RESUMO
The fungitoxic effect of aristolochic acids I and II on mycelial growth and conidial germination of Botrytis cinerea was analysed. Aristolochic acid I had a higher effect on mycelial growth of B. cinerea than aristolochic acid II with IC50 value of 18·7 and 57·0 µg ml-1 , respectively. These compounds did not affect the conidia germination. Also, the effect of both compounds on DNA and plasmatic membrane integrity of B. cinerea was studied. Only aristolochic acid II was able to cause damage to the integrity of the plasmatic membrane. When the fungus was incubated with a mixture of these compounds, degradation of DNA was observed. Finally, biotransformation products were not detected in the culture broth when B. cinerea was incubated in the presence of the aristolochic acids. Studies of structural characteristics that increase the antifungal effect of compounds against B. cinerea will permit to design new molecules to control this phytopathogenic fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungitoxic effect on Botrytis cinerea of aristolochic acids I and II was characterized. The only structural difference among these compounds is a methoxy group at carbon 8. However, despite their structural similarity, the fungitoxic effect of aristolochic acid I was higher than the effect of aristolochic acid II. This result suggests that the methoxy group is important for the fungitoxic activity of these compounds on B. cinerea.
Assuntos
Antifúngicos/farmacologia , Ácidos Aristolóquicos/farmacologia , Botrytis/crescimento & desenvolvimento , Micélio/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento , Botrytis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , DNA Fúngico/metabolismoRESUMO
Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.
Assuntos
Lactonas/farmacologia , Oócitos/efeitos dos fármacos , Fosfolipases A2/metabolismo , Sesquiterpenos/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Ácidos Aristolóquicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Bufo arenarum , Estrenos/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos , Neomicina/farmacologia , Norbornanos , Oócitos/enzimologia , Oócitos/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Pirrolidinonas/farmacologia , Quinacrina/farmacologia , Tiocarbamatos , Tionas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
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
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.