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.
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.
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.
The compound induces apoptosis in L. donovani and inhibits DNA topoisomerase I.
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
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.
The promastigotes of Leishmaniadonovani 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 (ΔΨm) is a characteristic feature of apoptosis.5 To determine the changes in ΔΨm, 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 ΔΨm 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 DeadEnd™ 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.
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
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, ΔΨm 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 ΔΨm 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 ΔΨm was achieved after 24 h of treatment with 1, which confirms that apoptosis is mediated via the loss of transmembrane potential.
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.
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.
Financial support was provided by the Department of Biotechnology, New Delhi, and Council of Scientific Research for fellowships of authors.
None to declare.
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.