Toxicon 93 (2015) 68e78
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom Ankanahalli N Nanjaraj Urs a, Manjunath Yariswamy a, Chandrasekaran Ramakrishnan c, Vikram Joshi a, Kanve Nagaraj Suvilesh a, Mysore Natarajan Savitha a, Devadasan Velmurugan b, c, Bannikuppe Sannanayak Vishwanath a, * a
Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka, India Bioinformatics Infrastructure Facility, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India c Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 September 2014 Received in revised form 25 October 2014 Accepted 4 November 2014 Available online 8 November 2014
Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn2þ) chelating agents; N,N,N0 ,N0 tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn2þ and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 mM) compared to DTPA and TTD. The specificity of TPEN towards Zn2þ was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn2þ chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Complementary therapy Hemorrhage Echis carinatus venom metalloproteases Local tissue damage Myotoxicity Zinc chelating agents
1. Introduction Chelation therapy is an important strategy employed to eliminate toxic heavy metals from the body. In addition, chelating agents are vital in restoring the physiological levels of metalloenzymes,
Abbreviations: DTPA, diethylene triamine pentaacetic acid; ECVMPs, Echis carinatus venom metalloproteases; MMPs, matrix metalloproteases; PPP, platelet poor plasma; TPEN, N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine; TTD, tetraethyl thiuram disulfide. * Corresponding author. Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka 570 006, India. E-mail address: [email protected] (B.S. Vishwanath). http://dx.doi.org/10.1016/j.toxicon.2014.11.224 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
particularly, the matrix metalloproteases (MMPs), as their dysregulated activity reflects in debilitating conditions such as cancer and arthritis (Gong et al., 2014; Shian et al., 2003). Similarly, upon envenomation, entry of snake venom metalloproteases (SVMPs) into victims causes dire consequences of local tissue damage such as hemorrhage, myonecrosis and in severe cases gangrene. Although anti-snake venoms are effective in neutralizing the snake venom induced systemic toxicity, they fail to neutralize local tissue damages often leading to permanent debilitated condition (Escalante et al., 2011; Markland and Swenson, 2013). In view of overcoming these persistent debilitating conditions, pharmacologically approved chelating agents such as ethylene diamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and 1,
A.N. Nanjaraj Urs et al. / Toxicon 93 (2015) 68e78
10-phenanthroline have been extensively studied for inhibition of SVMPs in addition to their role in regulating endogenous MMPs (Gowda et al., 2011; Howes et al., 2007; Kumar et al., 2010; Thompson et al., 2012). EDTA, EGTA and 1, 10 phenanthroline are non-specific divalent metal ion chelators and are very effective in neutralizing the activity of SVMPs in vitro. However, their nonspecific binding with physiologically vital divalent metal ions, particularly calcium, poses an obstacle for their pharmacological use (Pereanez et al., 2013). SVMPs being metzincin family proteases, possess a catalytically functional zinc (Zn2þ) at the active site bound to conserved Zn2þ-binding motif (Takeda et al., 2012). Chelating this Zn2þ metal ion by specific Zn2þ chelators rather than non-specific divalent metal ion chelators is more effective in the management of local toxicity as they can serve as adjunctive therapeutic molecules to aid antivenom therapy by limiting local tissue damage. In these lines, a high affinity membrane permeable Zn2þ chelator e TPEN (N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane1,2-diamine); an extracellular Zn2þ chelating agent e DTPA (Diethylene triamine pentaacetic acid) and an intracellular Zn2þ chelating agent e TTD (Tetraethyl thiuram disulfide) were selected for the study. Present study aims at evaluating the protective effects of afore said Zn2þ chelating agents against Echis carinatus venom (ECV) induced local tissue damage. Further, the specificity offered by these agents is an added advantage and will be of potential therapeutic value in management of viper snakebites.
2. Materials and methods 2.1. Venom Lyophilized powder of E. carinatus venom (ECV) was purchased from Irula Snake-Catchers Co-operative Society Ltd., (Chennai, India). Required amount of venom was re-dissolved in saline and centrifuged at 6000 g for 10 min to remove debris. Protein content of crude venom was determined according to the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. Aliquots were kept at 4 C until further use.
2.2. Chemicals N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD), gelatin, bovine fibrinogen, papain and bromelain were purchased from Sigma Aldrich (Saint Louis, USA). Fibri-prest® (thrombin reagent) was purchased from Diagnostica Stago Inc. (Paris, France). Casein, BSA, ethanol (HPLC grade), water (HPLC grade), dimethyl sulfoxide (DMSO), zinc chloride (ZnCl2), calcium chloride (CaCl2), phenyl methyl sulphonyl fluoride (PMSF), and iodo acetic acid (IAA) were purchased from Sisco Research Laboratories (Mumbai, India). Polyvalent equine anti-snake venom serum (ASV) specific towards venom of Naja naja, Bungarus caeruleus, Daoia russelii and E. carinatus snakes of India (batch number: A5310035; expiry date: 09/14) was kind gift from Bharat Serums and Vaccines Ltd., (Mumbai, India). All the other chemicals and reagents used in this study were of analytical grade.
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2.3. Animals Swiss Albino mice (either gender; 25e30 g) were obtained from Central Animal House Facility, University of Mysore (UOM), Mysore, India. Animal care and handling were in compliance with National Regulations for Animal Research and the experiments were performed according to the protocols reviewed by the Institutional Animal Ethical Committee, UOM, Mysore (No: UOM/IAEC/25/2011). 2.4. Human plasma Blood was drawn from healthy human volunteers (20e30 years; with consent), mixed with 3.2% trisodium citrate (9:1 v/v) and centrifuged at 250 g for 15 min. The plasma obtained was pooled and used as platelet poor plasma (PPP) for coagulation and fibrinolytic assays. The experiments conducted were in compliance with the protocols approved by the Institutional Human Ethical Committee, UOM, Mysore (IHEC-UOM No.62/Ph.D/2011-12). 2.5. Proteolytic activity Proteolytic activities of ECV, trypsin, and bromelain were assayed according to the method of Murata et al. (1963) using casein as a substrate. For inhibition studies, similar reactions were performed after pre-incubating 25 mg of venom with various concentrations of Zn2þ chelating agents (TPEN, DTPA and TTD) for 10 min at 37 C. Similarly, inhibition studies of trypsin and bromelain were performed after pre-incubating 500 mg of respective proteins with 1 mM chelating agents for 10 min at 37 C. One unit of enzyme activity was defined as the amount of enzyme required to increase an absorbance of 0.01 at 660 nm/h at 37 C. The proteolytic activity of ECV in the absence of inhibitors was considered as 100%. 2.6. UV-VIS spectral study Specificity of Zn2þ chelating nature of inhibitors was evaluated by determining the changes in their absorbance spectra with ZnCl2 and CaCl2. The complex formation between the chelating agents and metal ions was analyzed by UV spectral scanning (l ¼ 190e300 nm) using BioMate™ 3S UV-VIS Spectrophotometer (Waltham, USA). TPEN, DTPA and TTD (0.1 mM) were incubated with various concentrations of divalent metal ions (0.1 mMe10 mM) in 1 ml of HPLC grade water. The absorption spectra of the chelating agents, divalent metal ions, chelating agent and metal ion complexes were compared to determine their specificity. 2.7. Gelatinolytic activity Gelatinolytic activity of ECV was determined by substrate gel assay according to the method of Heussen and Dowdle (1980) with slight modifications. For inhibition studies, similar experiments were carried out with 10 mg ECV pre-incubated with various concentrations of TPEN, DTPA and TTD for 10 min prior to electrophoresis. Appropriate inhibitor and solvent controls were also processed in similar manner. 2.8. In vivo inhibition studies Inhibition studies were carried out by ‘co-injection method’ where ECV and various concentrations of Zn2þ chelators were pre-
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A.N. Nanjaraj Urs et al. / Toxicon 93 (2015) 68e78
incubated for different time points before administration and ‘independent injection method’ where, Zn2þ chelators were administered at different doses and time points after the administration of ECV. 2.8.1. Hemorrhagic activity Experimental animals were randomly divided into different groups (n ¼ 3) and hemorrhagic activity was determined as described in Gowda et al. (2011). Inhibition studies were carried out by both co-injection and independent injection methods. In coinjection experiments, various concentrations of TPEN, DTPA, and TTD were incubated with 3 mg venom for 10 min with appropriate inhibitor and solvent controls. In independent injection studies, 40 ml containing 5 mM TPEN or DTPA or TTD was injected 15 min following 3 mg of venom administration. Saline and 3 mg of venom served as negative and positive controls respectively. As a positive inhibitor control 1:100 w/w ASV (mg of ECV/mg of ASV) was administered. Area of hemorrhagic spot was measured using graph sheet and inhibition of hemorrhagic activity was measured in terms of decreased area of hemorrhagic spot in comparison to venom injected hemorrhagic spot. Further, the skin tissues were processed for histopathology. 2.8.2. Myotoxicity Myotoxicity studies were performed according to the method of Gutierrez et al. (1990). A group of mice (n ¼ 3) were injected (i. m.) with 5 mg of ECV in a total volume of 40 ml with saline and the mice which received saline alone served as controls. After 3 h, mice were anaesthetized; blood samples were collected by cardiac puncture and processed to obtain serum. A thin slice of skeletal muscle tissue from venom injected site was surgically removed and processed for histological studies. Initial screening of the chelating agents for inhibition of myotoxicity was carried out by co-injecting with ECV. Further, based on the efficacy 5, 10, 20 mM of TPEN and 1:100 w/w ASV were subjected for independent injection studies (15 min after venom administration), with appropriate inhibitor and solvent controls. Serum (1:20 v/v with saline) was used for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) activities according to manufacturer's instructions (AGAPPE kit, Ernakulam, India). Activity was expressed as U/L. 2.8.3. Histopathological studies Mice skin and thigh muscle from control and test groups were dissected out and 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 2 m thick sections were prepared using microtome (Leica, Solms, Germany). The sections were stained with hematoxylin and eosin stain and were observed under Axio Imager A2 Microscope with LED e Zeiss (Oberkochen, Germany) and photographed. 2.9. Fibrinogenolytic activity Fibrinogenolytic activity was performed according to the method of Ouyang and Teng (1976). For inhibition studies, similar experiments were carried out by pre-incubating 2 mg ECV with various concentrations of TPEN, DTPA and TTD for 10 min at 37 C. The degradation pattern of fibrinogen subunits was observed following electrophoresis on 10% SDS PAGE under reducing conditions and staining with Coomassie brilliant blue (Laemmli, 1970).
2.10. Plasma clot hydrolyzing activity Human plasma clot hydrolyzing activity was performed according to the method of Rajesh et al. (2007) with slight modifications. The cleavage pattern of washed plasma clot by ECV was analyzed by 10% SDS-PAGE (Laemmli, 1970). For inhibition studies, similar procedure was followed after pre-incubating 10 mg venom with various concentrations of TPEN, DTPA, and TTD for 10 min at 37 C. 2.11. Coagulant activity Plasma re-calcification time was determined according to the method of Condrea et al. (1983). Pre-warmed PPP (200 ml) was mixed with 20 ml of 10 mM Tris HCl (pH 7.4) for control, 20 ml 10 mM Tris HCl containing 1 mg of ECV for test. Samples were incubated for 1 min at 37 C and clot formation was initiated by adding 20 ml of 250 mM CaCl2. For inhibition studies, similar experiments were carried out after pre-incubating 1 mg of ECV with various concentrations of TPEN, TTD and DTPA for 2 min at 37 C. Inhibition of coagulant activity was expressed as percent recovery of normal coagulation time. Specificity of chelating agents towards metalloproteases was confirmed by thrombin time. Further, the chelating agents were supplemented to PPP prior to re-calcification time to evaluate the effect of these agents on normal re-calcification time and were compared with that of PPP alone. 2.12. Computational studies Structure of Zn2þ ECVMPs was modeled using Modeller (version: 9v7) software (Sali and Blundell, 1993). Sequence of the target protein was retrieved from Uniprot database (Q90495) and the coordinates of template structure (PDB ID: 2DW2) (Igarashi et al., 2007) with close homology was retrieved from protein data bank (PDB) (Berman et al., 2000). Target-template alignment was carried out to facilitate building of 3D structure of Zn2þ ECVMPs based on satisfaction of partial restraints. Out of 20 models generated, one model was selected based on the Discrete Optimized Protein Energy (DOPE) score and the model was validated using Ramachandran plot (Ramachandran et al., 1963). The model was then processed using protein preparation wizard, optimized with Root Mean Square Deviation (RMSD) cut off 0.3 Å and then used for docking analysis. The specific Zn2þ chelating agents TPEN (pubchem id: CID 5519), DTPA (pubchem id: CID 3053), and TTD (pubchem id: CID 3117) were selected for docking analysis based on in vitro and in vivo experimental data. The compounds were minimized using steepest descent and conjugate gradient methods and docked at the ECVMPs active site (Leu114, His148, 152, 158, Glu176, Pro179) using induced fit docking (IFD) (Sherman et al., 2006) module to understand their mode of binding at active site. The best pose was then chosen based on glide score, glide energy and the interactions of ligand with active site residues. The interactions between protein and ligand were portrayed using Ligplot (Wallace et al., 1995) software package. 2.13. Data analysis The results of the experiments are expressed as mean ± SD of three independent experiments performed in triplicates. Statistical analyses were carried out using Student's t-test. The comparison between the groups were considered significant if p 0.05. Data were analyzed using the statistical package GraphPad Prism® (La Jolla, USA).
A.N. Nanjaraj Urs et al. / Toxicon 93 (2015) 68e78
3. Results SVMPs are mainly accountable for local tissue damage in viper bites. In this study we have made an attempt to evaluate the neutralization abilities of three specific Zn2þ chelating agents against ECVMPs induced hemorrhage and myotoxicity in treatment mode and the results obtained are presented below.
3.1. Inhibition of proteolytic activity of ECVMPs by TPEN, DTPA and TTD ECV is known for its strong proteolytic nature and 90% of this activity is contributed by metalloproteases. We examined the effect of three specific Zn2þ chelators TPEN, DTPA and TTD on in vitro ECV proteolytic activity. The chelating agents significantly inhibited the proteolytic activity of ECV towards casein in a concentration dependent manner with IC50 values of 6.7 mM for TPEN, 44.5 mM for DTPA and 100 mM for TTD respectively (Fig. 1); TPEN was 6.6 and 14.9 times more potent than DTPA and TTD in inhibiting the proteolytic activity of ECV respectively. The specificity of chelating agents was confirmed by evaluating the effect of these inhibitors towards serine (trypsin) and cysteine (bromelain) proteases. TPEN, DTPA and TTD failed to inhibit the activities of either protease, indicating their specificity towards metalloproteases (Fig. 2). Further, specificity of chelating agents towards Zn2þ was confirmed by UV-VIS spectral studies. The spectral shift of Zn2þ with protease inhibitors confirmed that chelating agents preferentially chelate Zn2þ (Fig. 3). Based on these in vitro findings, in silico studies were conducted for TPEN, DTPA and TTD to determine their interacting sites in ECVMPs. TPEN and DTPA interacted strongly with ECVMPs upon docking studies however, on repeated trials TTD failed to interact with ECVMPs. Both TPEN and DTPA exhibited different interaction and the glide score of TPEN (9.30 ± 0.19) was 1.6 times better than that of DTPA (5.5 ± 0.20). Both TPEN and DTPA sequestered active site Zn2þ; however with active site amino acids, TPEN showed strong hydrophobic interaction whereas DTPA interacted by forming hydrogen bond (Fig. 4). Low glide score of TPEN towards active site Zn2þ in docking analysis was evidenced by its strong in vitro inhibitory effect towards ECVMPs.
Fig. 1. Inhibition of caseinolytic activity of ECVMPs by TPEN, DTPA and TTD: Reaction mixture (1 ml) contained 0.4 ml of casein (2%) in 0.2 M TriseHCl buffer pH 8.5 was incubated for 150 min at 37 ºC with 25 mg venom and various concentrations of specific Zn2þ chelating agents ranging from 10 pM to 100 mM.TPEN: N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine; DTPA: Diethylene triamine pentaacetic acid; TTD: Tetraethyl thiuram disulfide
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3.2. Inhibition of pro-coagulant activity of ECVMPs by TPEN, DTPA and TTD The strong proteolytic activity of ECV reflects in hemostatic alterations, particularly the pronounced pro-coagulant effect, evident by coagulation of citrated plasma even without addition of calcium chloride. The effects of TPEN, DTPA and TTD on ECVMPs induced pro-coagulant action was evaluated by plasma re-calcification time. The pro-coagulant nature of ECV was abolished and normal clotting time was restored by all Zn2þ chelating agents and the effect was concentration dependent. TPEN restored the same at 30 mM (98.5 ± 5%; p < 0.0001); whereas in case of DTPA and TTD, similar effect was achieved at 100 mM concentrations respectively. Further, chelating agents failed to inhibit procoagulant activity of thrombin. This inability of chelating agents towards thrombin inhibition, a vital serine protease of coagulation cascade, confirms their noninterference in vital physiological pathways mediated by serine proteases (Fig. 5). Further, the chelating agents did not affect normal re-calcification time, indicating their negligible interference with calcium homeostasis. 3.3. Inhibition of fibrino(geno)lytic activity of ECVMPs by TPEN, DTPA and TTD As a secondary effect to pronounced pro-coagulant nature, ECVMPs also mediate fibrin(ogen) degrading activities, resulting in exaggerated hemostatic alterations. ECVMPs specifically hydrolyze Aa, Bb chains of fibrinogen (Factor I); similarly they also selectively degrade a polymer and a chain of plasma fibrin (Factor Ia). The inhibitory effects of TPEN, DTPA and TTD on ECVMPs induced fibrino(geno)lytic activities were evaluated using electrophoresis. The inhibitions were concentration dependent and complete inhibition of fibrinogenolytic activity was observed at 0.25 mM for TPEN, 5 mM for DTPA and TTD. TPEN was 20 times more potent than DTPA and TTD towards the inhibition of fibrinogenolytic activity (Fig. 6A). Similarly 0.25 mM of TPEN, 2 mM of DTPA and 8 mM of TTD completely inhibited fibrinolytic activity and TPEN was 8 and 32 times potent than DTPA and TTD respectively towards inhibition of fibrinolytic activity of ECVMPs (Fig. 6B). 3.4. Inhibition of hemorrhage and myotoxicity of ECVMPs by TPEN, DTPA and TTD The action of ECV is not only restricted to hemostatic alterations, but also result in the induction of hemorrhage and subsequent progression to myotoxicity. Onset of hemorrhage is due to degradation of ECM and basement membrane proteins surrounding blood vessels by metalloproteases. ECVMPs induced hemorrhagic lesion was obtained 3 h after intra dermal injection of venom. The minimum hemorrhagic dose (MHD) was found to be 1 mg. For inhibition studies 3 MHD of venom was separately pre-incubated with specific Zn2þ chelating agents. Upon pre-incubation, TPEN, DTPA and TTD showed concentration dependent inhibition of hemorrhage and complete inhibition was observed at 0.2 mM for TPEN, 0.5 mM for DTPA and 2 mM for TTD. Based on the results of pre-incubation studies, a fixed dose (5 mM) of inhibitors was used for neutralization studies in treatment mode (15 min after venom administration). Among the inhibitors tested, TPEN showed maximum inhibition (65.5 ± 8%; p < 0.001), with 1.8 ± 0.5 fold (34.6 ± 10.5%; p ¼ 0.002) and 2.2 ± 0.8 fold (30 ± 10%; p ¼ 0.005) potent inhibition in comparison with DTPA and TTD respectively. In addition, TPEN was 1.7 ± 0.5 fold potent than ASV (39 ± 18.5%; p ¼ 0.001) in inhibiting the hemorrhagic activity of ECVMPs (Fig. 7). Further, upon independent injection (15 min after venom administration), 5,10, and 20 mM TPEN showed significant inhibition of
Fig. 2. Comparison of inhibitory potential of Zn2þ chelators towards (a) serine and (b) cysteine proteases: Reaction mixture (1 ml) contained 0.4 ml of casein (2%) in 0.2 M TriseHCl buffer pH 8.5 was incubated for 150 min at 37 ºC with 500 mg trypsin/bromelain þ 1 mM concentration of different protease inhibitors. ***p
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom Ankanahalli N Nanjaraj Urs a, Manjunath Yariswamy a, Chandrasekaran Ramakrishnan c, Vikram Joshi a, Kanve Nagaraj Suvilesh a, Mysore Natarajan Savitha a, Devadasan Velmurugan b, c, Bannikuppe Sannanayak Vishwanath a, * a
Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka, India Bioinformatics Infrastructure Facility, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India c Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 September 2014 Received in revised form 25 October 2014 Accepted 4 November 2014 Available online 8 November 2014
Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn2þ) chelating agents; N,N,N0 ,N0 tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn2þ and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 mM) compared to DTPA and TTD. The specificity of TPEN towards Zn2þ was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn2þ chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Complementary therapy Hemorrhage Echis carinatus venom metalloproteases Local tissue damage Myotoxicity Zinc chelating agents
1. Introduction Chelation therapy is an important strategy employed to eliminate toxic heavy metals from the body. In addition, chelating agents are vital in restoring the physiological levels of metalloenzymes,
Abbreviations: DTPA, diethylene triamine pentaacetic acid; ECVMPs, Echis carinatus venom metalloproteases; MMPs, matrix metalloproteases; PPP, platelet poor plasma; TPEN, N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine; TTD, tetraethyl thiuram disulfide. * Corresponding author. Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka 570 006, India. E-mail address: [email protected] (B.S. Vishwanath). http://dx.doi.org/10.1016/j.toxicon.2014.11.224 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
particularly, the matrix metalloproteases (MMPs), as their dysregulated activity reflects in debilitating conditions such as cancer and arthritis (Gong et al., 2014; Shian et al., 2003). Similarly, upon envenomation, entry of snake venom metalloproteases (SVMPs) into victims causes dire consequences of local tissue damage such as hemorrhage, myonecrosis and in severe cases gangrene. Although anti-snake venoms are effective in neutralizing the snake venom induced systemic toxicity, they fail to neutralize local tissue damages often leading to permanent debilitated condition (Escalante et al., 2011; Markland and Swenson, 2013). In view of overcoming these persistent debilitating conditions, pharmacologically approved chelating agents such as ethylene diamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and 1,
A.N. Nanjaraj Urs et al. / Toxicon 93 (2015) 68e78
10-phenanthroline have been extensively studied for inhibition of SVMPs in addition to their role in regulating endogenous MMPs (Gowda et al., 2011; Howes et al., 2007; Kumar et al., 2010; Thompson et al., 2012). EDTA, EGTA and 1, 10 phenanthroline are non-specific divalent metal ion chelators and are very effective in neutralizing the activity of SVMPs in vitro. However, their nonspecific binding with physiologically vital divalent metal ions, particularly calcium, poses an obstacle for their pharmacological use (Pereanez et al., 2013). SVMPs being metzincin family proteases, possess a catalytically functional zinc (Zn2þ) at the active site bound to conserved Zn2þ-binding motif (Takeda et al., 2012). Chelating this Zn2þ metal ion by specific Zn2þ chelators rather than non-specific divalent metal ion chelators is more effective in the management of local toxicity as they can serve as adjunctive therapeutic molecules to aid antivenom therapy by limiting local tissue damage. In these lines, a high affinity membrane permeable Zn2þ chelator e TPEN (N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane1,2-diamine); an extracellular Zn2þ chelating agent e DTPA (Diethylene triamine pentaacetic acid) and an intracellular Zn2þ chelating agent e TTD (Tetraethyl thiuram disulfide) were selected for the study. Present study aims at evaluating the protective effects of afore said Zn2þ chelating agents against Echis carinatus venom (ECV) induced local tissue damage. Further, the specificity offered by these agents is an added advantage and will be of potential therapeutic value in management of viper snakebites.
2. Materials and methods 2.1. Venom Lyophilized powder of E. carinatus venom (ECV) was purchased from Irula Snake-Catchers Co-operative Society Ltd., (Chennai, India). Required amount of venom was re-dissolved in saline and centrifuged at 6000 g for 10 min to remove debris. Protein content of crude venom was determined according to the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. Aliquots were kept at 4 C until further use.
2.2. Chemicals N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD), gelatin, bovine fibrinogen, papain and bromelain were purchased from Sigma Aldrich (Saint Louis, USA). Fibri-prest® (thrombin reagent) was purchased from Diagnostica Stago Inc. (Paris, France). Casein, BSA, ethanol (HPLC grade), water (HPLC grade), dimethyl sulfoxide (DMSO), zinc chloride (ZnCl2), calcium chloride (CaCl2), phenyl methyl sulphonyl fluoride (PMSF), and iodo acetic acid (IAA) were purchased from Sisco Research Laboratories (Mumbai, India). Polyvalent equine anti-snake venom serum (ASV) specific towards venom of Naja naja, Bungarus caeruleus, Daoia russelii and E. carinatus snakes of India (batch number: A5310035; expiry date: 09/14) was kind gift from Bharat Serums and Vaccines Ltd., (Mumbai, India). All the other chemicals and reagents used in this study were of analytical grade.
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2.3. Animals Swiss Albino mice (either gender; 25e30 g) were obtained from Central Animal House Facility, University of Mysore (UOM), Mysore, India. Animal care and handling were in compliance with National Regulations for Animal Research and the experiments were performed according to the protocols reviewed by the Institutional Animal Ethical Committee, UOM, Mysore (No: UOM/IAEC/25/2011). 2.4. Human plasma Blood was drawn from healthy human volunteers (20e30 years; with consent), mixed with 3.2% trisodium citrate (9:1 v/v) and centrifuged at 250 g for 15 min. The plasma obtained was pooled and used as platelet poor plasma (PPP) for coagulation and fibrinolytic assays. The experiments conducted were in compliance with the protocols approved by the Institutional Human Ethical Committee, UOM, Mysore (IHEC-UOM No.62/Ph.D/2011-12). 2.5. Proteolytic activity Proteolytic activities of ECV, trypsin, and bromelain were assayed according to the method of Murata et al. (1963) using casein as a substrate. For inhibition studies, similar reactions were performed after pre-incubating 25 mg of venom with various concentrations of Zn2þ chelating agents (TPEN, DTPA and TTD) for 10 min at 37 C. Similarly, inhibition studies of trypsin and bromelain were performed after pre-incubating 500 mg of respective proteins with 1 mM chelating agents for 10 min at 37 C. One unit of enzyme activity was defined as the amount of enzyme required to increase an absorbance of 0.01 at 660 nm/h at 37 C. The proteolytic activity of ECV in the absence of inhibitors was considered as 100%. 2.6. UV-VIS spectral study Specificity of Zn2þ chelating nature of inhibitors was evaluated by determining the changes in their absorbance spectra with ZnCl2 and CaCl2. The complex formation between the chelating agents and metal ions was analyzed by UV spectral scanning (l ¼ 190e300 nm) using BioMate™ 3S UV-VIS Spectrophotometer (Waltham, USA). TPEN, DTPA and TTD (0.1 mM) were incubated with various concentrations of divalent metal ions (0.1 mMe10 mM) in 1 ml of HPLC grade water. The absorption spectra of the chelating agents, divalent metal ions, chelating agent and metal ion complexes were compared to determine their specificity. 2.7. Gelatinolytic activity Gelatinolytic activity of ECV was determined by substrate gel assay according to the method of Heussen and Dowdle (1980) with slight modifications. For inhibition studies, similar experiments were carried out with 10 mg ECV pre-incubated with various concentrations of TPEN, DTPA and TTD for 10 min prior to electrophoresis. Appropriate inhibitor and solvent controls were also processed in similar manner. 2.8. In vivo inhibition studies Inhibition studies were carried out by ‘co-injection method’ where ECV and various concentrations of Zn2þ chelators were pre-
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incubated for different time points before administration and ‘independent injection method’ where, Zn2þ chelators were administered at different doses and time points after the administration of ECV. 2.8.1. Hemorrhagic activity Experimental animals were randomly divided into different groups (n ¼ 3) and hemorrhagic activity was determined as described in Gowda et al. (2011). Inhibition studies were carried out by both co-injection and independent injection methods. In coinjection experiments, various concentrations of TPEN, DTPA, and TTD were incubated with 3 mg venom for 10 min with appropriate inhibitor and solvent controls. In independent injection studies, 40 ml containing 5 mM TPEN or DTPA or TTD was injected 15 min following 3 mg of venom administration. Saline and 3 mg of venom served as negative and positive controls respectively. As a positive inhibitor control 1:100 w/w ASV (mg of ECV/mg of ASV) was administered. Area of hemorrhagic spot was measured using graph sheet and inhibition of hemorrhagic activity was measured in terms of decreased area of hemorrhagic spot in comparison to venom injected hemorrhagic spot. Further, the skin tissues were processed for histopathology. 2.8.2. Myotoxicity Myotoxicity studies were performed according to the method of Gutierrez et al. (1990). A group of mice (n ¼ 3) were injected (i. m.) with 5 mg of ECV in a total volume of 40 ml with saline and the mice which received saline alone served as controls. After 3 h, mice were anaesthetized; blood samples were collected by cardiac puncture and processed to obtain serum. A thin slice of skeletal muscle tissue from venom injected site was surgically removed and processed for histological studies. Initial screening of the chelating agents for inhibition of myotoxicity was carried out by co-injecting with ECV. Further, based on the efficacy 5, 10, 20 mM of TPEN and 1:100 w/w ASV were subjected for independent injection studies (15 min after venom administration), with appropriate inhibitor and solvent controls. Serum (1:20 v/v with saline) was used for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) activities according to manufacturer's instructions (AGAPPE kit, Ernakulam, India). Activity was expressed as U/L. 2.8.3. Histopathological studies Mice skin and thigh muscle from control and test groups were dissected out and 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 2 m thick sections were prepared using microtome (Leica, Solms, Germany). The sections were stained with hematoxylin and eosin stain and were observed under Axio Imager A2 Microscope with LED e Zeiss (Oberkochen, Germany) and photographed. 2.9. Fibrinogenolytic activity Fibrinogenolytic activity was performed according to the method of Ouyang and Teng (1976). For inhibition studies, similar experiments were carried out by pre-incubating 2 mg ECV with various concentrations of TPEN, DTPA and TTD for 10 min at 37 C. The degradation pattern of fibrinogen subunits was observed following electrophoresis on 10% SDS PAGE under reducing conditions and staining with Coomassie brilliant blue (Laemmli, 1970).
2.10. Plasma clot hydrolyzing activity Human plasma clot hydrolyzing activity was performed according to the method of Rajesh et al. (2007) with slight modifications. The cleavage pattern of washed plasma clot by ECV was analyzed by 10% SDS-PAGE (Laemmli, 1970). For inhibition studies, similar procedure was followed after pre-incubating 10 mg venom with various concentrations of TPEN, DTPA, and TTD for 10 min at 37 C. 2.11. Coagulant activity Plasma re-calcification time was determined according to the method of Condrea et al. (1983). Pre-warmed PPP (200 ml) was mixed with 20 ml of 10 mM Tris HCl (pH 7.4) for control, 20 ml 10 mM Tris HCl containing 1 mg of ECV for test. Samples were incubated for 1 min at 37 C and clot formation was initiated by adding 20 ml of 250 mM CaCl2. For inhibition studies, similar experiments were carried out after pre-incubating 1 mg of ECV with various concentrations of TPEN, TTD and DTPA for 2 min at 37 C. Inhibition of coagulant activity was expressed as percent recovery of normal coagulation time. Specificity of chelating agents towards metalloproteases was confirmed by thrombin time. Further, the chelating agents were supplemented to PPP prior to re-calcification time to evaluate the effect of these agents on normal re-calcification time and were compared with that of PPP alone. 2.12. Computational studies Structure of Zn2þ ECVMPs was modeled using Modeller (version: 9v7) software (Sali and Blundell, 1993). Sequence of the target protein was retrieved from Uniprot database (Q90495) and the coordinates of template structure (PDB ID: 2DW2) (Igarashi et al., 2007) with close homology was retrieved from protein data bank (PDB) (Berman et al., 2000). Target-template alignment was carried out to facilitate building of 3D structure of Zn2þ ECVMPs based on satisfaction of partial restraints. Out of 20 models generated, one model was selected based on the Discrete Optimized Protein Energy (DOPE) score and the model was validated using Ramachandran plot (Ramachandran et al., 1963). The model was then processed using protein preparation wizard, optimized with Root Mean Square Deviation (RMSD) cut off 0.3 Å and then used for docking analysis. The specific Zn2þ chelating agents TPEN (pubchem id: CID 5519), DTPA (pubchem id: CID 3053), and TTD (pubchem id: CID 3117) were selected for docking analysis based on in vitro and in vivo experimental data. The compounds were minimized using steepest descent and conjugate gradient methods and docked at the ECVMPs active site (Leu114, His148, 152, 158, Glu176, Pro179) using induced fit docking (IFD) (Sherman et al., 2006) module to understand their mode of binding at active site. The best pose was then chosen based on glide score, glide energy and the interactions of ligand with active site residues. The interactions between protein and ligand were portrayed using Ligplot (Wallace et al., 1995) software package. 2.13. Data analysis The results of the experiments are expressed as mean ± SD of three independent experiments performed in triplicates. Statistical analyses were carried out using Student's t-test. The comparison between the groups were considered significant if p 0.05. Data were analyzed using the statistical package GraphPad Prism® (La Jolla, USA).
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3. Results SVMPs are mainly accountable for local tissue damage in viper bites. In this study we have made an attempt to evaluate the neutralization abilities of three specific Zn2þ chelating agents against ECVMPs induced hemorrhage and myotoxicity in treatment mode and the results obtained are presented below.
3.1. Inhibition of proteolytic activity of ECVMPs by TPEN, DTPA and TTD ECV is known for its strong proteolytic nature and 90% of this activity is contributed by metalloproteases. We examined the effect of three specific Zn2þ chelators TPEN, DTPA and TTD on in vitro ECV proteolytic activity. The chelating agents significantly inhibited the proteolytic activity of ECV towards casein in a concentration dependent manner with IC50 values of 6.7 mM for TPEN, 44.5 mM for DTPA and 100 mM for TTD respectively (Fig. 1); TPEN was 6.6 and 14.9 times more potent than DTPA and TTD in inhibiting the proteolytic activity of ECV respectively. The specificity of chelating agents was confirmed by evaluating the effect of these inhibitors towards serine (trypsin) and cysteine (bromelain) proteases. TPEN, DTPA and TTD failed to inhibit the activities of either protease, indicating their specificity towards metalloproteases (Fig. 2). Further, specificity of chelating agents towards Zn2þ was confirmed by UV-VIS spectral studies. The spectral shift of Zn2þ with protease inhibitors confirmed that chelating agents preferentially chelate Zn2þ (Fig. 3). Based on these in vitro findings, in silico studies were conducted for TPEN, DTPA and TTD to determine their interacting sites in ECVMPs. TPEN and DTPA interacted strongly with ECVMPs upon docking studies however, on repeated trials TTD failed to interact with ECVMPs. Both TPEN and DTPA exhibited different interaction and the glide score of TPEN (9.30 ± 0.19) was 1.6 times better than that of DTPA (5.5 ± 0.20). Both TPEN and DTPA sequestered active site Zn2þ; however with active site amino acids, TPEN showed strong hydrophobic interaction whereas DTPA interacted by forming hydrogen bond (Fig. 4). Low glide score of TPEN towards active site Zn2þ in docking analysis was evidenced by its strong in vitro inhibitory effect towards ECVMPs.
Fig. 1. Inhibition of caseinolytic activity of ECVMPs by TPEN, DTPA and TTD: Reaction mixture (1 ml) contained 0.4 ml of casein (2%) in 0.2 M TriseHCl buffer pH 8.5 was incubated for 150 min at 37 ºC with 25 mg venom and various concentrations of specific Zn2þ chelating agents ranging from 10 pM to 100 mM.TPEN: N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine; DTPA: Diethylene triamine pentaacetic acid; TTD: Tetraethyl thiuram disulfide
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3.2. Inhibition of pro-coagulant activity of ECVMPs by TPEN, DTPA and TTD The strong proteolytic activity of ECV reflects in hemostatic alterations, particularly the pronounced pro-coagulant effect, evident by coagulation of citrated plasma even without addition of calcium chloride. The effects of TPEN, DTPA and TTD on ECVMPs induced pro-coagulant action was evaluated by plasma re-calcification time. The pro-coagulant nature of ECV was abolished and normal clotting time was restored by all Zn2þ chelating agents and the effect was concentration dependent. TPEN restored the same at 30 mM (98.5 ± 5%; p < 0.0001); whereas in case of DTPA and TTD, similar effect was achieved at 100 mM concentrations respectively. Further, chelating agents failed to inhibit procoagulant activity of thrombin. This inability of chelating agents towards thrombin inhibition, a vital serine protease of coagulation cascade, confirms their noninterference in vital physiological pathways mediated by serine proteases (Fig. 5). Further, the chelating agents did not affect normal re-calcification time, indicating their negligible interference with calcium homeostasis. 3.3. Inhibition of fibrino(geno)lytic activity of ECVMPs by TPEN, DTPA and TTD As a secondary effect to pronounced pro-coagulant nature, ECVMPs also mediate fibrin(ogen) degrading activities, resulting in exaggerated hemostatic alterations. ECVMPs specifically hydrolyze Aa, Bb chains of fibrinogen (Factor I); similarly they also selectively degrade a polymer and a chain of plasma fibrin (Factor Ia). The inhibitory effects of TPEN, DTPA and TTD on ECVMPs induced fibrino(geno)lytic activities were evaluated using electrophoresis. The inhibitions were concentration dependent and complete inhibition of fibrinogenolytic activity was observed at 0.25 mM for TPEN, 5 mM for DTPA and TTD. TPEN was 20 times more potent than DTPA and TTD towards the inhibition of fibrinogenolytic activity (Fig. 6A). Similarly 0.25 mM of TPEN, 2 mM of DTPA and 8 mM of TTD completely inhibited fibrinolytic activity and TPEN was 8 and 32 times potent than DTPA and TTD respectively towards inhibition of fibrinolytic activity of ECVMPs (Fig. 6B). 3.4. Inhibition of hemorrhage and myotoxicity of ECVMPs by TPEN, DTPA and TTD The action of ECV is not only restricted to hemostatic alterations, but also result in the induction of hemorrhage and subsequent progression to myotoxicity. Onset of hemorrhage is due to degradation of ECM and basement membrane proteins surrounding blood vessels by metalloproteases. ECVMPs induced hemorrhagic lesion was obtained 3 h after intra dermal injection of venom. The minimum hemorrhagic dose (MHD) was found to be 1 mg. For inhibition studies 3 MHD of venom was separately pre-incubated with specific Zn2þ chelating agents. Upon pre-incubation, TPEN, DTPA and TTD showed concentration dependent inhibition of hemorrhage and complete inhibition was observed at 0.2 mM for TPEN, 0.5 mM for DTPA and 2 mM for TTD. Based on the results of pre-incubation studies, a fixed dose (5 mM) of inhibitors was used for neutralization studies in treatment mode (15 min after venom administration). Among the inhibitors tested, TPEN showed maximum inhibition (65.5 ± 8%; p < 0.001), with 1.8 ± 0.5 fold (34.6 ± 10.5%; p ¼ 0.002) and 2.2 ± 0.8 fold (30 ± 10%; p ¼ 0.005) potent inhibition in comparison with DTPA and TTD respectively. In addition, TPEN was 1.7 ± 0.5 fold potent than ASV (39 ± 18.5%; p ¼ 0.001) in inhibiting the hemorrhagic activity of ECVMPs (Fig. 7). Further, upon independent injection (15 min after venom administration), 5,10, and 20 mM TPEN showed significant inhibition of
Fig. 2. Comparison of inhibitory potential of Zn2þ chelators towards (a) serine and (b) cysteine proteases: Reaction mixture (1 ml) contained 0.4 ml of casein (2%) in 0.2 M TriseHCl buffer pH 8.5 was incubated for 150 min at 37 ºC with 500 mg trypsin/bromelain þ 1 mM concentration of different protease inhibitors. ***p
