3-NAntC: A Potent Crotoxin B-Derived Peptide against the Triple-Negative MDA-MB-231 Breast Cancer Cell Line.

Breast cancer stands as the most prevalent type of tumor and a significant contributor to cancer-related deaths. Among its various subtypes, triple-negative breast cancer (TNBC) presents the worst prognosis due to its aggressive nature and the absence of effective treatments. Crotoxin, a protein found in the venom of Crotalus genus snakes, has demonstrated notable antitumor activity against aggressive solid tumors. However, its application has been hindered by substantial toxicity in humans. In efforts to address this challenge, Crotoxin B-derived peptides were synthesized and evaluated in vitro for their antitumor potential, leading to the discovery of 3-NAntC. Treatment with 3-NAntC at 1 µg/mL for 72 h notably reduced the viability of MDA-MB-231 cells to 49.0 ± 17.5% (p < 0.0001), while exhibiting minimal impact on the viability of HMEC cells (98.2 ± 13.8%) under the same conditions. Notably, 3-NAntC displayed superior antitumoral activity in vitro compared to cisplatin and exhibited a similar effect to doxorubicin. Further investigation revealed that 3-NAntC decreased the proliferation of MDA-MB-231 cells and induced G2/M phase arrest. It primarily prompted optimal cell death by apoptosis, with a lower incidence of the less desirable cell death by necrosis in comparison to doxorubicin. Additionally, 3-NAntC demonstrated low LDH release, and its cytotoxicity remained unaffected by the autophagy inhibitor 3-MA. In an in vivo zebrafish model, 3-NAntC exhibited excellent tolerability, showing no lethal effects and a low rate of malformations at high doses of up to 75 mg/mL. Overall, 3-NAntC emerges as a novel synthetic peptide with promising antitumor effects in vitro against TNBC cells and low toxicity in vivo.

Structural studies with crotoxin B from Crotalus durissus collilineatus venom suggest a heterodimeric assembly formed by two new isoforms.

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.

Cytotoxic effects of crotoxin from Crotalus durissus terrificus snake in canine mammary tumor cell lines

Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Results: Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Conclusion: Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

Crotoxin Modulates Macrophage Phenotypic Reprogramming.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 µg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 µg/animal: M1 and at 5.0 µg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 µg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Literature Review on Crotalus durissus terrificus Toxins: From a Perspective of Structural Biology and Therapeutic Applications.

BACKGROUND: The venom of Crotalus durissus terrificus, as well as its fractions, has intrigued research groups worldwide who are working to isolate, characterize, and find possible biotechnological applications. A number of studies have elucidated that these fractions and their derivatives possess pharmacological properties, which can enable the development of new drug prototypes with anti-inflammatory, antinociceptive, antitumor, antiviral, and antiparasitic applications. OBJECTIVE: This review presents a systematic study on Crotalus durissus terrificus, the most notable crotalid subspecies in South America, focusing on the composition, toxicological mechanisms, structural aspects, and applications of the main venom toxins (convulxin, gyroxin, crotamine, crotoxin, and their subunits). CONCLUSION: The authors have found that research on this snake and its toxins is still an area of focus, despite that almost a century has passed since the isolation of crotoxin. Several applications of these proteins in the development of novel drugs and bioactive substances have also been demonstrated.

Cytotoxic effect of crotoxin on cancer cells and its antitumoral effects correlated to tumor microenvironment: A review.

Cancer is the second leading cause of death worldwide, and despite the effort of standard treatments, the search for new tools against this disease is necessary. Importantly, it is known that the tumor microenvironment plays a crucial role in tumor initiation, progression, and response to therapies. Therefore, studies of potential drugs that act on these components are as critical as studies regarding antiproliferative substances. Through the years, studies of several natural products, including animal toxins, have been conducted to guide the development of medical compounds. In this review, we present the remarkable antitumor activities of crotoxin, a toxin from the rattlesnake Crotalus durissus terrificus, highlighting its effects on cancer cells and in the modulation of relevant elements in the tumor microenvironment as well as the clinical trials conducted with this compound. In summary, crotoxin acts through several mechanisms of action, such as activation of apoptosis, induction of cell cycle arrest, inhibition of metastasis, and decrease of tumor growth, in different tumor types. Crotoxin also modulates tumor-associated fibroblasts, endothelial cells, and immune cells, which contribute to its antitumoral effects. In addition, preliminary clinical studies confirm the promising results of crotoxin and support its potential future use as an anticancer drug.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions.

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

The antineoplastic potential of crotoxin isolated from Crotalus durissus terrificus snake venom on oral squamous cell carcinoma.

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 µg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients.

Design of Crotoxin-Based Peptides with Potentiator Activity Targeting the ΔF508NBD1 Cystic Fibrosis Transmembrane Conductance Regulator.

We have previously shown that the CBb subunit of crotoxin, a ß-neurotoxin with phospholipase A2 (PLA2) activity, targets the human ΔF508CFTR chloride channel implicated in cystic fibrosis (CF). By direct binding to the nucleotide binding domain 1 (NBD1) of ΔF508CFTR, this neurotoxic PLA2 acts as a potentiator increasing chloride channel current and corrects the trafficking defect of misfolded ΔF508CFTR inside the cell. Here, for a therapeutics development of new anti-cystic fibrosis agents, we use a structure-based in silico approach to design peptides mimicking the CBb-ΔF508NBD1 interface. Combining biophysical and electrophysiological methods, we identify several peptides that interact with the ΔF508NBD1 domain and reveal their effects as potentiators on phosphorylated ΔF508CFTR. Moreover, protein-peptide interactions and electrophysiological studies allowed us to identify key residues of ΔF508NBD1 governing the interactions with the novel potentiators. The designed peptides bind to the same region as CBb phospholipase A2 on ΔF508NBD1 and potentiate chloride channel activity. Certain peptides also show an additive effect towards the clinically approved VX-770 potentiator. The identified CF therapeutics peptides represent a novel class of CFTR potentiators and illustrate a strategy leading to reproducing the effect of specific protein-protein interactions.

Crotoxin modulates metabolism and secretory activity of peritoneal macrophages from Walker 256 tumor-bearing rats.

Crotoxin (CTX), the major toxin of Crotalus durissus terrificus snake venom, induces an inhibitory effect on tumor development and modulates the functions of macrophages (MØs), which play a key role as a defense mechanism against tumor growth. In early tumor progression stage, MØs are avidly phagocytic (inflammatory cell), releasing reactive nitrogen intermediates-RNI/ROI and cytokines TNF-α, IL-1ß, and IL-6. However, when the tumor has been developed, tumor-associated MØ (angiogenic cell) presents a decrease in the mentioned activities. We reported that CTX stimulates H2O2 release, NO production and secretion of cytokines by peritoneal MØs obtained from non-tumor-bearing rats. Considering that the mentioned mediators control tumor growth, it is mandatory to investigate whether CTX stimulates the production of these mediators by MØs obtained from tumor-bearing animals. The aim of this work was then to evaluate the CTX effect on metabolism and functions of peritoneal MØs obtained from Walker 256 tumor-bearing rats. For this purpose, male Wistar rats were subcutaneously inoculated in the right flank with 1 mL sterile suspension of 2 × 107 Walker 256 tumor cells. CTX (18 µg per animal) was subcutaneously administered in two protocols: a) on the 1st day of tumor cell injection and b) on the 4th day of tumor cell inoculation. In both protocols, MØs were obtaining on the 14th day of tumor cell inoculation to evaluate the release of H2O2, NO, and pro-inflammatory cytokines (IL-1ß, TNFα, and IL-6); maximal activity of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase, and 14CO2 production from [U-14C]-glucose and [U-14C]-glutamine. The treatment with CTX stimulated the release of NO, H2O2, and cytokines, and glucose and glutamine metabolism. Metabolic and functional changes induced by CTX were accompanied by a decrease of tumor growth as indicated by tumor fresh weight and diameter. These results indicate CTX not only as a scientific tool to investigate changes in metabolism and functions of peritoneal MØs but also for a better understanding of the mechanisms involved in tumor growth.

Crotoxin modulates inflammation and macrophages' functions in a murine sepsis model.

Sepsis is a syndrome of physiological and biochemical abnormalities induced by an infection that represents a major public health concern. It involves the early activation of inflammatory responses. Crotoxin (CTX), the major toxin of the South American rattlesnake Crotalus durissus terrificus venom, presents longstanding anti-inflammatory properties. Since immune system modulation may be a strategic target in sepsis management, and macrophages' functional and secretory activities are related to the disease's progression, we evaluated the effects of CTX on macrophages from septic animals. Balb/c male mice submitted to cecal ligation and puncture (CLP) were treated with CTX (0.9 µg/animal, subcutaneously) 1 h after the procedure and euthanized after 6 h. We used plasma samples to quantify circulating cytokines and eicosanoids. Bone marrow differentiated macrophages (BMDM) were used to evaluate the CTX effect on macrophages' functions. Our data show that CTX administration increased the survival rate of the animals from 40% to 80%. Septic mice presented lower plasma concentrations of IL-6 and TNF-α after CTX treatment, and higher concentrations of LXA4, PGE2, and IL-1ß. No effect was observed in IL-10, IFN-γ, and RD1 concentrations. BMDM from septic mice treated with CTX presented decreased capacity of E. coli phagocytosis, but sustained NO and H2O2 production. We also observed higher IL-6 concentration in the culture medium of BMDM from septic mice, and CTX induced a significant reduction. CTX treatment increased IL-10 production by macrophages as well. Our data show that the protective effect of CTX in sepsis mortality involves modulation of macrophage functions and inflammatory mediators' production.

Revisiting the potential of South American rattlesnake Crotalus durissus terrificus toxins as therapeutic, theranostic and/or biotechnological agents.

The potential biotechnological and biomedical applications of the animal venom components are widely recognized. Indeed, many components have been used either as drugs or as templates/prototypes for the development of innovative pharmaceutical drugs, among which many are still used for the treatment of human diseases. A specific South American rattlesnake, named Crotalus durissus terrificus, shows a venom composition relatively simpler compared to any viper or other snake species belonging to the Crotalus genus, although presenting a set of toxins with high potential for the treatment of several still unmet human therapeutic needs, as reviewed in this work. In addition to the main toxin named crotoxin, which is under clinical trials studies for antitumoral therapy and which has also anti-inflammatory and immunosuppressive activities, other toxins from the C. d. terrificus venom are also being studied, aiming for a wide variety of therapeutic applications, including as antinociceptive, anti-inflammatory, antimicrobial, antifungal, antitumoral or antiparasitic agent, or as modulator of animal metabolism, fibrin sealant (fibrin glue), gene carrier or theranostic agent. Among these rattlesnake toxins, the most relevant, considering the potential clinical applications, are crotamine, crotalphine and gyroxin. In this narrative revision, we propose to organize and present briefly the updates in the accumulated knowledge on potential therapeutic applications of toxins collectively found exclusively in the venom of this specific South American rattlesnake, with the objective of contributing to increase the chances of success in the discovery of drugs based on toxins.

Acidentes crotálicos no Brasil: atualidades e perspectivas / Crotalic accidents in Brazil: actualities and perspectives

Introdução: Os acidentes ofídicos são eventos negligenciados em países tropicais e em desenvolvimento, incluindo o Brasil. Serpentes do gênero Crotalus são aquelas que produzem os quadros de maior letalidade no país. Objetivos: Abordar os principais aspectos do acidente por Crotalus, com ênfase na biologia das serpentes, na condução clínica dos eventos mórbidos e nas propriedades terapêuticas da peçonha destes animais. Métodos: Revisão da literatura com estratégia de busca definida, a partir da utilização das bases PubMed, LILACS e SciELO. Resultados: A inoculação da peçonha crotálica produz sinais locais discretos, mas repercussões sistêmicas podem ocorrer, especialmente alterações neurológicas e insuficiência renal aguda. A avaliação laboratorial é importante para auxiliar na distinção de outros acidentes ofídicos e para estimar a gravidade do quadro. O soro anticrotálico precisa ser administrado o mais brevemente possível, a partir da estimativa da quantidade de peçonha inoculada. A maior parte dos agravos ocorre no período chuvoso, acometendo principalmente homens jovens que trabalham na zona rural. A adoção de medidas de proteção e a educação em saúde são estratégias pertinentes para a prevenção e a redução do número de casos. As peçonhas de Crotalus possuem ações antimicrobianas, antiagregantes plaquetárias e aplicabilidade em oftalmologia (estrabismo). Conclusão: O conhecimento dos diferentes aspectos dos acidentes crotálicos é essencial para a adequada abordagem diagnóstica e terapêutica de tais condições mórbidas. As propriedades farmacológicas de componentes da peçonha crotálica deverão ser melhor investigadas nos próximos anos, dadas as possibilidades de utilização para o tratamento de diferentes enfermidades humanas

Introduction: Ophidian accidents are neglected events in tropical and in developing countries, including Brazil. Serpents of the Crotalus genus (rattlesnakes) are those that produce the highest case-fatality in the country. Purpose: to address the main aspects of the accident caused by Crotalus, with emphasis on the snakes biology, the clinical approach to snake bites and the therapeutic properties of the venom of these animals. Methods: literature review with a defined search strategy, using the PubMed, LILACS and SciELO databases. Results: Inoculation of crotalic venom produces discrete local signs, but systemic repercussions can occur, especially neurological alterations and acute renal failure. Laboratory evaluation is important to help distinguish from other ophidian accidents and to estimate the severity of the condition. Anticrotalic serum must be administered as soon as possible, based on the estimated amount of inoculated venom. Most of the morbid events occur in the rainy season (higher temperature), mainly affecting young men who work in rural areas. The adoption of protective measures and health education, aimed at the population most commonly involved, are relevant strategies for preventing and reducing the number of cases. In addition, crotalic venoms have antimicrobial actions, antiplatelets functions and ophthalmological applicability (strabismus). Conclusion: Knowledge of the different aspects of crotalic injuries is essential for an adequate diagnostic and therapeutic approach to such morbid conditions. The pharmacological properties of crotalic venom components should be better investigated in the next few years, given the possibilities of their use for the treatment of different human diseases.

Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes.

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

Crotoxin Modulates Events Involved in Epithelial-Mesenchymal Transition in 3D Spheroid Model.

Epithelial-mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer.

The anti-cancer potential of crotoxin in estrogen receptor-positive breast cancer: Its effects and mechanism of action.

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 ß-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.

The Crotoxin:SBA-15 Complex Down-Regulates the Incidence and Intensity of Experimental Autoimmune Encephalomyelitis Through Peripheral and Central Actions.

Crotoxin (CTX), the main neurotoxin from Crotalus durissus terrificus snake venom, has anti-inflammatory, immunomodulatory and antinociceptive activities. However, the CTX-induced toxicity may compromise its use. Under this scenario, the use of nanoparticle such as nanostructured mesoporous silica (SBA-15) as a carrier might become a feasible approach to improve CTX safety. Here, we determined the benefits of SBA-15 on CTX-related neuroinflammatory and immunomodulatory properties during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis that replicates several histopathological and immunological features observed in humans. We showed that a single administration of CTX:SBA-15 (54 µg/kg) was more effective in reducing pain and ameliorated the clinical score (motor impairment) in EAE animals compared to the CTX-treated EAE group; therefore, improving the disease outcome. Of interest, CTX:SBA-15, but not unconjugated CTX, prevented EAE-induced atrophy and loss of muscle function. Further supporting an immune mechanism, CTX:SBA-15 treatment reduced both recruitment and proliferation of peripheral Th17 cells as well as diminished IL-17 expression and glial cells activation in the spinal cord in EAE animals when compared with CTX-treated EAE group. Finally, CTX:SBA-15, but not unconjugated CTX, prevented the EAE-induced cell infiltration in the CNS. These results provide evidence that SBA-15 maximizes the immunomodulatory and anti-inflammatory effects of CTX in an EAE model; therefore, suggesting that SBA-15 has the potential to improve CTX effectiveness in the treatment of MS.

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic ß-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 (CA1-4). 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.

Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes.

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.

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)