RESUMEN

This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.

Asunto(s)

Antibacterianos , Antifúngicos , Venenos de Crotálidos , Crotalus , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Antifúngicos/química , Antibacterianos/farmacología , Venenos de Crotálidos/farmacología , Animales , Staphylococcus aureus/efectos de los fármacos , Sinergismo Farmacológico , Candida albicans/efectos de los fármacos , Serpientes Venenosas

RESUMEN

Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.

Asunto(s)

Analgésicos , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.7 , Neuronas , Tetrodotoxina , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Ratones , Tetrodotoxina/farmacología , Analgésicos/farmacología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/farmacología , Masculino , Crotalus , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Péptidos

RESUMEN

Mexico has the highest diversity of snake species in the world, following Australia when considering just venomous snakes. Specifically, in Sonora, the second largest state in the country, more than 15 highly venomous species occur, including the northern black-tailed rattlesnake (Crotalus molossus). This specie's venom has not been as thoroughly researched in contrast with other Mexican vipers, nevertheless some studies report its biological activity and even pharmacological potential with antibacterial and cytotoxic activity. In this study we identified the main protein components from a pool of C. molossus venom through a gel-free proteomics approach, reporting ∼140 proteins belonging to the SVMP (38.76%), PLA2 (28.75%), CTL (11.93%), SVSP (6.03%) and LAAO (5.67%) toxin families. To study its biological activities, we evaluated its hemolytic, antibacterial, and cytotoxic activity in red blood cells, Gram positive and negative bacteria and a luminal A breast carcinoma cell line (T47D), respectively, in vitro. We report that concentrations <100 µg/mL are potentially not hemolytic and reduced the bacteria viability of E. coli and S. aureus with an IC50 of 10.27 and 11.51 µg/mL, respectively. Finally, we determined the C. molossus venom as cytotoxic against the T47D breast carcinoma cell line, with an IC50 of 1.55 µg/mL. We suggest that the evaluated cytotoxicity was due to a high abundance of SVMPs and PLA2s, since it's been reported that they affect the extracellular matrix and membrane permeation. This may provide a useful tool for pharmaceutical screening in the future.

Asunto(s)

Antibacterianos , Venenos de Crotálidos , Crotalus , Escherichia coli , Staphylococcus aureus , Animales , Venenos de Crotálidos/farmacología , Antibacterianos/farmacología , Staphylococcus aureus/efectos de los fármacos , Línea Celular Tumoral , Humanos , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Hemólisis/efectos de los fármacos , Femenino , Pruebas de Sensibilidad Microbiana , Eritrocitos/efectos de los fármacos , Serpientes Venenosas

RESUMEN

Eastern Diamondback Rattlesnake (Crotalus adamanteus) envenomation is a medical emergency encountered in the Southeastern United States. The venom contains a snake venom thrombin-like enzyme (SVTLE) that is defibrinogenating, causing coagulopathy without effects on platelets in humans. This investigation utilized thrombelastographic methods to document this coagulopathy kinetically on the molecular level in a rabbit model of envenomation via the analyses of whole blood samples without and with platelet inhibition. Subsequently, the administration of a novel ruthenium compound containing site-directed antivenom abrogated the coagulopathic effects of envenomation in whole blood without platelet inhibition and significantly diminished loss of coagulation in platelet-inhibited samples. This investigation provides coagulation kinetic insights into the molecular interactions and results of SVTLE on fibrinogen-dependent coagulation and confirmation of the efficacy of a ruthenium antivenom. These results serve as a rationale to investigate the coagulopathic effects of other venoms with this model and assess the efficacy of this site-directed antivenom.

Asunto(s)

Antivenenos , Coagulación Sanguínea , Venenos de Crotálidos , Crotalus , Animales , Conejos , Antivenenos/farmacología , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Coagulación Sanguínea/efectos de los fármacos , Tromboelastografía , Rutenio/química , Rutenio/farmacología , Mordeduras de Serpientes/tratamiento farmacológico , Masculino , Serpientes Venenosas

RESUMEN

Ruthenium chloride (RuCl3) is widely utilized for synthesis and catalysis of numerous compounds in academia and industry and is utilized as a key molecule in a variety of compounds with medical applications. Interestingly, RuCl3 has been demonstrated to modulate human plasmatic coagulation and serves as a constituent of a compounded inorganic antivenom that neutralizes the coagulopathic effects of snake venom in vitro and in vivo. Using thrombelastography, this investigation sought to determine if RuCl3 inhibition of the fibrinogenolytic effects of Crotalus atrox venom could be modulated by vehicle composition in human plasma. Venom was exposed to RuCl3 in 0.9% NaCl, phosphate-buffered saline (PBS), or 0.9% NaCl containing 1% dimethyl sulfoxide (DMSO). RuCl3 inhibited venom-mediated delay in the onset of thrombus formation, decreased clot growth velocity, and decreased clot strength. PBS and DMSO enhanced the effects of RuCl3. It is concluded that while a Ru-based cation is responsible for significant inhibition of venom activity, a combination of Ru-based ions containing phosphate and DMSO enhances RuCl3-mediated venom inhibition. Additional investigation is indicated to determine what specific Ru-containing molecules cause venom inhibition and what other combinations of inorganic/organic compounds may enhance the antivenom effects of RuCl3.

Asunto(s)

Antivenenos , Coagulación Sanguínea , Venenos de Crotálidos , Crotalus , Dimetilsulfóxido , Humanos , Dimetilsulfóxido/farmacología , Dimetilsulfóxido/química , Antivenenos/farmacología , Antivenenos/química , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/farmacología , Animales , Coagulación Sanguínea/efectos de los fármacos , Compuestos de Rutenio/farmacología , Compuestos de Rutenio/química , Cloruro de Sodio/farmacología , Cloruro de Sodio/química , Tromboelastografía , Serpientes Venenosas

RESUMEN

Snake venoms contain various bradykinin-potentiating peptides (BPPs). First studied for their vasorelaxant properties due to angiotensin converting enzyme (ACE) inhibition, these molecules present a range of binding partners, among them the argininosuccinate synthase (AsS) enzyme. This has renewed interest in their characterization from biological sources and the evaluation of their pharmacological activities. In the present work, the low molecular weight fraction of Bothrops moojeni venom was obtained and BPPs were characterized by mass spectrometry. Eleven BPPs or related peptides were sequenced, and one of them, BPP-Bm01, was new. Interestingly, some oxidized BPPs were detected. The three most abundant peptides were BPP-Bm01, BPP-Bax12, and BPP-13a, and their putative interactions with the AsS enzyme were investigated in silico. A binding cavity for these molecules was predicted, and docking studies allowed their ranking. Three peptides were synthesized and submitted to vasorelaxation assays using rat aortic rings. While all BPPs were active, BPP-Bm01 showed the highest potency in this assay. This work adds further diversity to BPPs from snake venoms and suggests, for the first time, a putative binding pocket for these molecules in the AsS enzyme. This can guide the design of new and more potent AsS activators.

Asunto(s)

Aorta , Bothrops , Oligopéptidos , Péptidos , Serpientes Venenosas , Animales , Ratas , Brasil , Aorta/efectos de los fármacos , Péptidos/farmacología , Péptidos/química , Bradiquinina/farmacología , Masculino , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/química , Ratas Wistar , Venenos de Serpiente/farmacología , Vasodilatadores/farmacología , Vasodilatadores/química , Estructura Molecular

RESUMEN

BACKGROUND: The Western diamondback rattlesnake ( Crotalus atrox ) is a medically important venomous snake in the Southwestern United States, injuring humans, and their companion animals. The goals of this investigation were to utilize a rabbit model of subcutaneous envenomation to assess Crotalus atrox venom coagulopathy and determine the efficacy of a ruthenium-containing antivenom (RA) in attenuating it. METHODS: Sedated New Zealand White rabbits had viscoelastic measurements of whole blood coagulation kinetics obtained from ear artery samples. Crotalus atrox venom (4 mg/kg) was injected subcutaneously and changes in coagulation determined over three hours and compared to samples obtained prior to envenomation. Other rabbits had site-directed RA injected 5 min after venom injection. RESULTS: A significant decrease in the velocity of clot growth and thrombus strength was observed in animals injected with venom alone. Site-directed administration of RA resulted in no change in coagulation over the 3 h following venom injection. The interaction of antivenom administration and time was significantly different in the cases of clot growth velocity and strength. CONCLUSIONS: A novel rabbit model was used to define the toxicodynamic profile of coagulopathy of Crotalus atrox venom and demonstrate the efficacy of RA. Future investigation is planned involving other medically important venoms and RA administration.

Asunto(s)

Antivenenos , Trastornos de la Coagulación Sanguínea , Venenos de Crotálidos , Crotalus , Serpientes Venenosas , Animales , Conejos , Antivenenos/farmacología , Antivenenos/uso terapéutico , Venenos de Crotálidos/farmacología , Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Mordeduras de Serpientes/tratamiento farmacológico , Coagulación Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

Asunto(s)

Plaquetas , Ratones Endogámicos C57BL , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Receptores de Trombina , Trombina , Animales , Plaquetas/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Trombina/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Modelos Animales de Enfermedad , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/toxicidad , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Selectina-P/metabolismo , Selectina-P/genética , Mutación Puntual , Técnicas de Sustitución del Gen , Transducción de Señal , Trombosis/genética , Trombosis/sangre , Masculino , Cloruros , Ratones , Activación Plaquetaria , Sistemas CRISPR-Cas , Humanos , Fenotipo , Compuestos Férricos , Oligopéptidos , Lectinas Tipo C , Receptores Proteinasa-Activados

RESUMEN

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A2 (PLA2s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor ß (TGF-ß) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings.

Asunto(s)

Venenos de Crotálidos , Mordeduras de Serpientes , Animales , Ratones , Antivenenos , Mordeduras de Serpientes/genética , Venenos de Serpiente , Venenos de Crotálidos/farmacología , Músculos , Colágeno

RESUMEN

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Asunto(s)

Antineoplásicos , Venenos de Crotálidos , Melanoma , Animales , Ratones , Modelos Animales de Enfermedad , Antineoplásicos/farmacología , Antineoplásicos/química , Venenos de Crotálidos/química , Venenos de Crotálidos/farmacología

RESUMEN

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.

Asunto(s)

Venenos de Crotálidos , Crotoxina , Animales , Crotoxina/farmacología , Ascitis , Macrófagos , Macrófagos Peritoneales , Crotalus , Venenos de Crotálidos/farmacología

RESUMEN

Ischemic heart disease (IHD), especially myocardial infarction (MI), is a leading cause of death worldwide. Although coronary reperfusion is the most straightforward treatment for limiting the MI size, it has nevertheless been shown to exacerbate ischemic myocardial injury. Therefore, identifying and developing therapeutic strategies to treat IHD is a major medical challenge. Snake venoms contain biologically active proteins and peptides that are of major interest for pharmacological applications in the cardiovascular system (CVS). This has led to their use for the development and design of new drugs, such as the first-in-class angiotensin-converting enzyme inhibitor captopril, developed from a peptide present in Bothrops jararaca snake venom. This review discusses the potential usefulness of snake venom toxins for developing effective treatments against IHD and related diseases such as hypertension and atherosclerosis. It describes their biological effects at the molecular scale, their mechanisms of action according to their different pharmacological properties, as well as their subsequent molecular pathways and therapeutic targets. The molecules reported here have either been approved for human medical use and are currently available on the drug market or are still in the clinical or preclinical developmental stages. The information summarized here may be useful in providing insights into the development of future snake venom-derived drugs.

Asunto(s)

Venenos de Crotálidos , Isquemia Miocárdica , Humanos , Venenos de Serpiente/uso terapéutico , Venenos de Serpiente/química , Péptidos/química , Proteínas , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/química , Venenos de Crotálidos/farmacología , Isquemia Miocárdica/tratamiento farmacológico

RESUMEN

Cancer is a global public health issue. Neuroblastoma (NB) originates from any tissue of the sympathetic nervous system, and the most affected site is the abdomen. The adrenal gland is the primary site in 38% of cases. Approximately 50% of patients have metastatic disease at diagnosis, and bone marrow is often affected. Metastatic disease is characterized by the spreading of cancer cells that are frequently resistant to chemotherapy and radiotherapy from the primary tumor to other specific parts of the body and is responsible for 90% of cancer-related deaths. Increasing evidence has indicated that nitric oxide (NO) signaling is implicated in the pathophysiology of many types of cancer, particularly in tumorigenesis and cancer progression. However, the effect of NO on metastasis cannot be easily classified as prometastatic or antimetastatic. An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. Here, the proline-rich decapeptide isolated from Bothrops jararaca venom (Bj-PRO-10c) that enhances and sustains the generation of NO was used to unravel the role of metabolic NO in steps of metastasis. Bj-PRO-10c showed an antimetastatic effect, mainly by interfering with actin cytoskeleton rearrangement, controlling cell proliferation, and decreasing the seeding efficiency of NB in metastatic niches. Therefore, we proposed that an approach for controlled NO induction with the right molecular strategies can hopefully inhibit metastasis and increase the lifespan of NB patients.

Asunto(s)

Venenos de Crotálidos , Neuroblastoma , Humanos , Argininosuccinato Sintasa/metabolismo , Óxido Nítrico/metabolismo , Venenos de Crotálidos/farmacología , Neuroblastoma/tratamiento farmacológico

RESUMEN

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.

Asunto(s)

Venenos de Crotálidos , Neoplasias de la Próstata , Humanos , Masculino , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/metabolismo , L-Aminoácido Oxidasa/farmacología , L-Aminoácido Oxidasa/química , L-Aminoácido Oxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular , Estrés Oxidativo

RESUMEN

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbß3, a fundamental platelet glycoprotein, and αvß3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of ß-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbß3 antagonist, capable of preventing arterial thrombosis.

Asunto(s)

Venenos de Crotálidos , Trombosis , Ratas , Animales , Desintegrinas/farmacología , Desintegrinas/química , Desintegrinas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/química , Venenos de Crotálidos/química , Venenos de Crotálidos/farmacología , Agregación Plaquetaria , Hemostasis , Integrinas/metabolismo , Trombosis/tratamiento farmacológico

RESUMEN

Dengue fever is considered a major public health problem in tropical and subtropical regions. Our study analyzed the effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with Dengue virus. In presence of BaltPLA2, the viability of infected cells increased significantly in virucidal, post-treatment, and adsorption assays. Although preliminary these results reveal the need for further studies to investigated whether BaltPLA2 has antiviral activity against Dengue virus.

Asunto(s)

Bothrops , Venenos de Crotálidos , Virus del Dengue , Animales , Fosfolipasas A2/farmacología , Venenos de Serpiente , Antivirales/farmacología , Venenos de Crotálidos/farmacología

RESUMEN

Snake toxins are widely studied owing to their importance in snakebite accidents, a serious public health issue in tropical countries, and their broad therapeutic potential. Isolated fractions from venom produced by snakes of the genus Crotalus sp. present a wide variety of pharmacological uses such as antifungal, antiviral, antibacterial, and antitumor properties, among other therapeutic potentialities. Given the direct effect of this venom on tumor cells, isolation of its compounds is important for the characterization of its anticarcinogenic actions. Crotalus durissus terrificus venom and its toxins have been widely evaluated as potential candidates for the development of new antineoplastic therapies that are efficient against different tumor lines and cellular targets. This review highlights the venom toxins of this species, with a focus on their antineoplastic properties.

Asunto(s)

Antineoplásicos , Venenos de Crotálidos , Mordeduras de Serpientes , Animales , Humanos , Venenos de Crotálidos/farmacología , Crotalus , Venenos de Serpiente/farmacología , Antineoplásicos/farmacología

RESUMEN

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated.

Asunto(s)

Bothrops , Venenos de Crotálidos , Neuroblastoma , Fármacos Neuroprotectores , Animales , Humanos , Antioxidantes/farmacología , Venenos de Crotálidos/química , Venenos de Crotálidos/farmacología , Péptidos , Venenos de Serpiente

RESUMEN

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Asunto(s)

Venenos de Crotálidos , Neurotoxinas , Animales , Humanos , Neurotoxinas/metabolismo , Tubulina (Proteína)/metabolismo , Crotalus/metabolismo , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/metabolismo , Péptidos/farmacología , Péptidos/metabolismo

RESUMEN

BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.

Asunto(s)

Bothrops , Venenos de Crotálidos , Hipertensión Renovascular , Animales , Ratas , Presión Sanguínea , Venenos de Crotálidos/farmacología , Frecuencia Cardíaca , Hipertensión Renovascular/tratamiento farmacológico , Metaloproteasas/farmacología , Ratas Wistar , Remodelación Ventricular