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Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.

Antitumor property of Crotoxin (CTX), the major toxin from Crotalus durissus terrificus snake venom, has been demonstrated in experimental animal models and clinical trials. However, the direct action of this toxin on the significant events involved in neovascularization, which are essential for tumor growth and survival, has not been confirmed. This study investigated the effects of CTX on the key parameters of neovascularization in two- and three-dimensional culture models. Murine endothelial cell lines derived from thymus hemangioma (t.End.1) were treated at different concentrations of CTX (6.25–200 nM). Endothelial cell proliferation, cell adhesion, and actin cytoskeletal dynamics on laminin (10 µg/ml), type I collagen (10 µg/ml), and fibronectin (3 µg/ml) were evaluated along with the endothelial cell migration and formation of capillary-like tubes in 3D Matrigel. CTX concentration of 50 nM inhibited tube formation on 3D Matrigel and impaired cell adhesion, proliferation, and migration under both culture medium and tumor-conditioned medium. These actions were not accountable for the loss of cell viability. Inhibition of cell adhesion to different extracellular matrix components was related to the reduction of αv and α2 integrin distribution and cytoskeletal actin polymerization (F-actin), accompanied by inhibition of focal adhesion kinase (FAK), Rac1 (GTPase) signaling proteins, and actin-related protein 2/3 (Arp 2/3) complex. This study proved that CTX inhibits the major events involved in angiogenesis, particularly against tumor stimuli, highlighting the importance of the anti-angiogenic action of CTX in inhibition of tumor progression.

Varespladib (LY315920) is a synthetic phospholipase A2 (PLA2) inhibitor that has been demonstrating antiophidic potential against snake venoms that present PLA2 neurotoxins. In this study, we evaluate the capacity of Varespladib to inhibit the neuromuscular effects of crotoxin (CTX), the main toxic component of Crotalus durissus terrificus snake venom, and its PLA2 subunit (CB). We performed a myographic study to compare the neuromuscular effects of CTX or CB and the mixture of these substances plus Varespladib in mice phrenic nerve-diaphragm muscle preparations. CTX (5 μg/mL), CB (20 μg/mL), or toxin-inhibitor mixtures pre-incubated with different concentration ratios of Varespladib (1:0.25; 1:0.5; 1:1; w/w) were added to the preparations and maintained throughout the experimentation period. Myotoxicity was assessed by light microscopic analysis of diaphragm muscle after myographic study. CTX and CB blocked the nerve-evoked twitches, and only CTX induced histological alterations in diaphragm muscle. Pre-incubation with Varespladib abolished the muscle-paralyzing activity of CTX and CB, and also the muscle-damaging activity of CTX. These findings emphasize the clinical potential of Varespladib in mitigating the toxic effects of C. d. terrificus snakebites and as a research tool to advance the knowledge of the mechanism of action of snake toxins.

14 páginas, 9 figuras, 3 tablas, Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots., This study was financially supported by DGAPA-PAPIIT (project IN211621), CONACYT (project264255); FORDECYT PRONACE (project 1715618/2020), FORDECYT (project 303045), Clemson University and the National Science Foundation (DEB 1822417) to CLP.

Snake venoms are mixtures of proteins whose physicochemical features confer them toxicity and immunogenicity. Animals (e.g., horses or sheep) immunized with snake venoms produce antibodies towards the venom proteins. Since these antibodies can neutralize the venom toxicity, they have been used to formulate snake antivenoms. The efficacy of the antivenoms is widely accepted, and standard venoms are expected to be representative of the snake's population that inhabit in the region where the antivenom is intended to be used. The representativeness of a single venom collected from a Crotalus simus snake, and its usefulness as standard venom to produce an antivenom is evaluated. The use of an “average venom” might be as representative of the population intended to be used, as the standard venom composed by many venom samples. Variations in the relative abundance concentration of crotoxin in the C. simus leads to different clinical manifestations, as well as differences in the neutralization efficacy of the antivenoms. A monovalent anti-Cs antivenom was produced from a single venom C. simus specimen, and its efficacy in neutralizing the lethal activity of 30 C. simus snakes was tested. Despite the variations in the relative abundance content of crotoxin found in the proteomes, the monovalent anti-Cs antivenom was successful in neutralize the toxicity caused by the variations on the venom composition of three different snake population used. Interestingly, it seems that the sex is not a key factor in the lethality of the venoms tested. The concept of representative venom mixtures for immunization should be revised for the case of C. simus on the populations found in Costa Rica, since it might use as less as one representative individual whose venom covers the mainly toxic enzymes.

For the past 80 years, Crotoxin has become one of the most investigated isolated toxins from snake venoms, partially due to its major role as the main toxic component in the venom of the South American rattlesnake Crotalus durissus terrificus. However, in the past decades, progressive studies have led researchers to shift their focus on Crotoxin, opening novel perspectives and applications as a therapeutic approach. Although this toxin acts on a wide variety of biological events, the modulation of immune responses is considered as one of its most relevant behaviors. Therefore, the present review describes the scientific investigations on the capacity of Crotoxin to modulate anti-inflammatory and immunosuppressive responses, and its application as a medicinal immunopharmacological approach. In addition, this review will also discuss its mechanisms, involving cellular and molecular pathways, capable of improving pathological alterations related to immune-associated disorders.

Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype of breast cancer. Currently, aromatase inhibitors (AIs) are used as first-line treatment option in this type of tumors, however they cause several side effects, which is why new therapeutic approaches are demanding. The South American rattlesnake Crotalus durissus terrificus produces a venom enriched in several bioactive substances, like phospholipases A2 (PLA2). One of those is crotoxin, a β-neurotoxin, that has already been reported for its anti-cancer properties in different cancers. Recently, its clinical interest has emerged and, in fact, a clinical trial in patients with advanced cancer is underway. Considering this, in this work, we studied the biological mechanisms behind the anti-cancer effects of crotoxin B (CTX) in an ER+ aromatase-overexpressing breast cancer cell line (MCF-7aro cells). Results revealed that CTX impairs MCF-7aro cells growth, through a cell cycle arrest at G2/M phase, inhibition of ERK1/2 pathway and by apoptosis through activation of caspase-8. In addition, it can be considered a safe natural compound as did not affect non-cancerous cells and only showed anti-growth effects in breast cancer cells. Therefore, this study represents an important landmark to better understand the effects and mechanisms of action of crotoxin in ER+ breast cancer.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic &beta, neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1&ndash, 4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

Crotalus Neutralizing Factor (CNF) was the first phospholipase A2 inhibitor isolated from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Previous biochemical and biophysical studies demonstrate an interaction of CNF with Crotoxin (CTX), the main toxic component in the venom of these snakes. CTX promotes the blockade of neuromuscular transmission by a sum of neurotoxic and myotoxic activities. However, the ability of CNF to inhibit these activities has not been shown until the present study. We performed a myographic study to compare the neuromuscular effects of CTX and the mixture CTX plus CNF in mice phrenic nerve-diaphragm muscle preparations. CTX (5 μg/mL) alone, or pre-incubated with CNF (5, 20 or 50 μg/mL) for 15 min was added to the preparations and maintained throughout the experimentation period. Myotoxicity was assessed by light microscopic analysis of diaphragm muscle after myographic study. CTX (5 μg/mL) blocked both indirectly and directly evoked twitches in neuromuscular preparations. In addition, CTX induced histological alterations in diaphragm muscle. Pre-incubation with CNF (50 μg/mL) abolished both the muscle-paralyzing and muscle-damaging activities of CTX. Therefore, the present study confirms, through functional studies, the antiophidic potential of CNF.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers, however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity, SVSPs, which deplete fibrinogen, and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection, however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

Snakebite in Mexico is commonly treated with an antivenom which uses Bothrops asper and Crotalus simus venoms as immunogens. Current taxonomic recommendations for the C. simus species complex suggest a novel endemic species from Mexico: Crotalus mictlantecuhtli. The aim of this report was to evaluate the immunogenic properties of C. mictlantecuhtli venom and its potential to generate polyclonal antibodies capable of neutralizing other pitviper venoms. We generated an experimental anti-Crotalus mictlantecuhtli serum, using the rabbit model, to test recognition and neutralizing capacity against the homologous venom as well as venoms from C. atrox, C.basiliscus, C. durissus terrificus, C. scutulatus salvini, C. tzabcan and Ophryacus sphenophrys. Pre-incubation neutralization experiments using our experimental serum showed positive results against venoms containing crotoxin, while venoms from two non-neurotoxic pit-vipers were not neutralized. Rescue experiments in mice showed that, when intravenously injected (i.v.), C. mictlantecuhtli venom is not neutralized by a maximum dose of Antivipmyn® and the experimental serum after 5 min of envenomation, albeit mice envenomated intraperitoneally (i.p.) and rescued i.v. with Antivipmyn® survived even at 50 min after envenomation. Our results highlight the importance of using the highly neurotoxic C. mictlantecuhtli venom to increase antivenom effectiveness against Mexican neurotoxic pitvipers.

Respiratory compromise in Crotalus durissus terrificus (C.d.t.) snakebite is an important pathological condition. Considering that crotoxin (CTX), a phospholipase A2 from C.d.t. venom, is the main component of the venom, the present work investigated the toxin effects on respiratory failure. Lung mechanics, morphology and soluble markers were evaluated from Swiss male mice, and mechanism determined using drugs/inhibitors of eicosanoids biosynthesis pathway and autonomic nervous system. Acute respiratory failure was observed, with an early phase (within 2 h) characterized by enhanced presence of eicosanoids, including prostaglandin E2, that accounted for the increased vascular permeability in the lung. The alterations of early phase were inhibited by indomethacin. The late phase (peaked 12 h) was marked by neutrophil infiltration, presence of pro-inflammatory cytokines/chemokines, and morphological alterations characterized by alveolar septal thickening and bronchoconstriction. In addition, lung mechanical function was impaired, with decreased lung compliance and inspiratory capacity. Hexamethonium, a nicotinic acetylcholine receptor antagonist, hampered late phase damages indicating that CTX-induced lung impairment could be associated with cholinergic transmission. The findings reported herein highlight the impact of CTX on respiratory compromise, and introduce the use of nicotinic blockers and prostanoids biosynthesis inhibitors as possible symptomatic therapy to Crotalus durissus terrificus snakebite.

11 páginas, 6 figuras y 2 yablas. Se puede consultar información complementaria en: https:// doi.org/10.1016/j.jprot.2020.103865., We report a structural and functional venomics characterization of the black-tailed horned pitviper, Mixcoatlus melanurus. The venom phenotype of this small and elusive pitviper endemic to México comprise peptides and proteins of 16 toxin families whose relative abundance mirror those of neurotoxic (type II) venoms described for some species within genera distributed in Central Asia (Gloydius) and the Americas (Sistrurus, Crotalus, Ophryacus, and Bothriechis). A novel β-neurotoxic heterodimeric PLA2, termed Melanurutoxin was characterized. With a relative abundance of 14.8% of the total M. melanurus venom proteome and a median lethal dose of 0.31 μg/g mouse body weight, Melanurutoxin accounted for 37.8% of the lethality of the whole venom (0.82 μg/g). The low percentage (1.1%) of snake venom metalloproteinases (PIII-SVMPs) and the high content of Melanurutoxin and bradykinin-potentiating peptides (BPP, 16%) found in the type-II venom proteome of M. melanurus correlate with the severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest observed in ex vivo neuromuscular junction experiments and in vivo experimental murine envenoming. Mexican antivenoms manufactured by Birmex and Bioclon showed low neutralization potency per vial (95 LD50s, Birmex; 114 LD50s, Antivipmyn®), and failed to reverse completely the paralysis and the hypotensive effect induced by the black-tailed horned pitviper, Mixcoatlus melanurus. We suggest that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom may be attributed to the use of immunization mixtures that include venom of taxa, C. basiliscus (Birmex) and C. simus (Antivipmyn®), that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s. BIOLOGICAL SIGNIFICANCE: This study represents the first proteomics and funcional investigations conducted on the venom of the black-tailed horned, Mixcoatlus melanurus, a pitviper species endemic to México. The venom's features unveiled through combination of bottom-up venomics and ex vivo and in vivo functional assays provided complementary evidence pointing to severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest as the predominant mechanism of murine prey immobilization and death caused by M. melanurus. A novel β-neurotoxic heterodimeric PLA2, coined Melanurutoxin, was identified as a major contributor to the lethality of the whole venom. Our study also showed the inefficacy of two commercial Mexican antivenoms to reverse competely the paralytic and hypotensive effects induced by M. melanurus venom in the murine model. We hypothesize that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom should be ascribed to the use as immunogens of venoms that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s., This research was partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain), grants PAPIIT-DGAPA IN207218 and CONACyT-FORjDECYT No. 303045 (“Venenos y Antivenenos”).

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

Neutrophils have a critical role in the innate immune response; these cells represent the primary line of defense against invading pathogens or tissue injury. Crotoxin (CTX), the major toxin of the South American rattlesnake (Crotalus durissus terrificus) venom, presents longstanding anti-inflammatory properties, inhibiting neutrophil migration and phagocytosis by peritoneal neutrophils for 14 days. Herein, to elucidate these sustained inhibitory effects induced by CTX, we performed in vitro and in vivo studies evaluating the functionality of bone marrow neutrophils and possible molecular mechanisms associated with these effects. CTX inhibited the processes of chemotaxis, adhesion to fibronectin, and phagocytosis of opsonized particles; however, it did not affect ROS production or degranulation in bone marrow neutrophils. To understand the molecular mechanisms that orchestrate this effect, we investigated the expression of CR3 on the neutrophil surface and the total expression and activity of signaling proteins from the Syk-GTPase pathway, which is involved in actin polymerization. CTX down-regulated both subunits of CR3, as well as, the activity of Syk, Vav1, Cdc42, Rac1 and RhoA, and the expression of the subunit 1B from Arp2/3. Together, our findings demonstrated that CTX inhibits the functionally of bone marrow neutrophils and that this effect may be associated with an impairment of the Syk-GTPase pathway. This study demonstrates, for the first time, that the sustained down-modulatory effect of CTX on circulating and peritoneal neutrophils is associated with functional modifications of neutrophils still in the bone marrow, and it also contributes to a better understanding of the anti-inflammatory effect of CTX.

Federal University of Para. Institute of Biological Sciences. Laboratory of Parasitology and Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Parasitology and Laboratory of Structural Biology. Bel?m, PA, Brazil. University of S?o Paulo. Department,Institute of Biomedical Sciences. Cell and Developmental Biology. S?o Paulo, SP, Brazil. Butantan Institute. Laboratory of Pathophysiology. S?o Paulo, SP, Brazil / University of S?o Paulo. Institute of Biomedical Sciences. Department of Pharmacology. S?o Paulo, SP, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Parasitology and Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22?86 ?g mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-? and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

Background Snakebite treatment requires administration of an appropriate antivenom that should contain antibodies capable of neutralizing the venom. To achieve this goal, antivenom production must start from a suitable immunization protocol and proper venom mixtures. In Brazil, antivenom against South American rattlesnake (Crotalus durissus terrificus) bites is produced by public institutions based on the guidelines defined by the regulatory agency of the Brazilian Ministry of Health, ANVISA. However, each institution uses its own mixture of rattlesnake venom antigens. Previous works have shown that crotamine, a toxin found in Crolatus durissus venom, shows marked individual and populational variation. In addition, serum produced from crotamine-negative venoms fails to recognize this molecule. Methods In this work, we used an antivenomics approach to assess the cross-reactivity of crotalic antivenom manufactured by IVB towards crotamine-negative venom and a mixture of crotamine-negative/crotamine-positive venoms. Results We show that the venom mixture containing 20% crotamine and 57% crotoxin produced a strong immunogenic response in horses. Antivenom raised against this venom mixture reacted with most venom components including crotamine and crotoxin, in contrast to the antivenom raised against crotamine-negative venom. Conclusions These results indicate that venomic databases and antivenomics analysis provide a useful approach for choosing the better venom mixture for antibody production and for the subsequent screening of antivenom cross-reactivity with relevant snake venom components.

Snake venom may vary in composition and toxicity across the geographic distribution of a species. In the case of the three species of the Neotropical rattlesnakes Crotalus simus, C. culminatus and C. tzabcan recent research has revealed that their venoms can contain a neurotoxic component (crotoxin homologs), but is not always the case. In the present work, we detected and quantified crotoxin homologs in venom samples from three species distributed across Mexico, to describe variation at the individual and subspecific levels, using slot blot and ELISA immunoassays. We found that all C. simus individuals analyzed had substantial percentages of crotoxin homologs in their venoms (7.6–44.3%). In contrast, C. culminatus lacked them completely and six of ten individuals of the species C. tzabcan had low percentages (3.0–7.7%). We also found a direct relationship between the lethality of a venom and the percentage of crotoxin homologs it contained, indicating that the quantity of this component influences venom lethality in the rattlesnake C. simus., Highlights • Monoclonal antibodies were produced that specifically recognized crotoxin homologs in venoms of Crotalus species. • Crotoxin homologs were quantified in three species of Crotalus: C. simus, C. culminatus and C. tzabcan. • All specimens of C. simus contained crotoxin homologs at different levels, while C. culminatus venoms lacked them completely. • In C. tzabcan, some venoms possess and other lack crotoxin homologs.

Accepted manuscript, Crotoxin (CTX), a heterodimeric phospholipase present in venom of snakes of the genus Crotalus, has demonstrated a broad spectrum of pharmacological properties, such as antimicrobial, hemostatic, and antitumoral. However, the precise mechanism of its cytotoxicity and antitumoral properties remains to be determined. Therefore, in the present study, we isolated crotoxin (F1 CTX) through two steps DEAE-Sepharose and Heparin-Sepharose FF chromatography. The C-terminal sequence of the A- and B-chain protein fragment was determined by LC-MS/MS mass spectrometry, which showed 100% identity to crotoxin structure. In order to investigate its cytotoxic effects, we demonstrated that the F1 CTX fraction at 0-30 μg/mL concentrations for 72 h presented a heterogeneous response profile on nine human cancer-derived cell lines from four tumor types (pancreatic, esophagus, cervical cancer, and glioma). The glioma (GAMG and HCB151) and pancreatic (PSN-1 and PANC-1) cancer cells showed a higher sensitivity with IC50 of, FAPESP, the Hermínio Ometto University Center, the Hospital de Cancer de Barretos and FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT). We acknowledge the Mass Spectrometry Laboratory of the Brazilian Biosciences National Laboratory, CNPEM, Campinas, Brazil, for providing support on mass spectrometry analysis, info:eu-repo/semantics/publishedVersion

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 &mu, g/mL) when compared to CTX-containing mouthwash (MIC = 750 &mu, g/mL) and nystatin (MIC = 375 &mu, g/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 &mu, g/mL) when compared to nystatin (IC100 &gt, 47 &mu, g/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 &micro, g/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

Venoms of the three species of Ophryacus (O. sphenophrys, O. smaragdinus, and O. undulatus), a viperid genus endemic to Mexico, were analyzed for the first time in the present work. The three venoms lacked procoagulant activity on human plasma, but induced hemorrhage and were highly lethal to mice. These venoms also displayed proteolytic and phospholipase A2 activities in vitro. The venom of O. sphenophrys was the most lethal and caused hind-limb paralysis in mice. Proteomic profiling of O. sphenophrys venom showed a predominance of metalloproteinase (34.9%), phospholipase A2 (24.8%) and serine protease (17.1%) in its composition. Strikingly, within its PLA2 components, 12.9% corresponded to a Crotoxin-like heterodimer, here named Sphenotoxin, which was not found in the other two species of Ophryacus. Sphenotoxin, like Crotoxin, is composed of non-covalently bound A and B subunits. Partial amino acid sequence was obtained for Sphenotoxin B and was similar (78–89%) to other subunits described. The mouse i.v. LD50 of Sphenotoxin at 1:1M radio was 0.16 μg/g. Also, like Crotoxin, Sphenotoxin induced a potent neuromuscular blockade in the phrenic nerve-diaphragm preparation. Ophryacus is the fifth genus and O. sphenophrys the third non-rattlesnake species shown to contain a novel Crotoxin-like heterodimeric β-neurotoxin. Biological significance: Ophryacus is an endemic genus of semi-arboreal pitvipers from Mexico that includes three species with restricted distributions. Little is known about the natural history of these species and nothing is known about the properties of their venoms. Research on these species' venoms could generate relevant information regarding venom composition of Mexican pitvipers. Additionally, research into the presence of neurotoxic Crotoxin-like molecules outside of rattlesnakes (genera Crotalus and Sistrurus) has identified this molecule in several new genera. Knowing which genera and species possess neurotoxic components is important to fully understand the repercussions of snakebites, the interaction with prey and predators, and the origin, evolution, and phylogenetic distribution of Crotoxin-like molecules during the evolutionary history of pitvipers. Our study expands current knowledge regarding venom's compositions and function from Mexican pitvipers, providing a comparative venom characterization of major activities in the three Ophryacus species. Additionally, the discovery and characterization of a novel Crotoxin-like molecule, here named Sphenotoxin, in O. sphenophrys, and the detailed protein composition of O. sphenophrys venom supports the hypotheses that Crotoxin-like -β-neurotoxins are more widespread than initially thought. Dirección General de Asuntos del Personal Académico/[DGAPA-PAPIIT: IN207218]/DGAPA/México Consejo Nacional de Ciencia y Tecnología/[703353]/CONACyT/Costa Rica Consejo Nacional de Ciencia y Tecnología/[254145]/CONACyT/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB throughWestern blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

Made available in DSpace on 2018-12-11T17:16:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-28. Added 1 bitstream(s) on 2021-07-15T15:21:55Z : No. of bitstreams: 1 S1678-91992017000100319.pdf: 1593656 bytes, checksum: d71e67eab0373767c2647904c1d05536 (MD5) Background: Classically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A2 and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported. Methods: The crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing. Results: The RP-HPLC profile of the isolated crotapotin chains already indicated that the α chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses. Conclusion: It was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the α chain, at positions 31 and 40. Moreover, substitutions could also be observed in β and γ chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom. Sao Paulo State University (UNESP) Postgraduate Program in Tropical Diseases Botucatu Medical School São Paulo State University (UNESP) Center for the Studies of Venoms and Venomous Animals (CEVAP) Butantan Institute Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500 Sao Paulo State University (UNESP) Postgraduate Program in Tropical Diseases Botucatu Medical School São Paulo State University (UNESP) Center for the Studies of Venoms and Venomous Animals (CEVAP)

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A2-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value

Human phospholipase A2 (hPLA2) of the IIA group (HGIIA) catalyzes the hydrolysis of membrane phospholipids, producing arachidonic acid and originating potent inflammatory mediators. Therefore, molecules that can inhibit this enzyme are a source of potential anti-inflammatory drugs, with different action mechanisms of known anti-inflammatory agents. For the study and development of new anti-inflammatory drugs with this action mechanism, snake venom PLA2 (svPLA2) can be employed, since the svPLA2 has high similarity with the human PLA2 HGIIA. Despite the high similarity between these secretory PLA2s, it is still not clear if these toxins can really be employed as an experimental model to predict the interactions that occur with the human PLA2 HGIIA and its inhibitors. Thus, the present study aims to compare and evaluate, by means of theoretical calculations, docking and molecular dynamics simulations, as well as experimental studies, the interactions of human PLA2 HGIIA and two svPLA2s, Bothrops toxin II and Crotoxin B (BthTX-II and CB, respectively). Our theoretical findings corroborate experimental data and point out that the human PLA2 HGIIA and svPLA2 BthTX-II lead to similar interactions with the studied compounds. From our results, the svPLA2 BthTX-II can be used as an experimental model for the development of anti-inflammatory drugs for therapy in humans.

A myographic study was performed to compare the neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis in mice phrenic-diaphragm preparation. It was concluded that both venoms present neurotoxic activity as a consequence of their crotoxin content. Furthermore, crotoxin from C.d. cumanensis is more potent than that from C.d. ruruima venom. At the concentration range in which both venoms express neurotoxic activity, only C.d. cumanensis venom also manifest a direct myotoxic effect that probably involves the synergic participation of other components than crotoxin.

artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado, 2015. Investigación apoyada por ICGEB-Italia, CYTED, Ministerio de Economía y Competitividad de España. Este documento es privado debido a limitaciones de derechos de autor. Bothriechis nigroviridis is an arboreal Neotropical pitviper found in Costa Rica and Panam a. A previous proteomic profiling of its venom revealed the presence of proteins with homology to the A and B subunits of crotoxin/Mojave toxin, a heterodimeric phospholipase A2 (PLA2) complex only described in rattlesnake venoms (genera Crotalus and Sistrurus). The native crotoxin-like heterodimer, named nigroviriditoxin, and its A and B subunits were isolated in the present work, and the complete amino acid sequence of the B subunit was determined. The purified A and B components were demonstrated to form a complex when reconstituted under native conditions. Nigroviriditoxin presents features similar to crotoxin, albeit displaying lower toxicity: the A component decreases the PLA2 activity of the B component, and increases its lethal potency in mice. Also in similarity to crotoxin B, nigroviriditoxin B induces myonecrosis. Its 122 amino acid sequence presents 81% identity with crotoxin B. Accordingly, nigroviriditoxin B was cross-recognized by equine antibodies from a Crotalus durissus terrificus antivenom. Phylogenetic analysis shows that the novel PLA2 from B. nigroviridis venom is basal to the branch including all the homologous PLA2 enzymes described in rattlesnakes, and more distant from PLA2s from Bothriechis species. Nigroviriditoxin is the first heterodimeric PLA2 complex found in a non-rattlesnake, Neotropical viperid venom, which displays structural, functional, and immunochemical similarities to crotoxin. The present findings are compatible with the existence of the particular structural trait of crotoxin-like molecules in New World pitvipers before the split of the Meso-South American and the Nearctic clades. Universidad de Costa Rica. ICGEB-Italia. CYTED. Ministerio de Economía y Competitividad de España. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Heme oxygenase-1 (HO-1, Hmox1 ) regulates viability, proliferation, and differentiation of many cell types; hence, it may affect regeneration of injured skeletal muscle. Here, we injected cardiotoxin into gastrocnemius muscle of Hmox1 +/+ and Hmox1 −/− animals and analyzed cellular response after muscle injury, focusing on muscle satellite cells (SCs), inflammatory reaction, fibrosis, and formation of new blood vessels. HO-1 is strongly induced after muscle injury, being expressed mostly in the infiltrating leukocytes (CD45 + cells), including macrophages (F4/80 + cells). Lack of HO-1 augments skeletal muscle injury, evidenced by increased creatinine kinase and lactate dehydrogenase, as well as expression of monocyte chemoattractant protein-1, IL-6, IL-1β, and insulin-like growth factor-1. This, together with disturbed proportion of M1/M2 macrophages, accompanied by enhanced formation of arterioles, may be responsible for shift of Hmox1 −/− myofiber size distribution toward larger one. Importantly, HO-1–deficient SCs are prone to activation and have higher proliferation on injury. This effect can be partially mimicked by stimulation of Hmox1 +/+ SCs with monocyte chemoattractant protein–1, IL-6, IL-1β, and is associated with increased MyoD expression, suggesting that Hmox1 −/− SCs are shifted toward more differentiated myogenic population. However, multiple rounds of degeneration/regeneration in conditions of HO-1 deficiency may lead to exhaustion of SC pool, and the number of SCs is decreased in old Hmox1 −/− mice. In summary, HO-1 modulates muscle repair mechanisms preventing its uncontrolled acceleration.

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

Crotoxin (CrTX) is the primary toxin in South American rattlesnake (Crotalus durissus terrificus) venom, and exhibits antitumor and other pharmacological actions in vivo and in vitro. Here, we investigated the molecular mechanisms of the antitumor action of CrTX in human lung carcinoma cells in vitro.Human lung squamous carcinoma SK-MES-1 cells were tested. The cytotoxicity of CrTX was evaluated in both MTT and colony formation assays. Cell cycle was investigated with flow cytometry. Cell apoptosis was studied with Hoechst 33258 and Annexin V-FITC staining. The levels of relevant proteins were analyzed using Western blot assays.CrTX (25, 50, 100 μmol/L) inhibited the growth and colony formation of SK-MES-1 cells in dose- and time-dependent manners. CrTX increased the proportion of S phase cells and dose-dependently induced cell apoptosis, accompanied by down-regulating the expression of proliferating cell nuclear antigen (PCNA), and increasing the level of cleaved caspase-3. Furthermore, CrTX dose-dependently increased the expression of autophagy-related proteins LC3-II and beclin 1, and decreased the level of p62 in the cells. Moreover, CrTX (50 μmol/L) significantly increased p38MAPK phosphorylation in the cells. Pretreatment of the cells with SB203580, a specific inhibitor of p38MAPK, blocked the inhibition of CrTX on cell proliferation, as well as CrTX-induced apoptosis and cleaved caspase-3 expression.The p38MAPK signaling pathway mediates CrTX-induced apoptosis and autophagy of human lung carcinoma SK-MES-1 cells in vitro.

Made available in DSpace on 2018-12-11T17:31:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-03 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation. Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP Departamento de Física Faculdade de Filosofia Ciências e Letras de Ribeirão Preto USP Departamento de Física Experimental Instituto de Física Universidade de São Paulo - USP Departamento de Farmacologia Instituto de Ciências Biológicas UFMG Diretoria de Pesquisa e Desenvolvimento Fundação Ezequiel Dias (FUNED) Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP CAPES: 063/2011 CAPES: 1592/2011 FAPESP: 2013/17864-8 FAPESP: 2014/26859-7 FAPESP: 2015/17286-0 CNPq: 300596/2013-8 CNPq: 300908/2012-1 CNPq: 304981/2012-5

Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom.

In this study, we evaluated the effect of the Crotalus durissus terrificus (Cdt) venom on the chronic paw edema induced by the injection of bacillus Calmette-Guerin (BCG) into the footpad of mice. The BCG injection evoked chronic edema, which was significantly diminished in animals treated subcutaneously (s.c.) with Cdt venom 1 h before or after the BCG injection. This inhibition persisted throughout the evaluation period (15 days). In mice injected with Cdt venom 6 or 11 days after injection of BCG, we observed a significant reduction in edema only in the period after the venom injection. While studying possible mechanisms involved in this inhibition, we observed that pre-treatment with dexamethasone, zileuton or Boc2 (a selective antagonist of formyl peptide receptors), but not with indomethacin, canceled out the inhibitory effect of Cdt venom on the edema induced by BCG. These results strongly suggest that this rattlesnake venom can stimulate the generation of mediators from the lipoxygenase pathway, which can down-regulate this chronic inflammatory edema. Using fractionated venom, the results indicated that crotoxin was the only component of Cdt venom responsible for this inhibitory effect. These results indicated that crotoxin, the main toxin of the C. durissus terrificus venom, has a significant inhibitory effect on BCG-induced chronic edema, possibly by generating anti-inflammatory mediators from the lipoxygenase pathway.

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca(2+) and Mg(2+)) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA(2) through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.

The venom of the Mexican west-coast rattlesnake ( Crotalus basiliscus ) was characterized for its protein composition, toxicological profile and immunogenic properties. This venom is composed of 68% Zn 2 + -dependent metalloproteinases (SVMPs), 14% phospholipases A 2 (PLA 2 s), 11% serine proteinases, 4% SVMPs-inhibitor tripeptides (SVMP-ITs), 2% bradykinin-potentiating peptides (BPPs), 0.6% cysteine-rich secretory proteins (CRISPs), and 0.2% l -amino acid oxidases (LAAOs). SVMPs present in the venom are responsible for azocasein hydrolysis and hemorrhagic activity, but their contribution to the lethal activity of the venom in mice is masked by the neurotoxic activity of PLA 2 s, which in addition are also responsible for myotoxic activity. Despite its relatively high content of serine proteinases, the venom of C. basiliscus did not exert in vitro coagulant or in vivo defibrinogenating activities. The ability of antivenoms raised against the venoms of C. basiliscus and C. simus (from Costa Rica) to neutralize homologous and heterologous venoms revealed antigenic similarities between toxins of both venoms. Preclinical evaluation of an antivenom produced by using the venom of C. basiliscus as immunogen demonstrated that it is able to neutralize not only the most relevant toxic activities of C. basiliscus venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. Biological significance The Central American rattlesnake ( Crotalus simus ) is widely distributed from Mexico to west central Costa Rica, and induces an important number of envenomations in this region. On the other hand, the immunogenic mixture used by Laboratorios de Biologicos y Reactivos de Mexico S.A. (Birmex) to produce the snake antivenom more frequently used in Mexico does not include the venom of C. simus . This immunogenic mixture is composed by the venoms of the Fer-de-lance ( Bothrops asper ) and the Mexican west-coast rattlesnake ( Crotalus basiliscus ). We studied the protein composition, toxicological profile and immunogenic properties of the venom of C. basiliscus , and evaluated the ability of the Birmex antivenom to neutralize the venom of C. basiliscus and whether it cross-neutralizes the venom of C. simus from Costa Rica. Using proteomics analysis, in combination with in vitro and mouse tests, we determined that the venom of C. basiliscus is mainly composed by SVMPs, which confer proteolytic and hemorrhagic activities to the venom. Other major components of the venom of C. basiliscus are PLA 2 s, which are responsible for the myotoxic activity and are the main contributors to the lethal activity. Non-clotting SVSPs correspond to 11% of the venom. Minor components include SVMP-ITs, BPPs, CRISPs and LAAOs, which have not been associated with toxicity. The antibodies induced in horses by the venom of C. basiliscus are able to neutralize not only the most relevant toxic activities of the homologous venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. Our preclinical evaluation suggests that Birmex antivenom can be used to treat envenomations by Costa Rican adult C. simus snakebites, despite this venom not being included in the immunizing mixture.

International audience; Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.

Made available in DSpace on 2022-04-29T07:32:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 Graduate Program in Tropical Diseases Botucatu Medical School Universidade Estadual Paulista Center for the Studies of Venoms and Venomous Animals Universidade Estadual Paulista Institute of Biosciences of Rio Claro Universidade Estadual Paulista Anaphylaxis Committee of the Brazilian Association of Allergy and Immunology National Institutes for Science and Technology Graduate Program in Tropical Diseases Botucatu Medical School Universidade Estadual Paulista Center for the Studies of Venoms and Venomous Animals Universidade Estadual Paulista Institute of Biosciences of Rio Claro Universidade Estadual Paulista

Crotoxin, one of the major toxins of South American rattlesnake Crotalus durissus subspecies, is an heterodimeric complex composed of two distinct subunits: a basic phospholipase A(2) (PLA(2), CB) and an acidic nontoxic catalytically inactive protein, crotapotin (CA). It's well known that CB has a high enzymatic activity; however the molecular aspects that determine this fact remain unknown. In this study, an in silico approach was used to predict the CA structure by homology modeling, and the crotoxin structure by means of molecular docking. CA structure was built using the software Modeller taking Crotalus atrox PLA(2) (1PP2:R) as a template. Different criteria measured by Procheck, Verify 3D and ProSA were indicative of the reliability and the proper fold for the predicted structural model of CA. Then, a combination of this model and CB crystal structure was used to build the structure of crotoxin complex through rigid-body protein-protein docking. The crotoxin-3D model suggested that by means of hydrophobic and π-π stacking interactions, CA-Y24 and CA-F119 interact with CB-F24 and CB-F119, respectively. Those interactions could prevent the interfacial adsorption of the CB onto the lipid/water interface by blocking part of the interfacial binding surface of the PLA(2). This fact could explain the differences regarding to enzymatic activity between the crotoxin complex and CB. In addition, the crotoxin-3D model showed solvent-exposed regions of CA that could bind the receptor expressed in target cells.

To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.Treatment of A549 cells with CrTX (25-200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC(50)=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G(1) arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.

The calcium-calmodulin-regulated protein phosphatase calcineurin plays an important regulatory role in muscle differentiation, fiber-type determination, hypertrophy, and muscle regeneration. Because calcineurin functions in numerous processes in muscle, multiple mechanisms are likely necessary to ensure that the activity of this phosphatase is appropriately regulated. Here we demonstrate that the muscle-specific scaffolding protein myospryn modulates calcineurin signaling by inhibiting calcineurin-dependent transcriptional activity in C2C12 myoblasts through direct interaction with the enzyme via its noncanonical tripartite motif (TRIM-like). Consistent with these data, transgenic mice overexpressing both the TRIM-like domain of myospryn and constitutively active calcineurin displayed a severe attenuation in the ability of calcineurin to induce a slow-fiber phenotype. Furthermore, transgenic mice overexpressing the TRIM-like domain of myospryn displayed attenuated muscle regeneration after cardiotoxin-induced muscle injury. These results indicate that myospryn functions as a novel inhibitor of the calcineurin signaling pathway in skeletal muscle.—Kielbasa, O. M., Reynolds, J. G., Wu, C-l., Snyder, C. M., Cho, M. Y., Weiler, H., Kandarian, S., Naya, F. J. Myospryn is a calcineurin-interacting protein that negatively modulates slow-fiber-type transformation and skeletal muscle regeneration.

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund’s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated.

Made available in DSpace on 2013-08-12T19:08:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-01 Made available in DSpace on 2013-09-30T17:41:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:12:23Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:12:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-01 Item merged in doublecheck by Felipe Arakaki (fe.arakaki@gmail.com) on 2015-11-17T12:58:55Z Item was identical to item(s): 72164, 335 at handle(s): http://hdl.handle.net/11449/72682, http://hdl.handle.net/11449/369 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) In this study, we evaluated the actions of Crotalus durissus cumanensis venom (CDCmV), and its crotoxin (Crtx) fraction, on renal and vascular functions in Wistar rats. In isolated perfused kidneys, CDCmV (10 mu g/mL) significantly increased the perfusion pressure (PP) from 110.7 +/- 2.4 to 125.3 +/- 2.8 mmHg after 30 minutes. This effect was accompanied by an increased renal vascular resistance (RVR) from 5.4 +/- 0.1 to 6.2 +/- 0.2 mmHg/mL. g(-1). min(-1). We observed decreases in urinary flow (UF) from 0.13 +/- 0.01 to 0.05 +/- 001 mL. g(-1). min(-1) and glomerular filtration rate (GFR) from 0.66 +/- 0.06 to 0.18 +/- 0.02 mL. g(-1). min(-1). Crtx did not change PP or RVR, but diminished GFR (from 0.65 +/- 0.05 to 0.26 +/- 003 mL. g(-1). min(-1)) and UF (from 0.11 +/- 0.008 to 0.09 +/- 0.008 mL. g(-1). min(-1)). Both CDCmV and Crtx reduced the percentage of tubular transport of sodium, chloride and potassium. The cytotoxicity of these substances against MDCK cells was tested by the MTT method: only CDCmV caused a decrease in the cell viability with an IC50 of 5.4 mu g/mL. In endothelium-intact isolated aortic rings, CDCmV (0.1 to 30 mu g/mL) increased the sustained phenylephrine-induced contraction to a value of 130.0 +/- 6.6% of its corresponding control, but showed a relaxant effect in endothelium-denuded preparations. Similar results were observed in aortic rings contracted with potassium (40 mM). Crtx was ineffective in aortic ring assays. Thus, it is reasonable to suggest that the renal effects induced by the CDCmV may be due to its influence on the endothelium's ability to release factors that can alter the contractile behavior of vascular smooth muscle. In conclusion, CDCmV is toxic to kidney cells. It changes parameters of the renal function including the glomerular filtration rate, renal vascular resistance and tubular transport. The actions induced by CDCmV also involve endothelium-dependent vasoactive properties. Their effects may be only partially attributed to Crtx. Universidade Federal do Ceará (UFC), Dept Anal Clin & Toxicol, Fac Farm, BR-60420970 Fortaleza, Ceara, Brazil Universidade Federal do Ceará (UFC), Dept Physiol & Pharmacol, BR-60420970 Fortaleza, Ceara, Brazil UNESP Univ Estadual Paulista, São Paulo State Univ, Sao Vicente, SP, Brazil Univ Estadual Campinas, Dept Biochem, Campinas, SP, Brazil UNESP Univ Estadual Paulista, São Paulo State Univ, Sao Vicente, SP, Brazil

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric beta-neurotoxin that consists of a weakly toxic basic phospholipase A(2) and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin.

In the present study, it was investigated which components are responsible for the anti-inflammatory properties of Crotalus durissus terrificus venom (CdtV). The effect of crotoxin, as well as of other CdtV components was evaluated on edema, cell migration and alterations in leukocyte-endothelium interactions induced by carrageenan. Crotoxin (40 microg kg(-1)) was injected at different time periods before or after the injection of carrageenan (15 mg kg(-1)) into the mouse hind paw, peritoneum or scrotum. Results showed that crotoxin, but not other CdtV components, significantly inhibited inflammatory edema and cell migration when administered before or after carrageenan injection in mice. This toxin also prevented the occurrence of alterations in leukocyte-endothelium interactions induced by carrageenan injection, such as the increase in adhered cells. In animals pretreated with Boc2 (a selective antagonist of formyl peptide receptors), crotoxin showed neither inhibitory effects on edema and cell migration, nor prevented alterations in leukocyte-endothelium interactions induced by carrageenan. These findings demonstrate that crotoxin is the component responsible for the long-lasting anti-inflammatory activity of crude C. durissus terrificus venom, and activation of formyl peptide receptors seems to play a major role in this effect.

The basic knowledge on neoplasms is increasing quickly; however, few advances have been achieved in clinical therapy against tumors. For this reason, the development of alternative drugs is relevant in the attempt to improve prognosis and to increase patients' survival. Snake venoms are natural sources of bioactive substances with therapeutic potential. The objective of this work was to identify and characterize the antitumoral effect of Crotalus durissus terrificus venom (CV) and its polypeptide, crotoxin, on benign and malignant tumors, respectively, pituitary adenoma and glioblastoma. The results demonstrated that CV possess a powerful antitumoral effect on benign (pituitary adenoma) and malignant (glioblastoma multiforme) tumors with IC50 values of 0.96 ± 0.11 µg/mL and 2.15 ± 0.2 µg/mL, respectively. This antitumoral effect is cell-cycle-specific and dependent on extracellular calcium, an important factor for crotoxin phospholipase A2 activity. The CV antitumoral effect can be ascribed, at least partially, to the polypeptide crotoxin that also induced brain tumor cell death. In spite of the known CV nephrotoxicity and neurotoxicity, acute treatment with its antitumoral dose established in vitro was not found to be toxic to the analyzed animals. These results indicate the biotechnological potential of CV as a source of pharmaceutical templates for cancer therapy.

We report a comparative venomic and antivenomic characterization of the venoms of newborn and adult specimens of the Central American rattlesnake, Crotalus simus, and of the subspecies cumanensis, durissus, ruruima, and terrificus of South American Crotalus durissus. Neonate and adult C. simus share about 50% of their venom proteome. The venom proteome of 6-week-old C. simus is predominantly made of the neurotoxic heterodimeric phospholipase A2 (PLA2 crotoxin) (55.9%) and serine proteinases (36%), whereas snake venom Zn2+-metalloproteinases (SVMPs), exclusively of class PIII, represent only 2% of the total venom proteins. In marked contrast, venom from adult C. simus comprises toxins from 7 protein families. A large proportion (71.7%) of these toxins are SVMPs, two-thirds of which belong to the PIII class. These toxin profiles correlate well with the overall biochemical and pharmacological features of venoms from adult (hemorrhagic) and newborn (neurotoxic) C. simus specimens. The venoms of the South American Crotalus subspecies belong to one of two distinct phenotypes. C. d. cumanensis exhibits high levels of SVMPs and low lethal potency (LD50), whereas C. d. subspecies terrificus, ruruima, and durissus have low SVMP activity and high neurotoxicity to mice. Their overall toxin compositions explain the outcome of envenomation by these species. Further, in all C. simus and C. durissus venoms, the concentration of neurotoxins (crotoxin and crotamine) is directly related with lethal activity, whereas lethality and metalloproteinase activity show an inverse relationship. The similar venom toxin profiles of newborn C. simus and adult C. durissus terrificus, ruruima, and durissus subspecies strongly suggests that the South American taxa have retained juvenile venom characteristics in the adult form (paedomorphism) along their North−South stepping-stone dispersal. The driving force behind paedomorphism is often competition or predation pressure. The increased concentration of the neurotoxins crotoxin and crotamine in South American rattlesnake venoms strongly argues that the gain of neurotoxicity and lethal venom activities to mammals may have represented the key axis along which overall venom toxicity has evolved during Crotalus durissus invasion of South America. The paedomorphic trend is supported by a decreasing LNC (lethal neurotoxicity coefficient, defined as the ratio between the average LD50 of the venom and the crotoxin + crotamine concentration) along the North−South axis, coincident with the evolutionary dispersal pattern of the Neotropical rattlesnakes. The indistinguisable immunoreactivity patterns of Costa Rican and Venezuelan polyvalent antivenoms toward C. simus and C. durissus venoms strongly suggest the possibility of using these antivenoms indistinctly for the management of snakebites by adult C. simus and by certain C. d. cumanensis populations exhibiting a hemorrhagic venom phenotype. The antivenomic results also explain why the antivenoms effectively neutralize the hemorrhagic activity of adult C. simus venoms but does not protect against adult C. durissus sp. and newborn C. simus envenomations. The identification of evolutionary trends among tropical Crotalus, as reported here, may have an impact in defining the mixture of venoms for immunization to produce an effective pan-American anti-Crotalus antivenom. Ministerio de Educación y Ciencia/[BFU2007-6156]//España Universidad de Costa Rica/[741-A7-611]/UCR/Costa Rica Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo/206AC0281/CYTED/España Acciones Integradas/2006CR001// Consejo Superior de Investigaciones Científicas/[2007CR0004]/CRUSA-CSIC/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A2 with high enzymatic activity. The phospholipases A2 are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A2. In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A2 (PLA2) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA2 inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF.