Original Article

Pharmacological perspectives of snake venoms from Viperidae family

C Paulchamy

Keywords

antibacterial peptide, phospholipase a2, snake venoms, therapeutic peptides, viper, viperidae

Citation

C Paulchamy. Pharmacological perspectives of snake venoms from Viperidae family. The Internet Journal of Pharmacology. 2009 Volume 8 Number 2.

Abstract

Snake venom is a combination of many different proteins and enzymes. Many of these proteins are harmless to humans, but some are toxins. Snake venoms effects include pro- and anti-blood coagulation, neurotoxicity, mycotoxicity, nephrotoxicity, cardiotoxicity and necrotoxicity. This article has focused only on the principle components of viperidae venoms and their corresponding pharmacological actions on cells. Is was also discussed the advantages of enzymes like phospholipase A2, L-amino acid oxidase, and metalloprotease while non-enzymatic proteins include warprin family proteins, platelet aggregation factors and C-type lectin-like protein. Most recently, few public-domain snake venoms database are released which contains natural history notes, pictures of venomous snakes, distribution maps and information about each individual snake and its venom. Thus, we conclude that the given information in this review will be more useful for researchers to investigate novel human therapeutics from snake venoms.

 

Introduction

Everyone assumes the snakes are more dangerous and threat to human society, but many of us have not been known that snakes have possessed a novel factory for human valuable medicines in their venoms. Snake venom contains a variety of chemicals including pharmacological properties. Snake venom is a mixture of different enzymes and having toxic and non-toxic activities. The mechanism of toxin secretion is highly conserved and diversification of matured toxin sequences shows existence of multiple protein isoforms in the venoms to adapt within prey environment1. The action of venom is the combined effect of all components present in the venoms and the snakes escape the effect of own toxins due to specific resistance mechanism and modulation of acetyl choline receptors. Through the recent development of technological knowledge in biotechnology and bioinformatics, snake venoms are widely used to develop anti-venom vaccines and medicine for rescuing snake venoms poisoning patients.

The Family

Snakes are taxonomically classified under phylum reptiles of vertebrates in animal kingdom. Laticaudae, elapidae, crotalidae and viperidae are the major families of snakes on which viperidae family is extensively studied for its taxonomy, physiology and ecological adaptation, and little is known for pharmacological action of venoms yet2. Azemiopinae, causinae, viperinae and crotalinae are subfamily of viperidae family. Vipera lebetina, Bothrops jararaca, Agkistrodon acutus, Daboia russellii siamensis, Deinag kistrodon acutus and Bothrops jararacussu of this viperidae family is well studied for the venoms properties and their components. The viperidae differ much among themselves in the toxicity of their venom1. Thus, this present review article was aimed to explore pharmacological knowledge of snake venoms of this family and their respective importance in medicine.

Geographic variations in snake venom toxicity

Toxin obtained from a given species may also differ depending on geographical regions. Snake venoms, for example, toxicity of South India would be different from snake venoms in North India. Variation in snake venom composition is a ubiquitous phenomenon at all taxonomic level3. Many factors including phylogeny, geographic origin, season, age and prey preference may influence venom composition. The composition of snake venom is also strongly influenced by environmental factors including habitat, climate and preys4. Sex-based differences in the venom proteome of sibling snakes (Bothrops jararaca) of a single litter raised under controlled conditions which seem to be genetically inherited and imposed by evolutionary forces5.

Classification of snake venoms

Snake venom proteins and polypeptides are classified into superfamilies of enzymes and non-enzymatic proteins. The members of each superfamily show similarity in their primary, secondary and tertiary structures, however, their biological functions are distinct still now. Among non-enzymatic proteins, superfamilies of three-finger toxins, serine proteinase inhibitors, C-type lectin-related proteins, atrial natriuretic peptides and nerve growth factors have already been well characterized6-8. L-Amino acid oxidase, phospholipase A2, metalloprotease and ribonuclease A are some examples of superfamilies of enzymes in this family9-13. Based on the structure, activity and components, crude venoms are also classified into cardiotoxin, neurotoxin, cytotoxin and myotoxin4, 14-15.

Snake venoms and drug discovery

The snake venom proteins are of biological interest because of their diverse and selective pharmacological and physiological effects through their interaction with various molecular targets. These proteins are prototypes for: (i) therapeutic agents (ii) pharmacological probes for the diagnosis of several diseases and (iii) research tools to decipher physiological and pathological processes16. Captopril, an antihypertensive drug, is a classic example that was designed based on the peptide inhibitor of angiotensin-converting enzyme from Bothrops jararaca venom8. Thrombin-like enzymes from snake venom are used for fibrinogen and fibrinogen-breakdown product assays as well as detecting dysfibrinogenaemias17 and also snake venom C-type lectins are routinely used to study platelet glycoprotein receptors18. Peptide neurotoxins from snake venoms are used for the identification and characterization of membrane ion channels and receptors in vertebrate cells, including human neurons19. Snake venom metalloproteinases and peptide neurotoxins can be used to study myoblast fusion. Fertilization and matrix metalloproteinase–cell interactions can be used for characterizing human cancers and small-cell carcinoma16. All of these studies demonstrate the significance of snake venom proteins as valuable tools for basic research, disease diagnosis and drug development. However, most snake venoms remain poorly characterized despite being a rich source of biologically active proteins with therapeutic potential. Hence, further studies are essential to identify and characterize novel venom proteins which can be used as leads or structural templates for developing new therapeutic agents.

Snake venoms and pharmacological perspectives

Cardiotoxin

Cardiotoxin forms a major toxic protein found in cobra venom and made up of 60-63 amino acid residues in a single polypeptide chain cross linked by four disulfide bonds. The mechanism of action of cardiotoxin peptide for inducing muscle contractions by Viper russelli russelli is not clear and much of the functional relationship and neutralization remains to be elucidated20. Snake venoms have not only the albumin like property, but also toxic effect on nerve tissue and blood. Venoms have antibacterial activity on the blood and active principle affecting the vascular tissue. Most recently, Chellapandi and Jebakumar (2008) reported the potential antibacterial activity of Vipera russelli venom proteins against Escherichia coli 21.

Warprin family proteins

Waprins is a second major family of venom proteins that shows structural similarity to WAPs (whey acidic proteins)22. The WAP domain inhibits Na+-K+ ATPase23, elafin and SLPI (secretory leucocyte proteinase inhibitor), which are proteinase inhibitors with potent antimicrobial activity24-25, ps20 with growth-inhibitory activity26, and SWAM1 and SWAM2 (single WAP motif proteins 1 and 2), which are antibacterial proteins27. Many of the reported WAP domain proteins are involved in the innate immune system. The three-dimensional structure of nawaprin, the first WAP domain protein from snake venom, shows significant similarity to that of elafin, with a flat disc-like shape, characterized by a spiral backbone configuration that forms outer and inner circular segments22. The biological function(s) of nawaprin has yet to be identified. A new member of waprin family, omwaprin from the venom of inland taipan snake (Oxyuranus microlepidotus) has been identified, purified and functionally characterized. It is cationic under physiological conditions and shows selective antibacterial activity against Gram-positive bacteria which was structure-dependent, salt–tolerant and specifically targets bacterial membranes28. Thus, the potential of this novel antibacterial protein as a research tool and as a prototype for the development of therapeutic agents would be highlighted. Similarly antifungal peptide from Indian Bothrops jararaca venom was purified and characterized that will replace classical drugs29.

Figure 1
Table 1: Some therapeutic peptides of snake venoms from

Platelet aggregation factors

Russelle vipers, Vipera russelli siamensis and Daboia russellii siamensis were studied for systemic symptoms of the action of venom, platelet aggregation factors, fibrinolytic and hemorrhagic factors, geographical variation in composition and lethal potency synergistic interaction of protease, and protease inhibitors. These venoms are also caused a significant increase in brain GABA content in mice. It suggested that they have a potent central nervous system depressant action6, 30. Saw scaled viper venom contains the assay factors like V, VII, X and platelet factor III and lupus coagulant so that it can be used for assaying lupus coagulant activity. Kallikrein like (kinin releasing) enzyme from viperidae, lebetase, a direct acting fibrinolytic enzyme from Vipera lebetina 31, pseudotoxin and coagulation factor X-binding protein from Deinag kistrodon acutus, have reported to induce changes in plasma membrane permeability32. Such approaches will be helpful to understand the anti-coagulation activity on the human blood cells and to develop blood-related therapeutics.

C-type lectin-like protein

A C-type lectin-like protein (CLP), agkisacutacin isolated from Agkistrodon acutus venom, had been identified as an antagonist of platelet aggregation induced by ristocetin, as well as a certain extent fibrinogenlytic activity. After three phase purification, agkisacutacin was retained the effect on ristocetin-induced, von Willebrand factor-dependent platelet aggregation, while it lost the fibrinogenlytic activity. FACS and ELISA assays showed that agkisacutacin belongs to membrane glycoprotein Ib-binding protein (GPIb-bp) for it could block and inhibit the binding of anti-GPIb antibody to GPIb. PAGE and ELISA was used to confirm anti-coagulant activity of agkisacutacin and the function as IX/X-binding protein. Thus, agkisacutacin binds to both platelet membrane receptors and coagulation factors, suggested to develop anti-coagulation agents7.

Phospholipase A

A novel phospholipase A2 (PLA2) from the snake venom of Protobothrops mucrosquamatus did not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice33-34. Phylogenetic analysis found that it combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup35. In viper venoms, phospholipase A2 activity was characterized to develop anti-bacterial compounds against pathogenic bacteria.

Metalloproteinase

The venom of Viperdae is a rich source of metalloproteinases, which have potential clinical applications for lowering plasma fibrinogen or dissolving thrombi. Recently, a novel proteinase from Formosan Agkistrodon acutus venom was purified that contained getatinolytic component, agkislysin, is a 22kDa-monomeric protein without Asn-linked sugar. Functional characterization showed that agkislysin possessed both fibronectin- and type IV collagen-cleaving activities. In addition, agkislysin preferentially cleaved the Aalpha chain of fibrinogen, followed by the beta chain, while the gamma chain was finally affected. Furthermore, agkislysin was also capable of cleaving prothrombin into various fragments, as well as suppressing ristocetin-induced platelet aggregation by hydrolyzing von Willebrand factor. Agkislysin is a P-I class metalloproteinase with unique biological properties12. Venom hemorrhagic metalloproteinase in Bothrops jararaca venom, jararafibrase I (JF I) play an important role in the development of coagulopathy through rapid spreading of venom coagulation components from the injected area into systemic circulation36.

L-Amino acid oxidase

Zn containing metallo-protease, apoptotic pathway inducing L-amino acid oxidase and hydrogen peroxides are identified and evaluated for their respective pharmacological perspectives. Antibacterial activity is found in snake venoms that may be due to L-amino acid oxidase. Antifungal and antiviral activities have also investigated in snake venom of Bothrops jararaca, thus other being adverse drugs may be replaced29, 37.

Conclusion

Though lethal toxin, nuclease, phasphodiesterase, cytotoxin, cardiotoxin and ribonuclease have been isolated and purified from cobra venoms, a little attempt have been made on the venoms of viperidae. Anti-platelet peptides (thrombin-like activity) in Peruvian Bothrops nad Lechesis genera38, platelet aggregation inducer in Cerastes cerastes 39, L-amino acid oxidase in Bothrops leucurus 40and cytotoxic L-amino acid oxidase from Bothrops moojeni 41, serine protease with anti-thrombic activity and bothojaracin from Bothrops jararaca 38, 42 are found in their venoms, whose pharmacological characteristics are well studied to become a novel medicine to human wealth. Recently, disintegrin (salmosinl), acts as a potent inhibitors of platelet aggregation, from Agkistrodon halys have been cloned on the perspectives of medicinal importance43. Amino acid sequence of phospholipase A-2 from sea krait, Laticuada semifaciata and pirayoxin-1, a myotoxin from Bothrops pirajai are predicted on the basis of Bioinformatics advantages15. The crystal structure of acutolysin, a three disulfide hemorrhagic zinc metallo-protease from snake venom of Agkistrodon acutus was solved by X-ray crystallography technique10. Venomdoc (www.venomdoc.com/), Natural Toxins Research Center - Snake Database (http://ntrc.tamuk.edu/cgi-bin/serpentarium/snake.query), and Australian Venom & Toxin Database (www.kingsnake.com/) are widely used databases for retrieving information regarding snake venoms. Such online resources and X-crystallographic structural information will also be strengthening the research activities towards developing anti-venom vaccines and snake venoms based therapeutics. Snake venom contains a novel medicinal properties and pharmacological active principles. Thus, this article suggests that snakes must be protected from their enemies and forming snake conservation, and venom bank are collectively essential need in world.

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Author Information

Chellapandi Paulchamy
Department of Bioinformatics, School of Life Sciences, Bharathidasan University

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