L. donovani DNA topoisomerase (Ld-topoI) crystal structure (PDB ID: 2B9S) has a vanadate ligand that is very small compared with toptecan in human DNA topoisomerase; thus, no cavity exists at this site in the protein–DNA interface sufficient to allow for docking studies in the Ld-topoI crystal structure. A site known to have important residues (Tyr-222, Arg-314, Lys-352 and Arg-410) can only be exposed by removing the DNA molecule as reported elsewhere.7 The protein was therefore prepared by removing the ligand and the DNA using Insight II software, and the hydrogens were optimized to a gradient of 0.01 kcal/mol. This structure was used for docking studies of 1 taking the corresponding ligand binding pocket defined as a 10 Å sphere around the vanadate ligand, using the software GOLD. Default GOLD settings were used and the generated docking poses were visually inspected. The best pose was selected on the basis of GoldScore and ChemScore and the scores were 31.21 and 12.12, respectively.

The peganine (best pose)–topoisomerase complex was minimized to a gradient of 0.01 kcal/mol using the CharmM force field, keeping a harmonic restraint of 25 kcal/mol on protein backbone atoms; thereafter, the constraints were removed and reminimization of the complex was carried out to a gradient of 0.01 kcal/mol. The resulting complex was then subjected to a molecular dynamics simulation at 300 K for 100 ps. The finally obtained complex was reminimized as earlier, and the binding energy between peganine and topoisomerase I was calculated using the following formula:

Binding Energy = Energy of complex – Energy of topoisomerase – Energy of peganine

Plasmid relaxation assay

The type I DNA topoisomerase was assayed by decreased mobility of the relaxed isomers of supercoiled pBluescript (SKþ) DNA in an agarose gel after treatment with enzyme. A relaxation assay was carried out as described3 with LdTOP1LS. The standard topoisomerase assay contained 25 mM Tris–HCl, pH 7.5, 5% glycerol, 50 mM KCl, 0.5 mM DTT, 10 mM MgCl2, 150 mg/L bovine serum albumin, 0.5 µg of pBS plasmid and 1 U of enzyme (1 U of topoisomerase I activity is the amount of enzyme that converts 0.5 µg of superhelical DNA to the relaxed state under the conditions of the assay). The reaction was carried out at 37°C for 30 min. Reactions were stopped by adding 1% SDS, 10 mM EDTA, 0.25 mg/L Bromophenol Blue and 15% glycerol. Samples were applied to a horizontal 1% agarose gel and subjected to electrophoresis in Tris–acetate–EDTA buffer (0.04 M Tris–acetate, 0.002 M EDTA, pH 8.0) at 1.5 V/cm for 14–16 h at room temperature. The gels were stained with ethidium bromide (5 mg/L), de-stained in water and photographed under UV illumination. Percent relaxation was measured by microdensitometry of negative photographs of supercoiled monomer DNA band fluorescence after ethidium bromide staining with a microdensitometer (LKB BROMMA 2202 Ultrascan), and the area under the peak was calculated.

Statistical analysis

The data are presented as mean ± SD. The statistical significance of differences in percentage between treated and untreated was analysed by one-way ANOVA using GraphPad Prism software.


   Results and discussion
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 Abstract
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Funding
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Compound 1 has exhibited considerably good antileishmanial activity in vitro (IC50 for promastigotes being 0.2 mM and for amastigotes 0.22 mM) without any cytotoxicity (P. Misra, T. Khaliq, K. P. Reddy, S. Gupta, R. Kant, P. R. Maulik, T. Narender and A. Dube, unpublished results). To clarify the mode of action of 1 against L. donovani, we have demonstrated using biochemical and morphological approaches that 1 induces a cell death that shares several phenotypic features with metazoan apoptosis,8 including phosphatidylserine exposure, in situ TUNEL staining of nicked DNA and loss of mitochondrial transmembrane potential. During apoptosis in metazoan and unicellular cells, phosphatidylserine is translocated from the inner side to the outer layer of the plasma membrane.4

Annexin V, a Ca2+-dependent phospholipid-binding protein with an affinity for phosphatidylserine, is routinely used to label externalization of phosphatidylserine. Since annexin V can also label necrotic cells, a PI stain was used to differentiate among apoptotic cells (annexin V-positive and PI-negative), necrotic cells (both annexin V- and PI-positive) and normal cells (both annexin V- and PI-negative). Promastigotes treated with 1 (0.2 mM for 48 h) were double-stained with FITC-conjugated annexin V and PI, and analysed by flow cytometry. A significant percentage (68.8%) of promastigotes were stained positive for annexin V (Figure 1b, lower right quadrant) at 48 h when compared with only 5.32% in control cells (Figure 1a, lower right quadrant). This was comparable with apoptosis triggered by miltefosine (77.9%; lower right quadrant of Figure 1c), which served as a positive control.9 Recently, the study of mitochondrial potential has become the focus of apoptosis regulation, as many investigations have demonstrated a major functional impact of mitochondrial alterations on apoptosis.10 Simultaneous measurement of the J-aggregate (indicative of intact mitochondria) and J-monomer (indicative of de-energized mitochondria) showed a slight increase at 4 h post-treatment followed by a sharp fall during 6–24 h post-treatment. After 4 h treatment with 1, {Delta}{Psi}m was increased by 16%, the ratio of 590/530 fluorescence of treated versus untreated being 5.81 ± 0.115 and 4.88 ± 0.12, respectively. A prominent drop in {Delta}{Psi}m by 43% occurred at 6 h post-treatment (ratio of treated versus untreated being 2.77 ± 0.39 and 4.93 ± 0.10, respectively), which further decreased at 12 h post-treatment (by 58%) and at 24 h by 65% (1.24 ± 0.04 versus 3.56 ± 0.35). From the above data, it can be inferred that 1 initially causes mitochondrial membrane hyperpolarization followed by a sustained hypopolarization thereafter. Maximal interference with {Delta}{Psi}m was achieved after 24 h of treatment with 1, which confirms that apoptosis is mediated via the loss of transmembrane potential.


Figure 1
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  		Figure 1. Detection of apoptosis in promastigotes by annexin V and PI double staining and in situ analysis (TUNEL staining) of apoptosis in L. donovani promastigotes (a colour version of this figure is available at JAC Online (http://jac.oxfordjournals.org/). (a) Untreated, (b) 1-treated and (c) miltefosine-treated promastigotes showing annexin V and PI staining. The lower-left quadrant indicates the percentage of unstained cells, the upper-left shows PI-positive cells, the lower-right shows annexin V stained cells and the upper-right shows PI- and annexin V-positive cells. In situ analysis (TUNEL staining) of apoptosis in L. donovani intracellular amastigotes. (d) Untreated and (e) treated promastigotes with an IC50 dose of 1 (results are representative of three independent experiments). Arrows indicate TUNEL-positive cells. (f) Untreated and (g) treated amastigote-infected macrophages with an IC50 dose of 1. (h) Merged image showing both TUNEL-stained treated amastigotes and PI-stained macrophage nuclei (results are representative of three independent experiments). A colour version of this figure is available at JAC Online (http://jac.oxfordjournals.org/).

 
In order to characterize the changes occurring in the nuclear material during cell death with 1, in situ TUNEL staining was performed to detect the free ends of DNA after breakage, which is one of the important biochemical hallmarks of eukaryotic apoptosis. Promastigotes treated with 0.2 mM of 1 showed TdT-labelled nuclei (brightly fluoresced yellowish green spots in Figure 1e) compared with untreated promastigotes, which, as compared with the control promastigotes, did not show any TUNEL-positive cells (Figure 1d) indicating DNA fragmentation, thus providing strong evidence for apoptosis in L. donovani. Amastigote-infected macrophages treated with or without 1 were subjected to in situ TUNEL assay. The nuclear DNA fragmentation of intracellular amastigotes, as determined by the green fluorescence, was clearly visible inside the treated macrophages (Figure 1g) when compared with the untreated amastigotes (Figure 1f), which did not show any green fluorescence. Macrophage nuclei were stained red, indicating that no damage was caused by treatment with 1 at this concentration, thus confirming that 1 induces apoptosis only in L. donovani without any toxicity to host cells.

While the above results support our proposed mechanism of action at a cellular level, the structural similarity of the compound with known topoisomerase I inhibitors (see the Supplementary data available at JAC Online, http://jac.oxfordjournals.org/) prompted us to carry out molecular modelling and molecular dynamics studies to further elucidate the structure-based mechanism of the compound. A docking study7,11 was carried out with 1 against the protein crystal structure of L. donovani topoisomerase I (Ld-topo I). It was inferred from the molecular dynamics studies that 1 forms a stable complex with Ld-topo I enzyme (binding energy –79.2 kcal/mol). We thus propose that its action may result from a direct interaction with the topoisomerase enzyme and it may mediate a conformational change in Ld-topo I. This was further validated by the relaxation assay carried out using DNA topoisomerase I of L. donovani. While studying the in vitro effect of 1 on L. donovani topoisomerase I, we found that the compound, when added together with the DNA and enzyme, did not inhibit relaxation (Figure 2b, lanes 13–16). Inhibition of enzyme activity is observed when the enzyme is pre-incubated with the compound for 5 min at 37° in the relaxation assay mixture before addition of the DNA substrate at same concentration. Figure 2(b) (lanes 6–8) shows the inhibition of catalytic activity by 1 in the above reaction condition. Lane 6 shows that the compound exerts 85% inhibition at 250 µM concentration. The results of this assay favours the mechanism proposed by the docking studies that peganine inhibits the DNA topoisomerase by directly interacting with the enzyme, as enzymatic inhibition was observed only in pre-incubation reactions.


Figure 2
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  		Figure 2. (a) Docking of 1 (yellow) in active site of L. donovani topoisomerase I. (b) Inhibition of catalytic activity of L. donovani DNA topoisomerase I. Lanes 1–8: pre-incubation of enzyme and 1; lanes 9–16: simultaneous addition of topoisomerase I, 1 and DNA. Lane 1: supercoiled pBS DNA; lane 2: DNA with 1 U of purified L. donovani topoisomerase I; lane 3: DNA with 1 U of purified L. donovani topoisomerase I and DMSO; lane 4: inhibition of catalytic activity with camptothecin (50 µM); lanes 5–8: inhibition of catalytic activity with increasing concentration of 1 (100, 250, 500 µM and 1 mM, respectively); lane 9: supercoiled pBS DNA; lane 10: DNA with 1 U of purified L. donovani topoisomerase I; lane 11: DNA with 1 U of purified L. donovani topoisomerase I and DMSO; lane 12: inhibition of catalytic activity with camptothecin (50 µM); lanes 13–16: inhibition of catalytic activity with increasing concentration of 1 (100, 250, 500 µM and 1 mM, respectively). A colour version of this figure is available at JAC Online (http://jac.oxfordjournals.org/).

 
Confirmation of our molecular modelling predictions by experimental proof shows that potential structure-based designing of selective, less toxic and potent peganine analogues can be done using in silico approaches. This study provides a novel approach to alternative computational strategies such as virtual screening or structure-based drug designing, which may be productively used without having complete information on the drug/DNA topoisomerase binding interaction, for identifying new and selective Leishmania topoisomerase inhibitors. Work in this direction is in progress.


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Financial support was provided by the Department of Biotechnology, New Delhi, and Council of Scientific Research for fellowships of authors.


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None to declare.


   Supplementary data
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Figure S1 and colour versions of Figures 1 and 2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We are thankful to Mr A. L. Vishwakarma, Technical Officer, SAIF division for flow cytometry analysis and spectral data. This is CDRI communication No. 7384.


   References
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1 Murray HW. Treatment of visceral leishmaniasis (kala-azar): a decade of progress and future approaches. Int J Infect Dis (2000) 4:158–77.[CrossRef][Medline]

2 Croft SL. Monitoring drug resistance in leishmaniasis. Trop Med Int Health (2001) 6:899–905.[CrossRef][Web of Science][Medline]

3 Das BB, Sen N, Ganguly A, et al. Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani. FEBS Lett (2004) 565:81–8.[CrossRef][Web of Science][Medline]

4 Mehta A, Shaha C. Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J Biol Chem (2004) 279:11798–813.[Abstract/Free Full Text]

5 Kroemer G. The mitochondrion as an integrator/coordinator of cell death pathways. Cell Death Differ (1998) 5:547.[CrossRef][Web of Science][Medline]

6 Dey R, Moraes CT. Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells. J Biol Chem (2000) 275:7087–94.[Abstract/Free Full Text]

7 Chhabra S, Sharma P, Ghoshal N. A computational docking study for prediction of binding mode of diospyrin and derivatives: inhibitors of human and leishmanial DNA topoisomerase-I. Bioorg Med Chem Lett (2007) 17:4604–12.[CrossRef][Medline]

8 Debrabant A, Lee N, Bertholet S, et al. Programmed cell death in trypanosomatids and other unicellular organisms. Int J Parasitol (2003) 33:257–67.[CrossRef][Web of Science][Medline]

9 Paris C, Loiseau PM, Bories C, et al. Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes. Antimicrob Agents Chemother (2004) 48:852–9.[Abstract/Free Full Text]

10 Gottlieb RA. Mitochondria and apoptosis. Biol Signals Recept (2001) 10:147–61.[CrossRef][Web of Science][Medline]

11 Wang R, Wang S. How does consensus scoring work for virtual library screening? An idealized computer experiment. J Chem Inf Comput Sci (2001) 41:1422–6.[CrossRef][Web of Science][Medline]


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