RESUMO
Snakebite is a neglected tropical disease that causes extensive mortality and morbidity in rural communities. Antivenim sera are the currently approved therapy for snake bites; however, they have some therapeutic limitations that have been extensively documented. Recently, small molecule toxin inhibitors have received significant attention as potential alternatives or co-adjuvant to immunoglobulin-based snakebite therapies. Thus, in this study, we evaluated the inhibitory effects of the phospholipase A2 inhibitor varespladib and the metalloproteinase inhibitor CP471474 and their synergistic effects on the lethal, edema-forming, hemorrhagic, and myotoxic activities of Bothrops asper and Crotalus durissus cumanensis venoms from Colombia. Except for the preincubation assay of the lethal activity with B. asper venom, the mixture showed the best inhibitory activity. Nevertheless, the mix did not display statistically significant differences to varespladib and CP471474 used separately in all assays. In preincubation assays, varespladib showed the best inhibitory activity against the lethal effect induced by B. asper venom. However, in independent injection assays, the mix of the compounds partially inhibited the lethal activity of both venoms (50%). In addition, in the assays to test the inhibition of edema-forming activity, the mixture exhibited the best inhibitory activity, followed by Varespladib, but without statistically significant differences (p > 0.05). The combination also decreased the myotoxic activity of evaluated venoms. In these assays, the mix showed statistical differences regarding CP471474 (p < 0.05). The mixture also abolished the hemorrhagic activity of B. asper venom in preincubation assays, with no statistical differences to CP471474. Finally, the mixture showed inhibition in studies with independent administration in a time-dependent manner. To propose a mode of action of varespladib and CP471474, molecular docking was performed. PLA2s and SVMPs from tested venoms were used as targets. In all cases, our molecular modeling results suggested that inhibitors may occupy the substrate-binding cleft of the enzymes, which was supported by specific interaction with amino acids from the active site, such as His48 for PLA2s and Glu143 for the metalloproteinase. In addition, varespladib and CP471474 also showed interaction with residues from the hydrophobic channel in PLA2s and substrate binding subsites in the SVMP. Our results suggest a synergistic action of the mixed inhibitors and show the potential of varespladib, CP471474, and their mixture to generate new treatments for snakebite envenoming with application in the field or as antivenom co-adjuvants.
Assuntos
Bothrops , Venenos de Crotalídeos , Mordeduras de Serpentes , Animais , Simulação de Acoplamento Molecular , Venenos de Crotalídeos/toxicidade , Antivenenos/farmacologia , Antivenenos/uso terapêutico , Mordeduras de Serpentes/tratamento farmacológico , Metaloproteases , Hemorragia/tratamento farmacológico , Edema/induzido quimicamente , Edema/tratamento farmacológicoRESUMO
Renealmia alpinia (Rottb.) MAAS, obtained by micropropagation (in vitro) and wild forms have previously been shown to inhibit some toxic activities of Bothrops asper snake venom if preincubated before injection. In this study, assays were performed in a murine model in which extracts were administered for three days before venom injection. R. alpinia extracts inhibited lethal activity of B. asper venom injected by intraperitoneal route. Median Effective Dose (ED50) values were 36.6 ± 3.2 mg/kg and 31.7 ± 5.4 mg/kg (p > 0.05) for R. alpinia wild and in vitro extracts, respectively. At a dose of 75 mg/kg, both extracts totally inhibited the lethal activity of the venom. Moreover, this dose prolonged survival time of mice receiving a lethal dose of venom by the intravenous route. At 75 mg/kg, both extracts of R. alpinia reduced the extent of venom-induced pulmonary hemorrhage by 48.0% (in vitro extract) and 34.7% (wild extract), in agreement with histological observations of lung tissue. R. alpinia extracts also inhibited hemorrhage in heart and kidneys, as evidenced by a decrease in mg of hemoglobin/g of organ. These results suggest the possibility of using R. alpinia as a prophylactic agent in snakebite, a hypothesis that needs to be further explored.
Assuntos
Antídotos/uso terapêutico , Bothrops , Venenos de Crotalídeos/toxicidade , Hemorragia/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Zingiberaceae , Animais , Rim/efeitos dos fármacos , Rim/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Miocárdio/patologia , Fitoterapia , Mordeduras de Serpentes/tratamento farmacológicoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. MATERIALS AND METHODS: We have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. RESULTS: Colorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. CONCLUSIONS: Our results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the enzymes nor protein aggregation, but is likely to depend on molecular interactions of secondary metabolites in the plant with these venom proteinases.
Assuntos
Venenos de Crotalídeos/antagonistas & inibidores , Metaloproteases/antagonistas & inibidores , Extratos Vegetais/farmacologia , Inibidores de Serina Proteinase/farmacologia , Zingiberaceae , Animais , Venenos de Crotalídeos/farmacologia , Edema/prevenção & controle , Fibrinogênio/antagonistas & inibidores , Hemorragia/prevenção & controle , Metaloproteases/farmacologia , Camundongos , Serina Proteases/metabolismoRESUMO
Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC50 of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²âº ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²âº, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.
Assuntos
Bothrops , Edema/tratamento farmacológico , Glicolatos/farmacologia , Metaloendopeptidases/toxicidade , Venenos de Serpentes/toxicidade , Animais , Caseínas/metabolismo , Domínio Catalítico/efeitos dos fármacos , Quelantes/química , Hemorragia/tratamento farmacológico , Concentração Inibidora 50 , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Simulação de Acoplamento Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteólise/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Mordeduras de Serpentes/tratamento farmacológico , Zinco/metabolismoRESUMO
Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 µg and 0.571 µg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.
Assuntos
Coagulantes/metabolismo , Venenos de Crotalídeos/enzimologia , Crotalus/metabolismo , Hemorragia/induzido quimicamente , Serina Proteases/metabolismo , Sequência de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Bovinos , Cromatografia Líquida de Alta Pressão , Coagulantes/química , Coagulantes/toxicidade , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Edema/induzido quimicamente , Edema/patologia , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Serina Proteases/química , Serina Proteases/toxicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Introducción. La medicina tradicional es una invaluable fuente de investigación de nuevos remedios como complemento para el tratamiento del accidente ofídico, considerado como un grave problema de salud pública a nivel mundial. Objetivo. Este trabajo de investigación pretende comprobar la capacidad de neutralizar los efectos hemorrágicos, coagulantes y proteolíticos, de los extractos de hojas de Renealmia alpinia, usada tradicionalmente por los indígenas del Chocó (Colombia) contra la mordedura de la serpiente Bothrops asper, causante de la gran mayoría de los accidentes ofídicos en nuestro país. Materiales y métodos. Se llevaron a cabo ensayos de toxicidad aguda y de actividad analgésica in vivo de R. alpinia. Además, se hicieron ensayos in vitro sobre inhibición de las actividades coagulante, hemolítica y proteolítica del veneno de B. asper. Resultados. El presente estudio demuestra que R. alpinia no produce efectos tóxicos en animales de experimentación; además, presenta efectos analgésicos in vivo y antiofídicos in vitro,y protege contra los efectos letales del veneno de B. asper, in vivo. Conclusión. Renealmia alpinia puede ser una buena alternativa terapéutica como complemento al tratamiento con antiveneno en el accidente ofídico, por sus efectos analgésicos y antiofídicos.
Introduction. Traditional medicine is an invaluable source of research into new medicines as a supplement for the treatment of snakebite, considered as a serious public health problem worldwide. The extracts of the medicinal plant, Renealmia alpina, have been used traditionally by indigenous people of Chocó (Colombia) against Bothrops asper snakebite, a snake responsible for the majority of snakebite accidents in Colombia. Objective. The ability of extracts of R. alpinia leaves was tested for its ability to neutralize the hemorrhagic, coagulant and proteolytic effects of the snakebite venom of B. asper. Materials and methods. The acute toxicity tests and analgesic activity of R. alpina were evaluated in vivo. In addition, tests were undertaken in in vitro conditions to demonstrate inhibition of coagulant, haemolytic and proteolytic activity of the B. asper venom. Results. Renealmia alpinia extracts had no toxic effects in experimental animals and also provided analgesic and antiophidian effects and protection against the lethal effects of the venom of B. asper. Conclusion. Renealmia. alpinia was an effective therapeutic alternative in association with antivenom treatment in the event of a B. asper snakebite accident. It was demonstrated to protect against the lethal effects and provided analgesic properties as well.
Assuntos
Animais , Feminino , Masculino , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Fitoterapia , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Mordeduras de Serpentes/tratamento farmacológico , Zingiberaceae , Acetatos , Analgésicos/uso terapêutico , Analgésicos/toxicidade , Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Etanol , Hexanos , Hemólise/efeitos dos fármacos , Metanol , Cloreto de Metileno , Camundongos Endogâmicos BALB C , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Proteólise/efeitos dos fármacos , SolventesRESUMO
Crotoxin, one of the major toxins of South American rattlesnake Crotalus durissus subspecies, is an heterodimeric complex composed of two distinct subunits: a basic phospholipase A(2) (PLA(2), CB) and an acidic nontoxic catalytically inactive protein, crotapotin (CA). It's well known that CB has a high enzymatic activity; however the molecular aspects that determine this fact remain unknown. In this study, an in silico approach was used to predict the CA structure by homology modeling, and the crotoxin structure by means of molecular docking. CA structure was built using the software Modeller taking Crotalus atrox PLA(2) (1PP2:R) as a template. Different criteria measured by Procheck, Verify 3D and ProSA were indicative of the reliability and the proper fold for the predicted structural model of CA. Then, a combination of this model and CB crystal structure was used to build the structure of crotoxin complex through rigid-body protein-protein docking. The crotoxin-3D model suggested that by means of hydrophobic and π-π stacking interactions, CA-Y24 and CA-F119 interact with CB-F24 and CB-F119, respectively. Those interactions could prevent the interfacial adsorption of the CB onto the lipid/water interface by blocking part of the interfacial binding surface of the PLA(2). This fact could explain the differences regarding to enzymatic activity between the crotoxin complex and CB. In addition, the crotoxin-3D model showed solvent-exposed regions of CA that could bind the receptor expressed in target cells.
Assuntos
Simulação por Computador , Crotoxina/química , Modelos Moleculares , Fosfolipases A2/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Domínio Catalítico , Dados de Sequência Molecular , Fosfatidilcolinas/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
INTRODUCTION: Traditional medicine is an invaluable source of research into new medicines as a supplement for the treatment of snakebite, considered as a serious public health problem worldwide. The extracts of the medicinal plant, Renealmia alpina, have been used traditionally by indigenous people of Chocó (Colombia) against Bothrops asper snakebite, a snake responsible for the majority of snakebite accidents in Colombia. OBJECTIVE: The ability of extracts of R. alpinia leaves was tested for its ability to neutralize the hemorrhagic, coagulant and proteolytic effects of the snakebite venom of B. asper. MATERIALS AND METHODS: The acute toxicity tests and analgesic activity of R. alpina were evaluated in vivo. In addition, tests were undertaken in in vitro conditions to demonstrate inhibition of coagulant, haemolytic and proteolytic activity of the B. asper venom. Results. Renealmia alpinia extracts had no toxic effects in experimental animals and also provided analgesic and antiophidian effects and protection against the lethal effects of the venom of B. asper. CONCLUSION: Renealmia. alpinia was an effective therapeutic alternative in association with antivenom treatment in the event of a B. asper snakebite accident. It was demonstrated to protect against the lethal effects and provided analgesic properties as well.
Assuntos
Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Fitoterapia , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Mordeduras de Serpentes/tratamento farmacológico , Zingiberaceae , Acetatos , Analgésicos/uso terapêutico , Analgésicos/toxicidade , Animais , Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Etanol , Feminino , Hemólise/efeitos dos fármacos , Hexanos , Masculino , Metanol , Cloreto de Metileno , Camundongos Endogâmicos BALB C , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Proteólise/efeitos dos fármacos , SolventesRESUMO
Bile acids, such as cholic acid (CA) and ursodeoxycholic acid (UDCA) have shown to decrease or increase the enzymatic activity of group IB pancreatic PLA(2), depending on the concentration used. Studies suggest that the inhibition of hydrolysis rate of the substrate is due to formation in aqueous phase of a complex between bile acid and PLA(2), which is catalytically inert. For this reason, we tested the inhibition of the enzymatic activity of group IIA snake venom PLA(2) by bile acids, using an aqueous phase model. In addition, we measured the ability of bile acids to inhibit the toxic effects caused by the mentioned toxin. UDCA and CA inhibited the enzymatic activity of the PLA(2) in a competitive mode. Moreover, these compounds inhibited myotoxic, cytotoxic and edema-forming activities induced by the toxin, but UDCA was more efficient than CA. It was demonstrated that bile acids interact directly with this protein by causing slight changes in the intrinsic fluorescence spectra. Preliminary molecular docking studies suggest that bile acids interact with amino acids at the active site of the PLA(2) through different interactions, CA showed hydrogen bonds with His48, whereas, UDCA displayed with Asp49. Results obtained herein may turn UDCA and CA into promising models for the development of new molecules with anti-inflammatory and anti-snake venom PLA(2) properties.