These authors contributed equally to this work.
Neutralization of Haemorrhagic Activity of Viper Venoms by 1-(3-Dimethylaminopropyl)-1-(4-Fluorophenyl)-3-Oxo-1,3-Dihydroisobenzofuran-5-Carbonitrile
Article first published online: 26 JUN 2011
© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society
Basic & Clinical Pharmacology & Toxicology
Volume 109, Issue 4, pages 292–299, October 2011
How to Cite
Sunitha, K., Hemshekhar, M., Gaonkar, S. L., Sebastin Santhosh, M., Suresh Kumar, M., Basappa, ., Priya, B. S., Kemparaju, K., Rangappa, K. S., Nanjunda Swamy, S. and Girish, K. S. (2011), Neutralization of Haemorrhagic Activity of Viper Venoms by 1-(3-Dimethylaminopropyl)-1-(4-Fluorophenyl)-3-Oxo-1,3-Dihydroisobenzofuran-5-Carbonitrile. Basic & Clinical Pharmacology & Toxicology, 109: 292–299. doi: 10.1111/j.1742-7843.2011.00725.x
- Issue published online: 12 SEP 2011
- Article first published online: 26 JUN 2011
- Accepted manuscript online: 16 MAY 2011 02:47PM EST
- (Received 9 January 2011; Accepted 26 April 2011)
Abstract: Viper envenomation undeniably induces brutal local manifestations such as haemorrhage, oedema and necrosis involving massive degradation of extracellular matrix at the bitten region and many a times results in dangerous systemic haemorrhage including pulmonary shock. Snake venom metalloproteases (SVMPs) are being considered to be the primary culprits for the venom-induced haemorrhage. As a consequence, the venom researchers and medical practitioners are in deliberate quest of SVMP inhibitors. In this study, we evaluated the inhibitory effect of 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3-dihydroisobenzofuran-5-carbonitrile (DFD) on viper venom-induced haemorrhagic and PLA2 activities. DFD effectively neutralized the haemorrhagic activity of the medically important viper venoms such as Echis carinatus, Echis ocelatus, Echis carinatus sochureki, Echis carinatus leakeyi and Crotalus atrox in a dose-dependent manner. The histological examinations revealed that the compound DFD effectively neutralizes the basement membrane degradation, and accumulation of inflammatory leucocytes at the site of Echis carinatus venom injection further confirms the inhibition of haemorrhagic activity. In addition, DFD dose dependently inhibited the PLA2 activities of Crotalus atrox and E. c. leakeyi venoms. According to the docking studies, DFD binds to hydrophobic pocket of SVMP with the ki of 19.26 × 10−9 (kcal/mol) without chelating Zn2+ in the active site. It is concluded that the clinically approved inhibitors of haemorrhagins could be used as a potent first-aid agent in snakebite management. Furthermore, a high degree of structural and functional homology between SVMPs and their relatives, the MMPs, suggests that DFD analogues may find immense value in the regulation of multifactorial pathological conditions like inflammation, cancer and wound healing.
Viper envenomation is characterized by brutal local manifestations including swelling at the bitten site, oedema, blistering and necrosis, which develop swiftly after the bite. In many instances, systemic effects such as haemorrhage of vital organs, coagulopathy, renal failure and pulmonary shock may occur [1–3]. Most of these pathological alterations are because of the action of metalloproteases of the venom, which are the zinc-dependent endopeptidases popularly called snake venom metalloproteases (SVMPs). They are one of the major enzymatic constituents of viperid snake venoms and are generally referred to as ‘Spreading factors’ as they facilitate diffusion of target-specific toxins into circulation by degrading proteins of the extracellular matrix (ECM) surrounding blood vessels at the envenomed region [3–5]. Therefore, inhibition of SVMPs perhaps serves dual functions, decreasing the magnitude of local tissue destruction as well as limiting the easy diffusion of systemic toxins into the circulating blood, hence increases in survival time of victims.
The only medically approved therapy for snake envenomation is immediate administration of equine or ovine-derived snake-specific or polyvalent antivenom. The antivenom is an effective treatment for systemic manifestations; however, it is comparatively ineffective against local tissue damage while the therapy is often associated with the risk of anaphylaxis and serum incompatibility [6,7]. Importantly, antivenoms are not readily available, particularly in rural areas of Africa and Asian countries, where the affected populations are living in resource-poor settings. In the past, SVMP inhibitory properties of chelating agents, inhibitors and antibiotics such as batimastat, EDTA, O-phenanthroline, TPEN (N,N,N,N’-tetrakis (2-pyridylmethyl) ethylenediamine), BAPTA bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid, biphosphonate clodrante and tetracycline doxycycline have been reported [8–12]. However, the efficacies of these molecules are not effective in the management and are associated with several limitations. Hence, in recent years, researchers are focusing on alternative/ancillary therapeutic agents such as plant-based therapeutics and clinically approved drugs/synthetic compounds to neutralize the local manifestations of individual venoms/toxins [13,14].
Citalopram is a clinically approved antidepressant drug that inhibits the reuptake of selective and centrally acting serotonin (5-hydroxytryptamine) and is used in the treatment against symptoms of major depression, social anxiety and panic disorders, post-stroke pathological crying and in pervasive developmental disorder. In addition, it has been observed to reduce the symptoms of diabetic neuropathy and premature ejaculation [15,16]. As part of an ongoing research programme on small molecular mass bioactive agents, a citalopram derivative 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3-dihydroisobenzo-furan-5-carbonitrile (DFD) was studied for its neutralizing effect on haemorrhage and PLA2 activity of selected viper venoms and further validated through structure–activity relationship (SAR) studies by molecular modelling approach.
Materials and Methods
Echis carinatus (EC) venom was obtained from Irula Snake Catchers, Chennai, India. Crotalus atrox (CA), Echis carinatus sochureki (ECS), Echis carinatus leakeyi (ECL) and Echis ocelatus (EO) venoms were a kind gift from Dr. R.M. Kini, National University of Singapore, Singapore; Prof. R. Harrison, Liverpool Institute for Tropical Diseases, UK; and Prof. K. Kemparaju, University of Mysore, India. Albino male mice weighing 20–25 g (from the Central Animal House Facility, Department of Zoology, University of Mysore, Mysore, India) were used for pharmacological studies. Animal care and handling complied with the National Regulation for Animal Research. Chemical reagents and all other solvents used in this study were purchased from SRL (Mumbai, India) and Merck (Mumbai, India).
Synthesis of DFD. The compound DFD was synthesized and characterized according to the published protocol .
Indirect haemolytic activity. Indirect haemolytic activity of individual venom was determined according to the method of Boman and Kaletta , using packed human erythrocytes (blood group A) washed several times with phosphate-buffered saline (145 mM NaCl in 150 mM phosphate buffer pH 7.2) and sedimented by gentle centrifugation (1000 × g) until a clear supernatant was obtained. For the assay, the stock was prepared by mixing packed erythrocytes (1 ml), egg yolk (1 ml) and phosphate-buffered saline (8 ml). One millilitre of suspension from stock was incubated with venom (10 μg) for 30 min. at 37°C. The reaction was terminated by adding 10 ml of ice-cold PBS and centrifuged at 4°C at 800 × g. The amount of haemoglobin released in the supernatant was measured at 540 nm. One millilitre of stock erythrocytes with 10 ml of ice-cold PBS alone is considered 0% lysis. For the inhibition study, individual venom (10 μg) was pre-incubated independently with different doses of the inhibitor (1:10, 1:20, 1:30, 1:40, 1:50; venom: inhibitor; w/w) for 10 min. at 37°C. The necessary controls were maintained in all respective cases.
Haemorrhagic activity. Haemorrhagic activity was assayed as described by Kondo et al. . Minimum haemorrhagic dose (MHD) was evaluated for individual venom as the amount of venom that produces a haemorrhagic halo of 10 mm. Two MHD (minimum haemorrhagic dose) of EC (1 μg), ECS (10 μg), ECL (5 μg), EO (5 μg) and CA (5 μg) were pre-incubated with different doses of inhibitor (1:10, 1:25, 1:50 and 1:75; venom: inhibitor; w/w) in a total volume of 50 μl at 37°C for 10 min. The pre-incubated samples were injected intradermally into groups of mice (n = 4) independently. The following control groups were included: (a) mice injected with venom alone dissolved in PBS, (b) mice injected with inhibitor alone dissolved in PBS and (c) mice injected with PBS alone.
For independent injection experiments, mice (n = 4) were subcutaneously injected with 2MHD of E. carinatus venom (1 μg in 25 μl of PBS) on their back. Then, at various time intervals (0, 2, 4, 8, 10, 15 and 20 min.), mice received an injection of DFD (45 μg in 50 μl in PBS) at the same site where venom had been administered. The dose was selected on the basis of the results obtained in experiments with pre-incubation of venom and the inhibitor. The mice were anaesthetized by diethyl ether inhalation after 3 hr and killed. The dorsal patch of skin was removed, the inner surface was observed for the haemorrhage and the diameter of the haemorrhagic spot was measured.
Histopathological studies. Skin tissues were dissected out from the site of injection of venom alone, and venom pre-incubated with DFD was fixed in Bouin’s solution overnight. The tissue samples were subjected to dehydration by processing with different grades of alcohol and chloroform: alcohol mixture. The processed tissues were embedded in molten paraffin wax, and 4-mm-thick sections were prepared using microtome (Leika, Solms, Germany). The sections were stained with haematoxylin–eosin staining for microscopic observations. The sections were observed under Leitz wetzlar Germany type-307-148.002 microscope, and photographs were taken using Photometrics colorsnap CF camera (made Roper Scientific Photometrics, Munich, Germany) attached to the microscope.
UV-Visible spectral study. The interaction between Zn2+/Ca2+ and DFD was studied by UV-VIS absorption spectroscopic scanning with a wavelength range of 190–300 nm, using a Beckman Coulter DU-730. The DFD (1.2 mM) was incubated with different concentrations of ZnCl2/CaCl2 (0–1.5 mM) in 1 ml of PBS.
Molecular modelling studies. The protein and the ligands were prepared using Autodock Tools. Docking runs were performed using the Lamarckian genetic algorithm (LGA) . The 3D grid box was created using the zinc atom (ZN503) as a grid centre. The number of grid points in the x, y and z axes was 60 × 60 × 60 with grid points separated by 0.375 Å. Each docking experiment was derived from 100 different runs that were set to terminate after a maximum of 1,500,000 energy evaluations, yielding 100 docked conformations. The population size was set to 50. Docked orientations within a root-mean square deviation of 0.5 Å were clustered together. The lowest free energy cluster returned for each compound was used for further analysis. Docking results were also carried out using discovery studio and visualized using Chimera [http://www.cgl.ucsf.edu/chimera/] and pymol [http://www.pymol.org/].
Protein estimation. The protein estimation was done according to the method of Lowry et al.  using bovine serum albumin (BSA) as standard.
Statistical analysis. All the experiments were repeated for five independent observations. The data are presented as mean ± S.E.M.
Inhibition of haemorrhagic activities.
Two MHD doses of viper venoms such as EC, ECS, ECL, EO and CA induced haemorrhagic spots with varied diameter. Table 1 presents the dose-dependent neutralization of haemorrhagic activity of viper venoms induced by DFD. Two MHD of ECS (10 μg), ECL (5 μg), EO (5 μg) and CA (5 μg) venoms induce a haemorrhagic spot with a diameter of 17 ± 1, 16 ± 2, 16 ± 3 and 13 ± 2 mm, respectively. The DFD inhibited the venom-induced haemorrhage, and complete inhibition was observed at a ratio of 1:50 (w/w) for the above said venoms. Further, 2MHD dose of EC venom (1 μg) induced a haemorrhagic spot with a diameter of 17 ± 2 mm. Haemorrhagic activity of EC venom was abrogated by DFD dose dependently (fig. 1). A ratio of 1:45 (venom: DFD; w/w) was found to abolish the haemorrhagic activity of EC venom. Further, histological examinations demonstrated an extensive degradation of ECM and basement membrane surrounding the blood vessels in venom-injected skin sections. The longitudinal section of skin tissue revealed prominent disorganized ECM and myofibroblasts at the venom-injected site (fig. 2B). In contrast, DFD completely inhibited the tissue damage and the histological sections revealed intact basement membrane and alignment of myofibroblasts (fig. 2C), similar to the control section (fig. 2A).
|Venom||2 MHD (μg)||Diameter of haemorrhagic spot (mm) Venom: DFD (w/w)|
|Echis carinatus||1||17 ± 2||9 ± 2||3 ± 1||0||0|
|Echis carinatus sochureki||10||17 ± 1||10 ± 3||4 ± 2||0||0|
|Echis carinatus leakeyi||5||16 ± 2||8 ± 1||2 ± 1||0||0|
|Echis oceollatus||5||16 ± 3||10 ± 1||4 ± 2||0||0|
|Crotalus atrox||5||13 ± 2||5 ± 1||2 ± 1||0||0|
In the independent injection experiment, EC venom (1 μg/mouse) induced haemorrhage spot with a diameter of 17 ± 2 mm. DFD significantly inhibited the haemorrhage when given within 2 min. after the venom injection. About 61% and 46% of haemorrhage inhibition was observed, respectively, when DFD was injected 4 and 8 min. after the venom administration. In contrast, no inhibition was observed when DFD was administered 15 min. later (fig. 3). The percentage inhibition of haemorrhage was found to decrease when the injection of DFD was delayed.
UV-Visible spectral study.
To investigate the possible interaction between DFD and cations (Ca2+ and Zn2+), spectral studies of DFD (1.2 mM) with different concentration of ZnCl2 and CaCl2 (0–1.5 mM) were performed independently (fig. 4). DFD showed maximal absorption between 210 and 220 nm, the absorbance intensity of DFD did not alter when ZnCl2 (fig. 4A) and CaCl2 (fig. 4B) were independently added. The spectral data confirmed the fact that DFD do not interact with the cations such as zinc and calcium.
Molecular modelling studies.
To know the mechanism of inhibition by DFD, docking studies of DFD and SVMP were performed using the available protein sequence [GI: 62468] in NCBI BLAST against Protein Data Bank (PDB). The sequence and NCBI conserved domain database analysis of the jararhagin revealed the presence of four domains. The template was found to have the structure of bothropasin (P-III class) from B. jararaca having a 95% identity against the jararhagin excluding the N-terminal domain. The bothropasin structure contains three domains, which are very much conserved (95% identity) with the jararhagin sequence. The main active site domains are well conserved, and thus, the available bothropasin experimental X-ray crystal structure was used for the docking purposes (PDBID 3DSL). The analysis of other SVMP crystal structures (PDB ID: 2ERO, 2ERP, 2ERQ, 2DW0, 2DW1 and 2DW2) revealed that the active site is present near the zinc-binding site of the bothropasin structure. The docking studies on DFD into the crystal structure of bothropasin were performed using Autodock. Out of 100 docked complexes, the lowest binding energy was selected and described. The observed binding free energy and the final docking energy for the DFD were −10.53 (kcal/mol) and −12.02 (kcal/mol), respectively. The Autodock calculated inhibition constant (ki) was found to be 19.26 × 10−9 (kcal/mol). The free energy of binding was calculated from the sum of the intermolecular and the torsional free energies and consequently converted into an inhibitory constant (ki) according to Hess’s law (fig. 5A). The docked complex forms two hydrogen bonds with Gly112 and Gly170 with bond lengths of 2.034 Å and 2.14 Å, respectively (fig. 5B). Surface representation in the figure clearly shows that the ligand was bound well into the hydrophobic pocket of SVMP (fig. 5C). The other amino acid residues such as ILE111, ILE 142, HIS145, GLU146, HIS 149, ILE168, THR 172 and ILE173 actively participate in van der Waals interactions (scaling factor = 1.00 Å), which is also the part of the zinc-binding motif.
To check the reliability of the docking studies, the obtained results were compared with the co-crystallized structure of SVMP (PDBID-2DW0) and ligand GM6001 [3-(n-hydroxycarboxamido)-2-isobutylpropanoyl-trp-methylamide] family. To predict the binding mode, redocking the GM6001 with the bothropasin structure was performed and results showed reasonable binding modes. The Autodock binding energy score for the docked complex was found to be −9.60 (kcal/mol), which is less than the binding energy for DFD. The superimposed co-crystal structure binding mode and docked binding mode are shown in fig. 6. The ligand GM6001 covalently bound to the three histidine residues such as His333, His337 and His343. In addition, the ligand forms a hydrogen bond with the amide groups of ILE361 and ILE299 residues (fig. 6A). In contrast, DFD binds to the hydrophobic pocket with the ki of 19.26 × 10−9 (kcal/mol) and forms a strong hydrogen bond with His333 (fig. 6B).
Inhibition of PLA2 activity.
The DFD significantly neutralized the indirect haemolytic activity and inhibition was found to be dose dependent (fig. 7). Both CA and ECL venoms produced 77% and 68% lysis, respectively, when compared to control (only water) on RBCs (Red Blood Cells). The percentage of lysis by venom was considered to be 100%. Complete inhibition of CA venom-induced PLA2 activity was observed at a concentration of 400 μg, and the ratio of venom to DFD at this point was found to be 1:40 (w/w), whereas a ratio of 1:50 abolishes the PLA2 activity of ECL venom. In contrast, EC, EO, ECS venoms did not induce indirect haemolytic activity.
Viper bites are one of the important causes of morbidity and mortality in the tropical and subtropical regions of the world and considered to be a neglected issue of public health. Amongst the bitten cases, about 75% of the victims suffer from brutal local effects including haemorrhage and tissue necrosis [22,23]. These remain as frequent and challenging complications and lead to permanent disability in many cases . The mounting knowledge on the snake venom-induced local effects has confirmed the participation of SVMPs . Because of the wide variety of SVMP-induced pathophysiological activities and inability of antivenoms in neutralizing local effects, these enzymatic toxins have received vast attention by venom researchers and medical practitioners [7,24]. The effective inhibition of these enzymes is being considered as the rate-limiting step in the significant reduction in local manifestations that reduces the diffusion of toxins and thereby extends the survival time of a victim. Therefore, the development of therapeutic strategies to neutralize SVMP-induced local effects and blocking the toxins swiftly after their entry constitutes an appropriate progress in the betterment of snakebite management. It has been assumed that the clinically approved low molecular mass bioactive agents with the superior diffusion rate to the affected tissues should be advantageous for this task. However, the information available in the literature regarding the efficacy of molecules to neutralize snake venom-induced local effects is inadequate.
Several studies reported the anti-haemorrhagic properties of small molecular mass molecules including chelating agents, plant phenolics and synthetic matrix metalloproteinase inhibitors (MMPIs). The cation (Zn2+ and Ca2+) chelating properties of these agents are considered to be the most probable mechanism for the inhibition of venom-induced proteolytic and haemorrhagic activities [8–12]. The inhibitory concentrations of DFD when tested against haemorrhagic activities of viper venoms are in the mM range, similar to the inhibitory concentration of chelating compounds such as EGTA, EDTA, CaNa2EDTA, TPEN, BAPTA, clodronate and doxycycline [9,10]. In contrast, batimastat, a hydroxamate peptidomimetic, was found to be effective at the μM range  because it mimics the cleavage site of collagen and its hydroxamate moiety chelates the zinc, and this could be the possible reason for the effective inhibition of haemorrhage compared to chelating agents. The result suggested that DFD-induced inhibition is not because of metal ion chelation as observed in spectral studies. However, molecular docking studies suggest high affinity of DFD towards the catalytic site of SVMP without chelating Zn2+. Recently, Leanpolchareanchai et al.  demonstrated the inhibition of hydrolytic enzymes and haemorrhagic activities of Calloselasma rhodostoma (CR) and Naja naja kaouthia (NK) venoms by MSKE (Thai mango seed kernels extract) and its isolated phenolic principles. The docking studies revealed the binding orientation of the phenolics in the binding pocket of rhodostoxin (P-I class SVMP) of CR and kaouthiagin (P-III class SVMP) of NK venoms. The phenolics formed hydrogen bonds with the three active histidine (His142, His146 and His152) residues in the conserved zinc-binding motif (HebxHxbGbxHD) and were able to chelate zinc which could result in the inhibition of the SVMPs.
Furthermore, DFD dose dependently inhibited the PLA2 activities of CA and ECL venoms. PLA2 enzymes are the most abundant group of enzymatic toxins in snake venoms, and they induce a wide spectrum of pharmacological activities such as hypotension, neurotoxicity, myotoxicity, cytotoxicity, oedema, cardiotoxicity, platelet aggregation (inhibition/activation) and coagulation (pro/anti) . A number of PLA2 inhibitors (PLIs) have been identified and characterized from marine organisms, microorganisms, snake’s sera/plasma and medicinal plants [13,27,28]. In addition, synthetic compounds and their derivatives including benzoyl phenyl benzoate and 1,2,3-trizole analogues have been reported as anti-PLA2 agents [29,30]. Recentlly, Oruch et al.  showed the inhibition of thrombin-induced platelet PLA2 activation by citalopram up to 24–35% and concluded that inhibition could be attributed to intercalation between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for PLA2. The dose-dependent inhibitory effect of the DFD against viper venom could be attributed to the modulation of the catalytic activity of PLA2 and not by Ca2+ chelation as suggested by spectral data. These results suggest that the ability of DFD to inhibit a wide spectrum of pharmacological/toxic effects could be a consequence of the primary inactivation of PLA2 of viper venoms.
Although several inhibitors of SVMPs have been identified and their efficacy towards locally acting toxins/enzymes using experimental mice/rat model has been evaluated, it cannot be extrapolated simplistically to human cases. However, clinically approved drugs with neutralizing capacities could be used for snakebite management. In conclusion, our observations demonstrate that the citalopram derivative neutralizes haemorrhagic and PLA2 activities of selected viper venoms. The compound was able to inhibit local haemorrhage induced by five selected viper venoms and neutralized the haemorrhagic activities of studied EC venom in both pre-incubation as well as independent injection assays. It would be interesting to know the efficacy of DFD in neutralizing myonecrosis and systemic haemorrhage induced by SVMPs and PLA2 of viper venoms. In addition, the potential use of a combination of SVMP and PLA2 inhibitors would complement the antivenom as an alternative therapy for snakebites.
This work was supported in part by UGC, New Delhi, for financial support under the major research project wide No: F. No. 38-220/2009 (SR). SNS thanks JSS Mahavidyapeetha for providing facilities to conduct this research work. MH thanks UGC, New Delhi, for the UGC Junior Research Fellowship.
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