Virtual analysis of structurally diverse synthetic analogs as inhibitors of snake venom secretory phospholipase A2.

Due to the toxic pathophysiological role of snake venom phospholipase A2 (PLA2 ), its compelling limitations to anti-venom therapy in humans and the need for alternative therapy foster considerable pharmacological interest towards search of PLA2 specific inhibitors. In this study, an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on VRV-PL-V (Vipera russellii venom phospholipase A2 fraction-V) belonging to Group II-B secretory PLA2 from Daboia russelli pulchella is carried out in order to study the structure-based inhibitor design. The accuracy of the model was validated using multiple computational approaches. The molecular docking study of this protein was undertaken using different classes of experimentally proven, structurally diverse synthetic inhibitors of secretory PLA2 whose selection is based on IC50 value that ranges from 25 µM to 100 µM. Estimation of protein-ligand contacts by docking analysis sheds light on the importance of His 47 and Asp 48 within the VRV-PL-V binding pocket as key residue for hydrogen bond interaction with ligands. Our virtual analysis revealed that compounds with different scaffold binds to the same active site region. ADME analysis was also further performed to filter and identify the best potential specific inhibitor against VRV-PL-V. Additionally, the e-pharmacophore was generated for the best potential specific inhibitor against VRV-PL-V and reported here. The present study should therefore play a guiding role in the experimental design of VRV-PL-V inhibitors that may provide better therapeutic molecular models for PLA2 recognition and anti-ophidian activity.

Albizia lebbeck seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus venom in a murine model.

CONTEXT AND OBJECTIVE: Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy. MATERIALS AND METHODS: Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme-toxin responsible for local- toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A2 (1:1-1:100 w/w). In addition, alleviation of ECV induced characteristic local- toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice. RESULTS: ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC50 = 36.32 µg, p < 0.0001) and hyaluronidase (IC50 = 91.95 µg, p < 0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED50 = 26.37 µg, p < 0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED50 = 37.5 µg, p < 0.0001) and lactate dehydrogenase (ED50 = 31.44 µg, p = 0.0021) levels at 1:50 w/w. DISCUSSION AND CONCLUSION: ALME demonstrated significant neutralization of ECV enzymes that contribute in local tissue damage and haemostatic alterations. The study scientifically supports the anecdotal use of A. lebbeck in complementary medicine and identifies ALME as principle fraction responsible for antivenom properties.

Evidence for existence of venom 5' nucleotidase in multiple forms through inhibition of concanavalin A.

Pharmacologically active 5' nucleotidase is a ubiquitously distributed enzyme in snake venoms. In this study the effect of concanavalin A (Con-A) on different snake venoms 5' nucleotidase activity is tested in order to know the protein nature which will ultimately help in purification of the enzyme with high yield. Con-A inhibited Naja naja, Naja kauthia, Naja melanoleuca, Naja naja sputatrix, Agistrodon halys blomhoffii, Bothrops asper and Oxyranus scutellas venom 5' nucleotidase activity at different concentrations. This indicates the presence of glycopart in the protein, thus glycoprotein in nature. Vipera russellii, Vipera plaestenae, Agistrodon contratrix, Bitis orientis, Echis carinatus and Trimeresures malabaricus was not inhibited by Con-A, indicating absence of glycopart in the protein. This study for the first time shows existence of 5' nucleotidase in multimeric forms.

Crystal structures of the free and anisic acid bound triple mutant of phospholipase A2.

Phospholipase A2 catalyses the hydrolysis of the ester bond of 3-sn-phosphoglycerides. Here, we report the crystal structures of the free and anisic acid-bound triple mutant (K53,56,120M) of bovine pancreatic phospholipase A2. In the bound triple mutant structure, the small organic molecule p-anisic acid is found in the active site, and one of the carboxylate oxygen atoms is coordinated to the functionally important primary calcium ion. The other carboxylate oxygen atom is hydrogen bonded to the phenolic hydroxyl group of Tyr69. In addition, the bound anisic acid molecule replaces one of the functionally important water molecules in the active site. The residues 60-70, which are in a loop (surface loop), are disordered in most of the bovine pancreatic phospholipase A2 structures. It is interesting to note that these residues are ordered in the bound triple mutant structure but are disordered in the free triple mutant structure. The organic crystallization ingredient 2-methyl-2,4-pentanediol is found near the active site of the free triple mutant structure. The overall tertiary folding and stereochemical parameters for the final models of the free and anisic acid-bound triple mutant are virtually identical.

Isolation and characterization of hyaluronidase a "spreading factor" from Indian cobra (Naja naja) venom.

Hyaluronidase, ubiquitous enzyme in snake venoms, known originally as "spreading factor", has not been well studied. The present study describes the purification and characterization of hyaluronidase from Indian cobra (Naja naja) venom and provides systematic evaluation of the spreading property of the enzyme. Hyaluronidase (NNH1) has been purified through gel permeation and ion exchange chromatography. The molecular mass was found to be 70.406 kDa by MALDI-TOF mass spectrometry and with the (p)i pI of 9.2. The amino acid sequence of the N-terminus was found to be NEQSTHGAYV. The enzyme shows absolute specificity for hyaluronan and belongs to the group of neutral active enzymes. Tetrasaccharides are the final product of hyaluronan digestion. The enzyme cleaves beta 1,4-glycosidic linkage and belongs to a group of endo-beta-N-acetyl hexosaminidases. Hyaluronidase indirectly potentiates the myotoxicity of VRV-PL-VIII, a phospholipolytic myotoxin, and also the hemorrhagic potency of a hemorrhagic complex-I. Localization of hyaluronan in human skin section and selective degradation by venom hyaluronidase (NNH1) corroborate the plausible in vivo degradation of hyaluronan in the extracellular matrix (ECM) resulting in easy dissemination of VRV-PL-VIII myotoxin and hemorrhagic complex-I.

Monoclonal antibodies to VRV-PL-VIIIa, a basic multitoxic phospholipase A2 from Vipera russelli venom.

VRV-PL-VIIIa, the most basic phospholipase A2 (PLA2) from the venom of Vipera russelli, induces multiple toxic effects, including neurotoxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal anti-serum was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzyme-linked immunosorbant assay (ELISA) with two other PLA2 from the same venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin and crotoxin. Twenty-two hybridoma cell lines secreting monoclonal antibodies against VRV-PL-VIIIa were isolated. The monoclonal antibodies exhibited apparent binding affinities in ELISA with VRV-PL-VIIIa ranging over two orders of magnitude. Most of the monoclonal antibodies cross-reacted moderately with VRV-PL-V and weakly with VRV-PL-VI. None of the antibodies cross-reacted with ammodytoxin, caudoxin or crotoxin. Reducing the disulfide bonds of VRV-PL-VIIIa lowered the ELISA signals of each monoclonal antibody to nonspecific levels, suggesting that all the antibodies recognize conformational epitopes. Four of the 22 antibodies neutralized the enzymatic activity of VRV-PL-VIIIa. Interestingly, two of the four exhibited the lowest affinities of the monoclonal antibody library for VRV-PL-VIIIa in ELISA, while the other two exhibited the highest. Each of the monoclonal antibodies was biotinylated and spatial binding relationships were evaluated by competition ELISA.

Effects of chemical modification on enzymatic and toxicological properties of phospholipases A2 from Naja naja naja and Vipera russelli snake venoms.

The effects of chemical modification with 4-NN-dimethyl amino azo benzene-4'-isothiocyanate on various biological activities of phospholipases A2, NN-XIII-PLA2 from Naja naja naja and VRV-PL-VIIIa from Vipera russelli snake venoms were investigated. Modification of the enzymes resulted in significant reduction of lethal, hemolytic, anticoagulant and enzymatic activities. The Km value of the modified enzymes was increased. The modified enzymes failed to induce edema in the foot pads of mice and were non-lethal up to 16 mg/kg body weight. However, considerable myotoxicity was retained, suggesting that the toxins have multiple sites for biological activities. The aggregated form obtained from modified NN-XIII-PLA2 exhibited decreased enzymatic activity and increased toxicity compared to the modified monomer. This aggregated form did not show pyrophosphatase/phosphomonoesterase activity in contrast to the aggregated form obtained from the native NN-XIII-PLA2 molecule.

Geographical variation in India in the composition and lethal potency of Russell's viper (Vipera russelli) venom.

Venom samples of Russell's viper (Vipera russelli) from three localities in India were analysed for their composition and toxicity. Column chromatographic fractionation on CM-Sephadex C-25 showed the absence of three fractions in the venom samples of southern India compared with the samples from northern and western India. The SDS-PAGE pattern of southern Indian venom samples also showed lack of three protein bands corresponding to molecular weights of 66,000, 39,000 and 9000. Venom samples from northern and western India possessed high acidic phospholipase activity while acidic phospholipase activity was absent in the samples from southern India, which in contrast showed large basic fractions with phospholipase activity. Proteolytic activity was present in all the venom samples; however, this activity, as well as trypsin inhibitor activity, was very low in the southern Indian samples. The ratio of proteolytic activity to inhibitor activity remained constant in most of the venom samples studied. LD50 values for most of the venom samples from northern and western India were twice as high as that of the samples from southern India. High phospholipase activity correlated with high lethal potency in the venom samples studied.

Effects of myonecrotic snake venom phospholipase A2 toxins on cultured muscle cells.

We have attempted to establish a cell culture model suitable for molecular mechanism of action studies of necrotic phospholipases A2 (PLA2). Three myonecrotic PLA2 were purified, one basic PLA2 from Naja nigricollis venom and two basic PLA2 (VRV-PL-V and VRV-PL-VIIIa) from Vipera russelli venom. The effects of these PLA2 on several established muscle cell lines were evaluated. As judged by light microscopy, some, but not all, cell lines detached from the culture plate in a time- and concentration-related fashion. Naja nigricollis PLA2 was the most potent at eliciting this effect, followed by VRV-PL-V and VRV-PL-VIIIa. The two most sensitive cell lines, 1447 and 1456, were chosen for further study using N. nigricollis PLA2. Cellular protein and nucleic acid syntheses were inhibited by the toxin in a time- and dose-related manner. However, it appeared that most, if not all, of the inhibition was due to toxin-induced reduction of precursor uptake, suggesting effects at the plasma membrane level. The putative membrane effects were specific, in that uptake of calcium, choline or glucose was not inhibited by the toxin. Moreover, treating the cells with toxin failed to significantly increase lactate dehydrogenase release into the medium. Polyclonal antiserum prepared against N. nigricollis basic PLA2 neutralized the toxicity completely with 1456 cells, but only partially with the 1447 cell line. Both the 1447 and 1456 lines appear to be suitable as cell culture models for necrotizing PLA2 molecular mechanism of action studies.

Comparative characterization of two toxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja naja) venom by column chromatography. The NN-XIa-and NN-XIb-PLA2s have mol. wts between 10,700 and 15,000. The NN-XIa-PLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. LD50 of 8.5 mg/kg body weight. The NN-XIa-PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. LD50 of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XIa-PLA2.

Interaction of aristolochic acid with Vipera russelli phospholipase A2: its effect on enzymatic and pathological activities.

Aristolochic acid, an alkaloid from the plant Aristolochia species, interacts with the major basic phospholipase A2 from Vipera russelli venom. It is an uncompetitive inhibitor with a Ki of 9.9 X 10(-4)M when phosphatidylcholine is used as substrate. The inhibition of direct and indirect hemolysis is higher compared to the inhibition of phosphatidylcholine hydrolysis. Edema-inducing activity of Vipera russelli phospholipase A2 is inhibited by aristolochic acid when injected either as a mixture or separately. Both i.m. and i.p. administration of aristolochic acid following phospholipase injection are equally effective in inhibiting edema. The alkaloid inhibits the edema-inducing activity as soon as it reaches the site, but does not aid in recovery. Aristolochic acid failed to inhibit other pathological activities of the enzyme.

Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli) venom.

A major phospholipase A2 (VRV PL-VIIIa) which constitutes 24% of the whole Vipera russelli venom was purified to homogeneity by CM-Sephadex C-25 column chromatography followed by gel filtration on Sephadex G-50. VRV PL-VIIIa is a basic protein with a molecular weight of 11,800 by SDS-PAGE. This enzyme contributes 45% of the total PLA2 activity of the venom, but it is least toxic compared to other purified basic PLA2 enzymes prepared from V. russelli venom. The LD50 value (i.p.) of VRV PL-VIIIa is 5.3 mg/kg body wt. It shows neurotoxic symptoms and damages vital organs such as lung, liver and kidney at LD50 doses. It induces myonecrosis when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads.

Dissociation of enzymatic activity from toxic properties of the most basic phospholipase A2 from Vipera russelli snake venom by guanidination of lysine residues.

The most basic phospholipase A2 VRV-PL-VIIIa purified from Russell's viper venom is a toxic enzyme. It induced neurotoxicity, myotoxicity, and oedema and was lethal to mice at 5.3 micrograms/g body weight. It also inhibited the coagulation of the human plasma. The epsilon-amino groups of lysine residues of the toxic enzyme VRV-PL-VIIIa were guanidinated with o-methylisourea. Guanidination of the enzyme did not alter the enzymatic activity markedly. The guanidinated enzyme became non-lethal in doses up to 16 micrograms/g body weight, and failed to elicit neurotoxic symptoms in experimental animals and oedema in the foot pads of mice. Also, its myotoxic and anticoagulant potencies were decreased significantly.

Isolation and characterization of an endogenous inhibitor of phospholipase A2 from Indian cobra (Naja naja naja) venom.

A PLA2-inhibitor has been purified from Indian cobra (Naja naja naja) venom by the combination of ion-exchange and gel-filtration chromatography. The inhibitor, NN-I3 was a peptide with mol.wt 6500 and has a fluorescence emission maxima ca 340 nm. NN-I3 specifically inhibited the enzyme activity of the three acidic PLA2 from the same venom. The inhibition of NN-I2d-PLA2 and NN-I2c-PLA2 by NN-I3 was of mixed type and NN-I2c-PLA2 was of uncompetitive type. Neither the inhibitor nor the individual mixtures of acidic PLA2 with the inhibitor (1:1 w/w or 1:2 mol:mol) were lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight.

Purification and characterization of three acidic, cytotoxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Three acidic phospholipases A2 (NN-I2c-PLA2, NN-I2d-PLA2 and NN-I2c-PLA2) were purified by successive chromatography of Indian cobra (Naja naja naja) venom on CM-Sephadex C-25, Sephadex G-50 and QAE Sephadex A-25 columns. They have molecular weights of 13,000-14,500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (approximately 345 nm). All the three phospholipases A2 were enzymatically highly active with specific activities 9-17 micromol min(-1) mg(-1). They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities.

Synergistic interaction of a protease and protease inhibitors from Russell's viper (Vipera russelli) venom.

An acidic proteolytic enzyme, RVVX, was purified from Vipera russelli venom by successive chromatography on CM-Sephadex C-25, DEAE-cellulose and Sephadex G-100 columns. RVVX is a glycoprotein with a mol. wt of 79,000. It exhibited caseinolytic and factor X activating properties. Two trypsin inhibitors, TI-I and TI-II, were purified from V. russelli venom in a single step by CM-Sephadex C-25 column chromatography. The trypsin inhibitors interacted with the proteolytic enzyme RVVX. TI-I inhibited only the factor X activating property of RVVX while TI-II inhibited both, the caseinolytic and also factor X activating properties of RVVX. The edema inducing activity of RVVX increased markedly in the presence of non-edema inducing doses of TI-I and TI-II. RVVX, TI-I and TI-II were non-lethal in mice. The combination of RVVX and TI-II demonstrated enhanced toxicity.

Detection, using antibodies, of pharmacologically active sites, apart from the catalytic site, on venom phospholipase A2.

Polyclonal antibodies to a purified neurotoxic phospholipase A2 VRV PL-V from Vipera russelli venom were raised in rabbits. The gamma globulin fraction from the serum of the rabbits injected with VRV PL-V-toxoid (anti VRV PL-V antibodies) is termed anti PL-V Ig. Anti PL-V Ig neutralized the neurotoxic symptoms and lethal effects of neurotoxic PLA2s (VRV PL-V, VRV PL-VI and VRV PL-VIIIa) and neurotoxic symptoms of whole V. russelli venom, without affecting their phospholipase A2 activities. Their edema inducing activity was also unaffected. Anti VRV PL-V-antibodies inhibited the anticoagulant potencies of the neurotoxic PLA2s (VRV PL-V, VRV PL-VI and VRV PL-VIIIa) from V. russelli venom and VRV PL-V-toxoid. The myonecrotic activity of VRV PL-V, VRV PL-VIIIa and whole venom remain unaffected in presence of anti PL-V Ig. The hemoglobinuria induced in rabbits by injection of VRV PL-V-toxoid was inhibited by anti VRV PL-V antibodies. These results indicate the presence of multiple pharmacological sites apart from the catalytic site on V. russelli PLA2 molecules.

Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom.

A major phospholipase A2 (NN-XIII-PLA2) which constitutes 20% of the whole Naja naja naja venom was purified to homogeneity on CM-Sephadex C-25 column chromatography. NN-XIII-PLA2 is a basic protein with a mol. wt of 11,200 by SDS-PAGE. This enzyme has low enzymatic activity but is more toxic to mice than the whole venom. The LD50 value (i.p.) of NN-XIII-PLA2 is 2.4 mg/kg body weight (whole venoms LD50 is 2.8 mg/kg body weight). It induces neurotoxic-like signs in experimental animals. It induces myotoxicity when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads of mice. This enzyme has a fluorescence maxima between 310-316 nm which is typical of tyrosine residues.

Structure-function relationships among neurotoxic phospholipases: NN-XIII-PLA2 from Indian cobra (Naja naja naja) and VRV PL-V from Russell's viper (Vipera russelli) venoms.

Though venom phospholipases induce various pharmacological effects their mechanism of action is in some cases unclear. There may be separate pharmacological sites on the venom phospholipase molecule. In order to understand the structure-function relationships among venom phospholipases, studies on interaction of venom phospholipases with its antibodies and various alkaloids were carried out. The alkaloids aristolochic acid, ajmaline and reserpine were incapable of inhibiting the phospholipase A2 activity of NN-XIII-PLA2 but inhibited its edema inducing potency and partially inhibited the symptoms of neurotoxicity. The direct and indirect hemolytic activity remain unaffected. Polyclonal antibodies (anti PL-V Ig) to a neurotoxic PLA2 VRV PL-V neutralized the neurotoxic symptoms and lethality of VRV PL-V without affecting its in vitro phospholipase A2 activity when egg PC was used as the substrate. However, they inhibited the catalytic activity of VRV PL-V when synaptosomes were used as the substrate. Our results indicate the presence of multiple pharmacologically active sites apart from catalytic site.

Dissociation of catalytic activity and neurotoxicity of a basic phospholipase A2 from Russell's viper (Vipera russelli) venom.

A neurotoxic phospholipase A2, VRV PL-V was purified from Vipera russelli venom in a single step by CM-Sephadex C-25 column chromatography. VRV PL-V is a basic PLA2 with a mol. wt of approximately 10,000. The lethal potency of VRV PL-V was greater than that of the crude V. russelli venom. VRV PL-V showed anticoagulant activity and induced edema in the foot pad of the mouse. VRV PL-V undergoes aggregation at pH 4.8. The size of the aggregate increased as the temperature at which the enzyme was incubated was raised. A highly aggregated form with a mol. wt of 53,100 was formed at 96 degrees C. This aggregate showed a two-fold increase in its catalytic activity, while its neurotoxic activity disappeared. The aggregate also showed a significant increase in its anticoagulant activity when compared to the monomeric form. Edema-inducing activity decreased upon association to higher molecular form.