Bioprospecção de compostos inibidores da atividade catalítica de homólogos das enzimas conversoras de angiotensina (ECA 1 e ECA 2) de insetos e de mamíferos
Data
2022-07-29
Autores
Pederiva, Victoria Perini [UNIFESP]
Orientadores
Lapa, Antonio José [UNIFESP]
Tipo
Trabalho de conclusão de curso de graduação
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Mosquitos transmissores de arboviroses (dengue, zika, chikungunya, febre amarela), ou de malária e filariose, têm apresentado resistência progressiva aos inseticidas sintéticos clássicos de baixa toxicidade humana. A (quase) inexistência de vacinas eficazes e de medicamentos específicos contra essas enfermidades soma-se ao insucesso na pesquisa de novos inseticidas para prevenir surtos de doenças vetoriais. As enzimas conversoras de angiotensina (ECAs) de mamíferos têm importantes ações reguladoras da pressão arterial e foram encontrados homólogos dessas enzimas em insetos, cuja função ainda é pouco conhecida, porém vital. Esta pesquisa visa a obtenção de compostos naturais inibidores das ECAs de insetos que podem agir como inseticidas. A Cecropia glazioui foi a planta selecionada para este estudo.
As atividades enzimáticas das ECAs foram determinadas por fluorimetria utilizando o substrato FRET do tipo Abz-FRK(Dnp)P-OH para a ECA-1, e Mca-APK(Dnp) para a ECA-2. A atividade enzimática foi determinada incubando o homogenato de larvas (ou o plasma de ratos) com a fração butanólica (Fbut) (10-1000 ug/mL) ou com os compostos majoritários (0,1-100 uM) da C. glazioui. A Fbut inibiu não seletivamente as ECAs de plasma de rato e de homogenato de larvas, com valores de IC50 muito próximos. Apesar dos compostos serem menos ativos que a Fbut, o ácido clorogênico, as procianidinas B5, B3 e B2, a orientina, a isoorientina e a isovitexina (100 uM) reduziram significativamente a fluorescência emitida pelo substrato Abz para ECA-1 (p<0,0001)
Para a prova de conceito in vivo, foram testados a Fbut (0,3-30 mg/mL) e os compostos (0,03-0,3 mg/mL) que apresentaram maior atividade nos testes in vitro: orientina, isoorientina e isovitexina. Nesse ensaio, as larvas de Aedes aegypti foram expostas a diferentes concentrações desses compostos, registrando a mortalidade após 24, 48 e 72h. O desenvolvimento da larva até pupa foi avaliado por duas semanas. A letalidade para os primeiros estágios larvais foi maior e significativamente diferente da observada no estágio final de desenvolvimento da larva. Apesar dos flavonóides não terem mostrado uma ação inseticida in vivo, esses compostos inibiram significativamente o crescimento nos primeiros ínstares. O efeito foi persistente durante o tempo de exposição das larvas aos compostos. É possível que essa atividade esteja relacionada à inibição dos homólogos das ECAs em insetos, porém mecanismos adicionais ainda não foram estudados.
Mosquitoes that transmit arboviruses (dengue, zika, chikungunya, yellow fever), or malaria and filariasis, are becoming more resistant to insecticides of low human toxicity. The lack of vaccines and specific drugs against those vectorial diseases is aggravated by the low output in the search for new insecticides to prevent outbreaks of these diseases. Mammalian ACEs have important blood pressure-regulating actions and homologs of these enzymes have been found in insects, whose function is still not very well known, but vital. This research aims to obtain natural compounds that inhibit ACEs from insects that can act as insecticides. Cecropia glazioui was the plant selected for this study. ACE activity was determined by the quenched fluorescence method (FRET) using the substrates: Abz-FRK (Dnp)P-OH for ACE-1 and Mca-APK(Dnp) for ACE-2. The enzymatic activity was determined by incubation of the larvae homogenate (or rat plasma) with the butanolic fraction (Fbut) (10-1000 ug/mL) or the major compounds (0,1-100 uM) of C. glazioui. Fbut inhibited non-selectively ACE activity of the rat plasma and larvae homogenate, with similar IC50 values. Although less active than Fbut, chlorogenic acid, procyanidins B5, B3 and B2, orientin, isoorientin and isovitexin (100 uM) reduced significantly the fluorescence emitted by the substrate Abz for ACE-1(p<0,0001). For the in vivo test, it was tested Fbut (0,3-30 mg/mL) and the compounds (0,03-0,3 mg/mL) that showed higher activity in the in vitro ACE activity: orientin, isoorientin and isovitexin. For the larvicidal test, Aedes aegypti larvae were exposed to different concentrations of these compounds and lethality was recorded after 24, 48 and 72h. The larvae development into pupae was followed up for 2 weeks. The lethality for the first larval stages was higher and significantly different from that observed in the final stage of larval development. Although the flavonoids did not show an insecticidal activity in vivo, these compounds significantly inhibited the growth in the first instars. The effect was persistent during the time of exposure of the larvae to the compounds. It is possible that this activity is related to the inhibition of ACE homologs in insects, but additional mechanisms have not yet been studied.
Mosquitoes that transmit arboviruses (dengue, zika, chikungunya, yellow fever), or malaria and filariasis, are becoming more resistant to insecticides of low human toxicity. The lack of vaccines and specific drugs against those vectorial diseases is aggravated by the low output in the search for new insecticides to prevent outbreaks of these diseases. Mammalian ACEs have important blood pressure-regulating actions and homologs of these enzymes have been found in insects, whose function is still not very well known, but vital. This research aims to obtain natural compounds that inhibit ACEs from insects that can act as insecticides. Cecropia glazioui was the plant selected for this study. ACE activity was determined by the quenched fluorescence method (FRET) using the substrates: Abz-FRK (Dnp)P-OH for ACE-1 and Mca-APK(Dnp) for ACE-2. The enzymatic activity was determined by incubation of the larvae homogenate (or rat plasma) with the butanolic fraction (Fbut) (10-1000 ug/mL) or the major compounds (0,1-100 uM) of C. glazioui. Fbut inhibited non-selectively ACE activity of the rat plasma and larvae homogenate, with similar IC50 values. Although less active than Fbut, chlorogenic acid, procyanidins B5, B3 and B2, orientin, isoorientin and isovitexin (100 uM) reduced significantly the fluorescence emitted by the substrate Abz for ACE-1(p<0,0001). For the in vivo test, it was tested Fbut (0,3-30 mg/mL) and the compounds (0,03-0,3 mg/mL) that showed higher activity in the in vitro ACE activity: orientin, isoorientin and isovitexin. For the larvicidal test, Aedes aegypti larvae were exposed to different concentrations of these compounds and lethality was recorded after 24, 48 and 72h. The larvae development into pupae was followed up for 2 weeks. The lethality for the first larval stages was higher and significantly different from that observed in the final stage of larval development. Although the flavonoids did not show an insecticidal activity in vivo, these compounds significantly inhibited the growth in the first instars. The effect was persistent during the time of exposure of the larvae to the compounds. It is possible that this activity is related to the inhibition of ACE homologs in insects, but additional mechanisms have not yet been studied.