RESUMO
BACKGROUND: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. METHODS: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. RESULTS: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. CONCLUSION: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. GENERAL SIGNIFICANCE: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.
Assuntos
Acetatos/farmacologia , Bothrops/metabolismo , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Indóis/farmacologia , Cetoácidos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/química , Proteínas de Répteis/antagonistas & inibidores , Animais , Cristalografia por Raios X , Fosfolipases A2 do Grupo II/toxicidade , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas de Répteis/toxicidadeRESUMO
Snakebite is a neglected disease with a high impact in tropical and subtropical countries. Therapy based on antivenom has limited efficacy in local tissue damage caused by venoms. Phospholipases A2 (PLA2) are enzymes that abundantly occur in snake venoms and induce several systemic and local effects. Furthermore, sulfur compounds such as thioesters have an inhibitory capacity against a snake venom PLA2. Hence, the objective of this work was to obtain a carbodithioate from a thioester with known activity against PLA2 and test its ability to inhibit the same enzyme. Benzyl 4-nitrobenzenecarbodithioate (I) was synthesized, purified, and characterized using as precursor 4-nitrothiobenzoic acid S-benzyl ester (II). Compound I showed inhibition of the enzymatic activity a PLA2 isolated from the venom of the Colombian rattlesnake Crotalus durissus cumanensis with an IC50 of 55.58 µM. This result is comparable with the reported inhibition obtained for II. Computational calculations were performed to support the study, and molecular docking results suggested that compounds I and II interact with the active site residues of the enzyme, impeding the normal catalysis cycle and attachment of the substrate to the active site of the PLA2.
Assuntos
Venenos de Crotalídeos/química , Crotalus , Simulação de Acoplamento Molecular , Inibidores de Fosfolipase A2/química , Fosfolipases A2/química , Proteínas de Répteis , Compostos de Enxofre/química , Animais , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/químicaRESUMO
A need exists to develop specific and clinically useful inhibitors of toxic enzymes present in snake venoms, responsible for severe tissue damage and life-threatening effects occurring in thousands of people suffering envenomations globally. LY315920 (Varespladib, S-5920, A-001), a low molecular weight drug developed to inhibit several human secreted phospholipases A2 (PLA2s), was recently shown to also inhibit PLA2s in whole snake venoms with high potency, yet no studies have examined its direct effect on purified snake venom PLA2s. This work evaluated the ability of LY315920 to neutralize the enzymatic and toxic activities of three isolated PLA2 toxins of structural groups I (pseudexin) and II (crotoxin B and myotoxin I), and their corresponding whole venoms. In vitro, LY315920 inhibited the catalytic activity of these three enzymes upon a synthetic substrate. The drug also blocked their cytotoxic effect on cultured murine myotubes. In mice, preincubation of the toxins or venoms with LY315920, followed by their intramuscular injection, resulted in significant inhibition of muscle damage. Finally, immediate, independent injection of LY315920 at the site of toxin or venom inoculation also resulted in a large reduction of myonecrosis in the case of pseudexin and myotoxin-I, and of Pseudechis colletti and Bothrops asper whole venoms, suggesting a possible method of drug delivery in emergency situations. Present findings add evidence to suggest the possibility of using LY315920 as a field antidote in snakebites, aiming to limit the myonecrosis induced by many venom PLA2s in the clinical setting.
Assuntos
Acetatos/farmacologia , Fosfolipases A2 do Grupo I/efeitos dos fármacos , Fosfolipases A2 do Grupo II/efeitos dos fármacos , Indóis/farmacologia , Acetatos/administração & dosagem , Animais , Células Cultivadas , Venenos de Crotalídeos , Crotoxina/antagonistas & inibidores , Venenos Elapídicos/antagonistas & inibidores , Feminino , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Indóis/administração & dosagem , Cetoácidos , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/patologia , Proteínas de Répteis/antagonistas & inibidoresRESUMO
In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10⯵g of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37⯰C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100â¯kDa, 45-70â¯kDa, 21-24â¯kDa and ~15â¯kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68â¯kDa (seen with 0.05-0.25⯵g of venom). Caseinolytic activity occurred at 50-66â¯kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.
Assuntos
Colubridae/fisiologia , Peptídeo Hidrolases/metabolismo , Fosfolipases A2/metabolismo , Proteínas de Répteis/metabolismo , Venenos de Serpentes/enzimologia , Animais , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Brasil , Colubridae/crescimento & desenvolvimento , Esterases/química , Esterases/metabolismo , Esterases/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Metaloproteases/química , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Peso Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/química , Fosfolipases A2/farmacologia , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Ratos Wistar , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Venenos de Serpentes/metabolismo , Venenos de Serpentes/farmacologia , Especificidade por Substrato , TemperaturaRESUMO
While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.
Assuntos
Venenos de Crotalídeos/metabolismo , Crotalus/fisiologia , Evolução Molecular , Músculo Esquelético/efeitos dos fármacos , Bloqueadores Neuromusculares/metabolismo , Neurotoxinas/metabolismo , Proteínas de Répteis/metabolismo , Animais , Antivenenos/farmacologia , Arizona , Galinhas , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Crotalus/crescimento & desenvolvimento , Clima Desértico , Feminino , Técnicas In Vitro , Dose Letal Mediana , Masculino , México , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/inervação , Bloqueadores Neuromusculares/antagonistas & inibidores , Bloqueadores Neuromusculares/química , Bloqueadores Neuromusculares/toxicidade , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/química , Neurotoxinas/toxicidade , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Fosfolipases A2/toxicidade , Proteômica/métodos , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/química , Proteínas de Répteis/toxicidade , Especificidade da Espécie , Especificidade por Substrato , TexasRESUMO
Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammation; therefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.
Assuntos
Anti-Inflamatórios/química , Fosfolipases A2 Secretórias/química , Quercetina/análogos & derivados , Proteínas de Répteis/química , Animais , Anti-Inflamatórios/farmacologia , Bothrops , Linhagem Celular , Venenos de Crotalídeos/enzimologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Camundongos , Fosfolipases A2 Secretórias/antagonistas & inibidores , Fosfolipases A2 Secretórias/isolamento & purificação , Quercetina/química , Quercetina/farmacologia , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/isolamento & purificaçãoRESUMO
The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs) began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig) supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using "gold-standard" methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted.
Assuntos
Antídotos/uso terapêutico , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/uso terapêutico , Proteínas de Répteis/antagonistas & inibidores , Mordeduras de Serpentes/tratamento farmacológico , Venenos de Serpentes/antagonistas & inibidores , Animais , Antídotos/história , História do Século XVIII , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/história , Metaloendopeptidases/metabolismo , Inibidores de Proteases/história , Conformação Proteica , Proteínas de Répteis/química , Proteínas de Répteis/história , Proteínas de Répteis/metabolismo , Mordeduras de Serpentes/enzimologia , Mordeduras de Serpentes/história , Venenos de Serpentes/química , Venenos de Serpentes/enzimologia , Venenos de Serpentes/história , Relação Estrutura-AtividadeRESUMO
One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims.
Assuntos
Antídotos/farmacologia , Ácidos Aristolóquicos/farmacologia , Bothrops/metabolismo , Ácidos Cafeicos/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Proteínas de Répteis/antagonistas & inibidores , Animais , Antídotos/química , Ácidos Aristolóquicos/química , Ácidos Cafeicos/química , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Fosfolipases A2 do Grupo II/química , Fosfolipases A2 do Grupo II/metabolismo , Camundongos , Modelos Moleculares , Músculos/efeitos dos fármacos , Músculos/patologia , Músculos/fisiopatologia , Conformação Proteica , Proteínas de Répteis/química , Proteínas de Répteis/metabolismoRESUMO
Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, 'Asp49' and 'Lys49' myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/toxicidade , Fibras Musculares Esqueléticas/efeitos dos fármacos , Neurotoxinas/toxicidade , Fosfolipases A2/toxicidade , Proteínas de Répteis/toxicidade , Acetofenonas/química , Sequência de Aminoácidos , Animais , Ácido Aspártico/química , Morte Celular/efeitos dos fármacos , Linhagem Celular , Creatina Quinase/sangue , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/toxicidade , Lisina/química , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/química , Proteínas de Répteis/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A2 activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A2 activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd was administered in combination with BA. The SDS-PAGE profiles showed that several of the BaV protein (SDS-PAGE) and enzyme (zymography) bands were not detected when the venom was pre-incubated, and Western blot revealed that this was not caused by the AEBd enzymes observed in the zymogram. The "pseudo inhibition" observed after pre-incubation in this study may be due to the presence of tannins in the extract, which could act as chelating agents, removing metalloproteins and Ca²âº ions and thus inhibiting hemorrhagin and PLA2 activity. However, when administered according to traditional methods, B. dichotoma extract was effective in blocking BaV-induced edematogenic activity and had an additional effect on inhibition of this activity by BA.
Assuntos
Antivenenos/uso terapêutico , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Melastomataceae/química , Casca de Planta/química , Extratos Vegetais/uso terapêutico , Mordeduras de Serpentes/tratamento farmacológico , Animais , Antivenenos/química , Antivenenos/isolamento & purificação , Antivenenos/farmacologia , Brasil , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/toxicidade , Edema/etiologia , Edema/prevenção & controle , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Etnofarmacologia , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/metabolismo , Hemorragia/etiologia , Hemorragia/prevenção & controle , Hemostáticos/química , Hemostáticos/isolamento & purificação , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Masculino , Medicina Tradicional , Camundongos , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/metabolismo , Pele/efeitos dos fármacos , Mordeduras de Serpentes/fisiopatologia , Taninos/análise , Taninos/farmacologia , Taninos/uso terapêuticoRESUMO
Crotoxin (Crtx), the main toxin in the venom of Crotalus durissus terrificus snake, is a heterodimer with a basic subunit, CB, and an acidic subunit, CA. CB is a phospholipase A2 that depends on CA to specifically bind to the cell membrane. This toxin acts in the central nervous system (CNS) causing chronic seizure effects and other cytotoxic effects. Here, we report its action on glutamate release in rat cerebral cortex synaptosomes. Aiming at a better understanding of the mechanism of action of Crtx, calcium channel blockers were used and internalization studies were performed in cerebellar granule neurons. Our results show that Crtx induces calcium-dependent glutamate release via N and P/Q calcium channels. In addition, the CB subunit of Crtx is shown to be internalized. This internalization does not depend on the presence of CA subunit neither on the PLA2 activity of CB. A correlation between CB internalization and glutamate release remains to be established.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Córtex Cerebral/efeitos dos fármacos , Venenos de Crotalídeos/química , Crotalus , Crotoxina/farmacologia , Sinaptossomos/efeitos dos fármacos , Animais , Agonistas dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/química , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/metabolismo , Venenos de Crotalídeos/enzimologia , Crotoxina/antagonistas & inibidores , Crotoxina/metabolismo , Ácido Glutâmico/metabolismo , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo II/farmacologia , Masculino , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Subunidades Proteicas/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos Wistar , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/metabolismo , Proteínas de Répteis/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
The venoms of Bothrops (Rhinocerophis) alternatus (B.a.) from different regions of Argentina have shown biochemical, toxicological and immunological variations. Considering these variations, we produced nine experimental antisera (rabbit, IgG) against venoms from snakes of nine different regions and a pool of venom, comprised of equal amounts of venoms from each region. The immunologic studies (ELISA, Westernblot) showed significant cross reactivity among all regional antivenoms with all regional venoms, with no significant differences regarding the specificity of the immunogens used for the production of antivenom. Neutralization of hemorrhage was variable (although all the antivenoms neutralized this activity in all venoms) and the neutralization of coagulant and phospholipase activities were evident in all cases. Some antivenoms neutralized toxic activities that were absent or very low in the venoms used as immunogen, on other non-homologous venoms (e.g. thrombin like activity). Despite the different toxic potencies of regional venoms, antivenoms developed using venoms of snakes from a particular region showed high immunochemical reactivity and cross-neutralizing capacity on snake venoms from different and distant regions, in occasions over those of the homologous antivenoms. These findings could be used to improve the generation of pools of venoms for the production of antivenoms.
Assuntos
Antivenenos/farmacologia , Bothrops , Coagulantes/antagonistas & inibidores , Venenos de Crotalídeos/antagonistas & inibidores , Drogas em Investigação/farmacologia , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Anticoagulantes/farmacologia , Argentina , Coagulação Sanguínea/efeitos dos fármacos , Western Blotting , Coagulantes/farmacologia , Reações Cruzadas , Venenos de Crotalídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Fosfolipases A2 do Grupo II/metabolismo , Hemólise/efeitos dos fármacos , Hemostáticos/farmacologia , Cavalos , Humanos , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/metabolismoRESUMO
BACKGROUND: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs. METHODS: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated. RESULTS: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 µg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid. CONCLUSION: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.
Assuntos
Benzodioxóis/farmacologia , Bothrops , Venenos de Crotalídeos/enzimologia , Inibidores Enzimáticos/farmacologia , Fabaceae/química , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Isoflavonas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Acetofenonas/farmacologia , Animais , Ácidos Aristolóquicos/farmacologia , Benzodioxóis/isolamento & purificação , Benzodioxóis/uso terapêutico , Brasil , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/uso terapêutico , Fosfolipases A2 do Grupo II/química , Humanos , Isoflavonas/isolamento & purificação , Isoflavonas/uso terapêutico , Nitrobenzoatos/metabolismo , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Folhas de Planta , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/química , Mordeduras de Serpentes/tratamento farmacológico , Mordeduras de Serpentes/enzimologiaRESUMO
Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C. sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)s (crotoxin from C. durissus terrificus, bothropstoxin-I from B. jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 microM) and all other PLA(2) toxins (1.0 microM) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA(2) toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C) significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1:10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA(2) inhibitors.