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1.
PLoS One ; 15(2): e0229657, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106235

RESUMO

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.


Assuntos
Boidae/sangue , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Inibidores de Fosfolipase A2/sangue , Sequência de Aminoácidos , Animais , Bothrops/metabolismo , Brasil , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/química , Fosfolipases A2 do Grupo IV/química , Peso Molecular , Inibidores de Fosfolipase A2/química , Domínios e Motivos de Interação entre Proteínas , Venenos de Serpentes/antagonistas & inibidores , Venenos de Serpentes/química , Espectrometria de Massas em Tandem
2.
Artigo em Inglês | MEDLINE | ID: mdl-31634575

RESUMO

Dispholidus typus and Thelotornis mossambicanus are closely related rear-fanged colubrid snakes that both possess strongly procoagulant venoms. However, despite similarities in overall venom biochemistry and resulting clinical manifestations, the underlying venom composition differs significantly between the two species. As a result, the only available antivenom-which is a monovalent antivenom for D. typus-has minimal cross reactivity with T. mossambicanus and is not a clinically viable option. It was hypothesised that this lack of cross reactivity is due to the additional large metalloprotease protein within T. mossambicanus venom, which may also be responsible for faster coagulation times. In this study, we found that T. mossambicanus venom is a more powerful activator of prothrombin than that of D. typus and that the SVMP transcripts from T. mossambicanus form a clade with those from D. typus. The sequences from D. typus and T. mossambicanus were highly similar in length, with the calculated molecular weights of the T. mossambicanus transcripts being significantly less than the molecular weights of some isoforms on the 1D SDS-PAGE gels. Analyses utilising degylcosylating enzymes revealed that T. mossambicanus SVMPs are glycosylated during post-translational modification, but that this does not lead to the different molecular weight bands observed in 1D SDS-PAGE gels. However, differences in glycosylation patterns may still explain some of the difference between the enzymatic activities and neutralization by antivenom that have been observed in these venoms. The results of this study provide new information regarding the treatment options for patients envenomated by T. mossambicanus as well as the evolution of these dangerous snakes.


Assuntos
Colubridae/fisiologia , Metaloproteases/metabolismo , Protrombina/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Animais , Colubridae/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicosilação , Metaloproteases/genética , Filogenia , Protrombina/química , Protrombina/farmacologia , Transcriptoma
3.
PLoS Negl Trop Dis ; 13(12): e0007899, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31805055

RESUMO

BACKGROUND: Snakebite in India causes the highest annual rates of death (46,000) and disability (140,000) than any other country. Antivenom is the mainstay treatment of snakebite, whose manufacturing protocols, in essence, have remained unchanged for over a century. In India, a polyvalent antivenom is produced for the treatment of envenomations from the so called 'big four' snakes: the spectacled cobra (Naja naja), common krait (Bungarus caeruleus), Russell's viper (Daboia russelii), and saw-scaled viper (Echis carinatus). In addition to the 'big four', India is abode to many other species of venomous snakes that have the potential to inflict severe clinical or, even, lethal envenomations in their human bite victims. Unfortunately, specific antivenoms are not produced against these species and, instead, the 'big four' antivenom is routinely used for the treatment. METHODS: We characterized the venom compositions, biochemical and pharmacological activities and toxicity profiles (mouse model) of the major neglected yet medically important Indian snakes (E. c. sochureki, B. sindanus, B. fasciatus, and two populations of N. kaouthia) and their closest 'big four' congeners. By performing WHO recommended in vitro and in vivo preclinical assays, we evaluated the efficiencies of the commercially marketed Indian antivenoms in recognizing venoms and neutralizing envenomations by these neglected species. FINDINGS: As a consequence of dissimilar ecologies and diet, the medically important snakes investigated exhibited dramatic inter- and intraspecific differences in their venom profiles. Currently marketed antivenoms were found to exhibit poor dose efficacy and venom recognition potential against the 'neglected many'. Premium Serums antivenom failed to neutralise bites from many of the neglected species and one of the 'big four' snakes (North Indian population of B. caeruleus). CONCLUSIONS: This study unravels disturbing deficiencies in dose efficacy and neutralisation capabilities of the currently marketed Indian antivenoms, and emphasises the pressing need to develop region-specific snakebite therapy for the 'neglected many'.


Assuntos
Antitoxinas/farmacologia , Antivenenos/uso terapêutico , Mordeduras de Serpentes/terapia , Venenos de Serpentes/química , Venenos de Serpentes/toxicidade , Animais , Modelos Animais de Doenças , Índia , Masculino , Camundongos , Análise de Sobrevida , Resultado do Tratamento
4.
Dokl Biochem Biophys ; 487(1): 251-255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31559591

RESUMO

Three-finger snake neurotoxins are selective antagonists of some nicotinic acetylcholine receptor subtypes and are widely used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective blocker of muscle-type nicotinic acetylcholine receptor. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Neurotoxinas/química , Antagonistas Nicotínicos/química , Polissacarídeos/química , Receptores Nicotínicos/metabolismo , Sulfatos/química , Cápsulas , Neurotoxinas/toxicidade , Antagonistas Nicotínicos/toxicidade , Tamanho da Partícula , Venenos de Serpentes/química
5.
Molecules ; 24(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370142

RESUMO

Hypertension is considered a major public health issue due to its high prevalence and subsequent risk of cardiovascular and kidney diseases. Thus, the search for new antihypertensive compounds remains of great interest. Snake venoms provide an abundant source of lead molecules that affect the cardiovascular system, which makes them prominent from a pharmaceutical perspective. Such snake venom components include bradykinin potentiating peptides (proline-rich oligopeptides), natriuretic peptides, phospholipases A2, serine-proteases and vascular endothelial growth factors. Some heparin binding hypotensive factors, three-finger toxins and 5' nucleotidases can also exert blood pressure lowering activity. Great advances have been made during the last decade regarding the understanding of the mechanism of action of these hypotensive proteins. Bradykinin potentiating peptides exert their action primarily by inhibiting the angiotensin-converting enzyme and increasing the effect of endogenous bradykinin. Snake venom phospholipases A2 are capable of reducing blood pressure through the production of arachidonic acid, a precursor of cyclooxygenase metabolites (prostaglandins or prostacyclin). Other snake venom proteins mimic the effects of endogenous kallikrein, natriuretic peptides or vascular endothelial growth factors. The aim of this work was to review the current state of knowledge regarding snake venom components with potential antihypertensive activity and their mechanisms of action.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Hipotensão/tratamento farmacológico , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Anti-Hipertensivos/química , Bradicinina/química , Bradicinina/uso terapêutico , Humanos , Peptídeos/química , Peptídeos/uso terapêutico , Venenos de Serpentes/química
6.
Curr Top Med Chem ; 19(22): 2041-2048, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31340737

RESUMO

BACKGROUND: Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential. METHODS: A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated. RESULTS: Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 µg/mL against Leishmania infantum promastigotes and of 13.8 µg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 µg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 µg/mL. CONCLUSION: Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.


Assuntos
Antiparasitários/farmacologia , Leishmania infantum/efeitos dos fármacos , Fosfolipases A2/farmacologia , Venenos de Serpentes/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiparasitários/química , Antiparasitários/isolamento & purificação , Bothrops , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Camundongos , Paraguai , Testes de Sensibilidade Parasitária , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Relação Estrutura-Atividade
7.
Curr Top Med Chem ; 19(22): 2032-2040, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31340738

RESUMO

BACKGROUND: Phospholipases A2 (PLA2) from snake venoms have a broad potential as pharmacological tools on medicine. In this context, strongyloidiasis is a neglected parasitic disease caused by helminths of the genus Strongyloides. Currently, ivermectin is the drug of choice for treatment, however, besides its notable toxicity, therapeutic failures and cases of drug resistance have been reported. BnSP-6, from Bothorps pauloensis snake venom, is a PLA2 with depth biochemical characterization, reporting effects against tumor cells and bacteria. OBJECTIVE: The aim of this study is to demonstrate for the first time the action of the PLA2 on Strongyloides venezuelensis. METHODS: After 72 hours of treatment with BnSP-6 mortality of the infective larvae was assessed by motility assay. Cell and parasite viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, autophagic vacuoles were labeled with Monodansylcadaverine (MDC) and nuclei of apoptotic cells were labeled with Propidium Iodide (PI). Tissue degeneration of the parasite was highlighted by Transmission Electron Microscopy (TEM). RESULTS: The mortality index demonstrated that BnSP-6 abolishes the motility of the parasite. In addition, the MTT assay attested the cytotoxicity of BnSP-6 at lower concentrations when compared with ivermectin, while autophagic and apoptosis processes were confirmed. Moreover, the anthelmintic effect was demonstrated by tissue degeneration observed by TEM. Furthermore, we report that BnSP-6 showed low cytotoxicity on human intestinal cells (Caco-2). CONCLUSION: Altogether, our results shed light on the potential of BNSP-6 as an anthelmintic agent, which can lead to further investigations as a tool for pharmaceutical discoveries.


Assuntos
Anti-Helmínticos/farmacologia , Venenos de Crotalídeos/farmacologia , Fosfolipases A2/farmacologia , Venenos de Serpentes/farmacologia , Strongyloides/efeitos dos fármacos , Animais , Anti-Helmínticos/química , Anti-Helmínticos/isolamento & purificação , Bothrops , Células CACO-2 , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Relação Dose-Resposta a Droga , Feminino , Humanos , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Strongyloides/parasitologia , Relação Estrutura-Atividade
8.
Curr Top Med Chem ; 19(22): 1962-1980, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31345151

RESUMO

Among the ophidians that inhabit the Northeast of Argentina, the genus Bothrops such as B. alternatus and B. diporus species (also known as yararás) and Crotalus durisus terrificus (named cascabel), represent the most studied snake venom for more than thirty years. These two genera of venomous snakes account for the majority of poisonous snake envenomations and therefore, constitute a medical emergency in this region. This review presents a broad description of the compiled knowledge about venomous snakebite: its pathophysiological action, protein composition, isolated toxins, toxin synergism, toxin-antitoxin cross-reaction assays. Properties of some isolated toxins support a potential pharmacological application.


Assuntos
Venenos de Serpentes/farmacologia , Toxinas Biológicas/farmacologia , Animais , Argentina , Bothrops , Crotalus , Humanos , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Toxinas Biológicas/química , Toxinas Biológicas/isolamento & purificação
9.
Artigo em Inglês | MEDLINE | ID: mdl-31181499

RESUMO

Snake venoms are complex mixtures of a large number of distinct proteins and peptides with biological activity. Peptide spectral libraries are compilations of previously identified MS/MS spectra obtained from proteomics experiments. Here we present the generation and use of a Venom Peptidome and a Venom Proteome spectral library for the analysis of venom proteomes and peptidomes from distinct snake species.


Assuntos
Peptídeos/química , Proteínas de Répteis/química , Venenos de Serpentes/química , Espectrometria de Massas em Tandem , Animais , Bothrops/metabolismo , Venenos de Crotalídeos/química , Bases de Dados de Proteínas , Proteoma/química , Proteômica/métodos , Serpentes/metabolismo , Espectrometria de Massas em Tandem/métodos
10.
Zebrafish ; 16(4): 379-387, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31145051

RESUMO

Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 µg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.


Assuntos
Cardiotoxicidade/embriologia , Cardiotoxinas/farmacologia , Crotalinae , Lectinas Tipo C , Proteínas de Répteis/química , Venenos de Serpentes/química , Peixe-Zebra/embriologia , Animais , Cardiotoxinas/química , Crotalinae/embriologia , Embrião não Mamífero/efeitos dos fármacos , Lectinas Tipo C/química , Proteínas de Répteis/farmacologia
11.
Int J Biol Macromol ; 135: 261-273, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31128190

RESUMO

This work shows the antitumor and antimetastatic effects of BthTX-II, an Asp-49 PLA2 from Bothrops jararacussu venom, on MDA-MB-231 human triple negative breast cancer cells. BthTX-II caused a dose-dependent cell death of MDA-MB-231 cells when compared with the non-tumorigenic breast cells by inducing apoptosis and autophagy. BthTX-II was also able to decrease the proliferation and to inhibit cell cycle progression. We also observed an upregulation of the ATM gene, which is responsible for cell-cycle arrest and DNA repair such as CCND1, CCNE1, CDC25A, E2F1, AKT1 and AKT3. Interestingly, BthTX-II inhibited invasion, migration and 3D cell growth of MDA-MB-231 cells, as well as inhibited the epithelial-mesenchymal transition (EMT) of this cell by increasing E-cadherin (CDH-1) and decreasing TWIST1, CTNNB1, vimentin and cytokeratin-5 expression. In conclusion, these results showed that BthTX-II displays antitumor and antimetastatic effects on MDA-MB-231 cells and may be useful for the development of new approaches and therapeutic strategies to manage triple negative breast cancer.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Bothrops , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Fosfolipases A2 do Grupo II/química , Fosfolipases A2 do Grupo II/farmacologia , Animais , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Biomarcadores Tumorais , Adesão Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Fosfolipases A2 do Grupo II/isolamento & purificação , Humanos , Venenos de Serpentes/química , Venenos de Serpentes/farmacologia
12.
Int J Biol Macromol ; 134: 613-621, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31071401

RESUMO

Vascular endothelium plays an important modulatory role due to the production of molecules that mediate vasomotricity, inflammation, and leukocyte adhesion and rolling. Here we addressed whether crotoxin (25-200 µg/mL) - the main component of Crotalus durissus terrificus snake venom - interferes with cell viability, apotosis/necrosis, and cell response to oxidative stress in human umbilical vein endothelial cells (HUVEC) in vitro. We also examined whether crotoxin alters the levels of interleukins, adhesion molecules, and endothelial vasoactive factors in HUVEC cells treated or not with lipopolysaccharide (LPS; 1 µg/mL; 24 h). Crotoxin was not cytotoxic towards HUVEC cells, and downregulated the LPS-induced production of adhesion molecules (VCAM-1, ICAM-1, and E-selectin), vasoactive factors (endothelin-1 and prostaglandin I2), and interleukins (IL-6, IL-8, and IL1ß), as well as protected cells against H2O2-induced oxidative stress. Hence, crotoxin played anti-inflammatory, antioxidant, immunomodulating, and vasoactive actions on HUVEC cells, in vitro. Considering that the initial stages of atherosclerosis is characterized by vasoconstriction, increased levels of adhesion molecules, inflammatory cytokines, and oxidative stress in the vascular endothelium; and crotoxin downmodulated all these events, our findings indicate that the actions of crotoxin here demonstrated suggest that it may have an anti-atherogenic action in vivo, which deserves to be tested in future studies.


Assuntos
Crotoxina/química , Crotoxina/farmacologia , Células Endoteliais/efeitos dos fármacos , Neurotoxinas/química , Neurotoxinas/farmacologia , Venenos de Serpentes/química , Venenos de Serpentes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Citocinas/biossíntese , Humanos , Mediadores da Inflamação/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos
13.
Toxicon ; 164: 71-81, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30998944

RESUMO

Most colubrid snake venoms have been poorly studied, despite the fact that they represent a great resource for biological, ecological, toxinological and pharmacological research. Herein, we explore the venom delivery system of the Aesculapian False Coral Snake Erythrolamprus aesculapii as well as some biochemical and toxicological properties of its venom. Its Duvernoy's venom gland is composed of serous secretory cells arranged in densely packed secretory tubules, and the most striking feature of its fang is their double-curved shape, exhibiting a beveled bladelike appearance near the tips. Although E. aesculapii resembles elapid snakes of the genus Micrurus in color pattern, this species produces a venom reminiscent of viperid venoms, containing mainly tissue-damaging toxins such as proteinases. Prominent hemorrhage developed both locally and systemically in mice injected with the venom, and the minimum hemorrhagic dose was found to be 18.8 µg/mouse; the lethal dose, determined in mice, was 9.5 ±â€¯3.7 µg/g body weight. This work has toxicological implications that bites to humans by E. aesculapii could result in moderately severe local (and perhaps systemic) hemorrhage and gives insight into future directions for research on the venom of this species.


Assuntos
Colubridae/anatomia & histologia , Venenos de Serpentes/química , Venenos de Serpentes/toxicidade , Animais , Antivenenos/imunologia , Glândulas Exócrinas/anatomia & histologia , Feminino , Hemorragia/induzido quimicamente , Humanos , Masculino , Maxila/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Proteólise , Mordeduras de Serpentes , Venenos de Serpentes/imunologia , Dente/ultraestrutura
14.
Mater Sci Eng C Mater Biol Appl ; 100: 23-29, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948057

RESUMO

Emulsions are crucial in the treatment of snake bites to bust the antibody response of the inmunogen. The widely used Freund's emulsion typically combines 50/50 water-oil (W/O) phase. However, its use is limited because it is associated with tissue damage. We formulated and characterized a Pickering Emulsion 70/30 (W/O) that uses a chemically modified hydrophobic hydroxyapatite as surfactant. This Pickering emulsion has similar rheologic behavior to Freund's emulsion 50/50, but with lower oil and surfactant concentration. Evaluation of cell recruitment, antibody response and adhering tissue in mice immunized with B. asper of Pacific venom and treated with Freund's and Pickering 70/30 emulsions resulted in similar adjuvant activity (only 18% lower in Pickering 70/30 emulsion). However, Pickering 70/30 emulsions minimized negative side effects in the host animals and showed better ease of flow that favors injection of the host. Our results open up room for optimization and improvement of Pickering emulsion based on modified nanoparticles for medical applications.


Assuntos
Adjuvantes Imunológicos/química , Anticorpos/metabolismo , Durapatita/química , Emulsões/química , Nanopartículas/química , Venenos de Serpentes/imunologia , Animais , Camundongos , Venenos de Serpentes/química , Serpentes/metabolismo , Tensoativos/química
15.
J Mol Model ; 25(4): 88, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30847632

RESUMO

Alternative treatments from plant-derived small molecules for neutralizing the venom lethality in snake envenomation are prevalent now. Elephantopus scaber, a tropical plant species has been recognized for its various pharmacological activities and especially anti-snake venom property; however, the molecular basis for this property is not understood. It is reported that snake venom PLA2 is a toxic factor with pharmacological effects independent of their catalytic activity. Here we report the inhibition of catalytic property of Cobra and Viper (group I and group II) snake venom PLA2 by the phytocompounds from E. scaber through molecular docking and dynamics studies. Initially, Lipinski's rule, ADMET, and molecular docking studies were carried out. Our results show that among 124 phytocompounds, crepiside E (deacylcynaropicrin-3' beta-glucopyranoside) has shown interactions with the conserved catalytic active site residues, His 48 and Asp 49, in both the PLA2s. Further, molecular dynamic simulations for 60 ns confirmed the stability of crepiside E in the active site of PLA2s and were found to be stable throughout the simulation. In order to understand the drug-likeness of crepiside E, pIC50 and MMGBSA scores were correlated by performing a linear regression analysis. Crepiside E was found to have similar chemical features to that of doxycycline, a known PLA2 inhibitor as indicated by a similarity score of 64.15%. Hence, it is concluded that crepiside E beta glucopyranoside present in Elephantopus scaber contributes to neutralizing the snake venom.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Fitoquímicos/química , Venenos de Serpentes/química , Asteraceae/química , Sítios de Ligação , Domínio Catalítico , Concentração Inibidora 50 , Ligantes , Modelos Moleculares , Conformação Molecular , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/toxicidade , Venenos de Serpentes/antagonistas & inibidores , Relação Estrutura-Atividade
16.
Toxins (Basel) ; 11(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823637

RESUMO

Snake venoms contain an astounding variety of different proteins. Among them are numerous C-type lectin family members, which are grouped into classical Ca2+- and sugar-binding lectins and the non-sugar-binding snake venom C-type lectin-related proteins (SV-CLRPs), also called snaclecs. Both groups share the robust C-type lectin domain (CTLD) fold but differ in a long loop, which either contributes to a sugar-binding site or is expanded into a loop-swapping heterodimerization domain between two CLRP subunits. Most C-type lectin (-related) proteins assemble in ordered supramolecular complexes with a high versatility of subunit numbers and geometric arrays. Similarly versatile is their ability to inhibit or block their target molecules as well as to agonistically stimulate or antagonistically blunt a cellular reaction triggered by their target receptor. By utilizing distinct interaction sites differentially, SV-CLRPs target a plethora of molecules, such as distinct coagulation factors and receptors of platelets and endothelial cells that are involved in hemostasis, thrombus formation, inflammation and hematogenous metastasis. Because of their robust structure and their high affinity towards their clinically relevant targets, SV-CLRPs are and will potentially be valuable prototypes to develop new diagnostic and therapeutic tools in medicine, provided that the molecular mechanisms underlying their versatility are disclosed.


Assuntos
Lectinas Tipo C/química , Venenos de Serpentes/química , Animais , Humanos , Conformação Proteica
17.
Int J Biol Macromol ; 131: 127-133, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30867125

RESUMO

Among the activities triggered by Crotalus durissus terrificus snake venom, coagulation is intriguing and contradictory since the venom contains both coagulant and anticoagulant precursor proteins. This work describes the in vitro effects of crude venom and purified proteins from snake Crotalus durissus terrificus as they affect coagulation factors of clotting pathways. Coagulant and/or anticoagulant activities of crude venom, and purified proteins were all analyzed directly in human plasma. Clots formed by crude venom and Gyroxin presented as flexible hyaline masses in punctiform distribution. Clot formation time evaluation of isolated proteins with PT and APTT assays made it possible to infer that these proteins interfere in all coagulation pathways. However, regarding ophidism by C. d. terrificus, Gyroxin acts directly, breaking down fibrinogen to fibrin and increasing the amount plasminogen activator, which results in the formation of thrombi. Crotoxin complex, Crotoxin A and Crotoxin B proteins can act in prothrombinase complex formation; Crotoxin B can inhibit prothrombinase complex formation by direct interaction with Factor Xa. Crotamine interacts with negatively charged regions of differing coagulation factors in all coagulation pathways, and possesses a whole set of activities causing dysfunction, activation and/or inhibition of natural anticoagulants and disturbing hemostasis.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Crotalus , Venenos de Serpentes/química , Venenos de Serpentes/farmacologia , Sequência de Aminoácidos , Animais , Testes de Coagulação Sanguínea , Humanos , Modelos Moleculares , Conformação Molecular , Fenômenos Físicos , Venenos de Serpentes/isolamento & purificação
18.
Arch Virol ; 164(4): 1159-1171, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30809709

RESUMO

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/farmacologia , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Venenos de Serpentes/enzimologia , Animais , Antivirais/química , Cromatografia de Afinidade , Crotalus , Vírus da Dengue/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/farmacologia , Fosfolipases A2/química , Fosfolipases A2/genética , Dobramento de Proteína , Proteínas de Répteis/química , Proteínas de Répteis/genética , Venenos de Serpentes/química , Vírus da Febre Amarela/efeitos dos fármacos , Zika virus/efeitos dos fármacos
19.
J Biol Inorg Chem ; 24(2): 171-178, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30673877

RESUMO

In this work, we have studied the effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin (Mb), and by means of electrochemical techniques, we have shown that this reaction is undoubtedly affected following the interaction with the venom. Surface plasmon resonance, electrophoresis, UV-Vis, and circular dichroism showed that the interaction involves the attachment of some constituent of the venom to the protein, although not affecting its first and secondary structures. Mass spectra support this suggestion by showing the appearance of signals assigned to the Mb dimer and to a new species resulting from the interaction between Mb and the venom proteins. In addition, the mass spectra suggest the aromatic amino acids of myoglobin, mainly tryptophan and phenylalanine, are more exposed to the solvent medium upon the exposure to the venom solution. The results altogether indicate that the harmful effects of the venom of Crotalus basiliscus snake are likely connected to the blocking of the redox site of Mb.


Assuntos
Mioglobina/antagonistas & inibidores , Venenos de Serpentes/farmacologia , Animais , Crotalus , Técnicas Eletroquímicas , Humanos , Mioglobina/metabolismo , Oxirredução , Venenos de Serpentes/química
20.
J Biol Chem ; 294(4): 1250-1256, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30504218

RESUMO

Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal ß-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.


Assuntos
Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas Secretadas pela Próstata/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Viperidae/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Proteínas Secretadas pela Próstata/química , Conformação Proteica , Homologia de Sequência
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