Snake Venom, A Natural Library of New Potential Therapeutic Molecules: Challenges and Current Perspectives.

BACKGROUND: Research involving snake venom has gradually surpassed the simple discovery of new molecules using purification and structural characterization processes, and extended to the identification of their molecular targets and the evaluation of their therapeutic potential. Nevertheless, this only became possible due to constant progress in experimental biology and protein purification approaches. OBJECTIVE: This review aims to discuss the main components of snake venoms that have been investigated for biotechnological purposes, and to discover how these promising biomolecules were obtained with the satisfactory degree of purity that have enabled such studies. Advances in purification technologies of various snake venom molecules have allowed for important discoveries of proteins and peptides with different biomedical and biotechnological applications. RESULT AND CONCLUSION: It is believed that significant experimental and computational advances will arise in similar proportions in the coming years that will allow researchers to map the molecular regions responsible for their pharmacological actions, their respective mechanisms of action and their cell targets.

BbrzSP-32, the first serine protease isolated from Bothrops brazili venom: Purification and characterization.

Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated.

Isolation and biochemical characterization of a new thrombin-like serine protease from Bothrops pirajai snake venom.

This paper presents a novel serine protease (SP) isolated from Bothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu(2+) significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated from Bothrops pirajai snake venom.

Antitumoral activity of snake venom proteins: new trends in cancer therapy.

For more than half a century, cytotoxic agents have been investigated as a possible treatment for cancer. Research on animal venoms has revealed their high toxicity on tissues and cell cultures, both normal and tumoral. Snake venoms show the highest cytotoxic potential, since ophidian accidents cause a large amount of tissue damage, suggesting a promising utilization of these venoms or their components as antitumoral agents. Over the last few years, we have studied the effects of snake venoms and their isolated enzymes on tumor cell cultures. Some in vivo assays showed antineoplastic activity against induced tumors in mice. In human beings, both the crude venom and isolated enzymes revealed antitumor activities in preliminary assays, with measurable clinical responses in the advanced treatment phase. These enzymes include metalloproteases (MP), disintegrins, L-amino acid oxidases (LAAOs), C-type lectins, and phospholipases A2 (PLA2s). Their mechanisms of action include direct toxic action (PLA2s), free radical generation (LAAOs), apoptosis induction (PLA2s, MP, and LAAOs), and antiangiogenesis (disintegrins and lectins). Higher cytotoxic and cytostatic activities upon tumor cells than normal cells suggest the possibility for clinical applications. Further studies should be conducted to ensure the efficacy and safety of different snake venom compounds for cancer drug development.

Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

ESI-MS/MS identification of a bradykinin-potentiating peptide from Amazon Bothrops atrox snake venom using a hybrid Qq-oaTOF mass spectrometer.

A bradykinin-potentiating peptide (BPP) from Amazon Bothrops atrox venom with m/z 1384.7386 was identified and characterized by collision induced dissociation (CID) using an ESI-MS/MS spectra obtained in positive ion mode on a hybrid Qq-oaTOF mass spectrometer, Xevo G2 QTof MS (Waters, Manchester, UK). De novo peptide sequence analysis of the CID fragmentation spectra showed the amino acid sequence ZKWPRPGPEIPP, with a pyroglutamic acid and theoretical monoisotopic m/z 1384.7378, which is similar to experimental data, showing a mass accuracy of 0.6 ppm. The peptide is homologous to other BPP from Bothrops moojeni and was named as BPP-BAX12.

Local and systemic biochemical alterations induced by Bothrops atrox snake venom in mice.

The local and systemic alterations induced by Bothrops atrox snake venom (BaV) injection in mice were studied. BaV induced superoxide production by migrated neutrophils, mast cell degranulation and phagocytosis by macrophages. Moreover, BaV caused hemorrhage in dorsum of mice after 2hr post- injection. Three hours post-injection in gastrocnemius muscle, we also observed myonecrosis, which was assessed by the determination of serum and tissue CK besides the release of urea, but not creatinine and uric acid, indicating kidney alterations. BaV also induced the release of LDH and transaminases (ALT and AST) indicating tissue and liver abnormalities. In conclusion, the data indicate that BaV induces events of local and systemic importance.

Snake venom L-amino acid oxidases: some consideration about their functional characterization.

Snake Venom L-amino acid oxidases (LAAOs E.C. 1.4.3.2) are flavoenzymes broadly found in various snake venom compositions. LAAOs have become an attractive subject for molecular biology, biochemistry, physiology and medicine due to their actions on various cells and biological effects on platelets, apoptosis, hemorrhage and others. In this review we try to summarize some of these reports, with special emphasis on apoptosis, anti-protozoa, bactericidal and anti-viral activities.