Sreening de uma biblioteca com potenciais moléculas Acaricidas

Introdução: Carrapatos são ectoparasitos hematófagos responsáveis pela transmissão de diversos agentes patogênicos. O controle químico continua sendo o método mais eficaz para a supressão do parasita, mas o uso de acaricidas leva a seleção de populações resistentes de carrapatos. O aumento da biodegradação do acaricida por enzimas detoxificadoras, como as glutationa S-transferases (GST), é um dos mecanismos de resistência, por biotransformar compostos tóxicos, facilitando a eliminação dessas moléculas. Objetivo: Analisar in vitro o efeito de compostos sintéticos sobre a atividade da enzima GST de cinco diferentes espécies de carrapato: Rhipicephalus microplus (RmGST), Haemaphysalis longicornis (HlGST), Amblyomma variegatum (AvGST), Rhipicephalus decoloratus (RdGST) e Rhipicephalus appendiculatus (RaGST). Métodos: Para expressar as enzimas GST em sistema heterólogo de Escherichia coli (E. coli) na forma recombinante, foi utilizado plasmídeo pET-43 contendo as ORF (open reading frames) que codificam as GST de cada espécie. As proteínas foram purificadas através de cromatografia de afinidade à glutationa e testadas para os substratos CDNB (1,2-dicloro-4-nitrobenzeno) e DCNB (3,4-dicloronitrobenzeno) na presença do co-substrato glutationa. Inicialmente, foram testadas 60 moléculas com relação aos efeitos na atividade da enzima rRmGST. Os compostos que apresentaram inibição maior que 50% foram testados contra as GST de outras espécies de carrapato: rHlGST, rAvGST, rRdGST e rRaGST. Resultados e discussão: As GST recombinantes com massa molecular de 25 kDa foram expressas a 30 oC com 1 mM de IPTG e purificadas por cromatografia de afinidade à glutationa. Todas as enzimas tiveram atividade com o substrato CDNB, mas não contra DCNB. O composto 795 apresentou uma capacidade de inibição de 58,1% para rRmGST, 27,8% para rAvGST, 2,3% para rHlGST, 23,9% para rRdGST e 31,9% para rRaGST, enquanto o composto 1258 inibiu em 59,4% a atividade da rRmGST, 6,2% darAvGST, 24,3% da rHlGST, 1,9% da rRdGST e 100% da rRaGST. A moléculas sintéticas 795 e 1258 pertencem às classes químicas dos curcominoides e esteroides, respectivamente. Conclusão: Os resultados identificaram um grupo de possíveis compostos com potencial de ser usado para o controle do carrapato, necessitando de mais estudos para melhor avaliação deste efeito.Introduction: Ticks are hematophagous ectoparasites responsible for the transmission of numerous diseases. Chemical control remains the most effective method for suppressing the parasite, but the use of acaricides leads to the selection of resistant tick populations. The increased biodegradation of the acaricide by detoxifying enzymes, such as glutathione S- transferase (GSTs), is a mechanism of resistance mediated by the biotransformation of toxic compounds facilitating the excretion of these molecules. Objective: To analyze the in vitro effects of synthetic compounds about the activity of the enzyme GST of five different tick species: Rhipicephalus microplus (RmGST), Haemaphysalis longicornis (HlGST), Amblyomma variegatum (AvGST), Rhipicephalus decoloratus (RdGST) and Rhipicephalus appendiculatus (RaGST). Methods: For expression of recombinant GSTs enzymes in heterologous E. coli system, the plasmid pET-43 containing GST ORFs (open reading frames) that codify GSTs of different species were used. Proteins were purified by glutathione affinity chromatography and activity tested against the substrates CDNB (1,2- dichloro-4-nitrobenzene) and DCNB (3,4-dichloronitrobenzene) in the presence of the co- substrate glutathione. Firstly, 60 molecules were tested against the rRmGST enzyme. Compounds that inhibited the enzyme activity in more than 50% were test with rHlGST, rAvGST, rRdGST and rRaGST. Result and discussion: Recombinant proteins with a molecular weight of 25 kDa were expressed at 30oC with 1mM of IPTG and purified with glutathione affinity chromatography. All enzymes showed activity against the CDNB substrate, but not against DCNB. Compound 795 inhibited the enzymatic activity in 58.1% for rRmGST, 27.8% for rAvGST, 2.3% for rHlGST, 23.9% for rRdGST and 31.9% for rRaGST, while compound 1258 inhibited 59.4% of rRmGST, 6.2% of rAvGST, 24.3% of rHlGST, 1.9% of rRdGST, 100% of rRaGST activity. The synthetic molecule 795 and 1258 belong to the chemical class of curcominoids and steroids, respectively. Conclusion: These resultsidentified a group of possible compounds with the potential to be used for tick control, requiring further studies for a better evaluation of these effect

Novel tick glutathione transferase inhibitors as promising acaricidal compounds

Ticks are important ectoparasites with a worldwide distribution. The most commonly used method for tick control involves the use of acaricides. The main problem is that its indiscriminate use has led to the selection of resistant tick populations. Glutathione transferases (GSTs) are enzymes that play an important role in the detoxification of several types of compounds used in commercial tick control products. This work aims to find new bioactive molecules through in vitro assays with a panel of 160 molecules with putative inhibitory activity on the Rhipicephalus microplus GST enzyme (RmGST). Also, selected molecules were tested against GSTs from other tick species; Rhipicephalus decoloratus, Amblyomma variegatum, Rhipicephalus appendiculatus, and Haemaphysalis longicornis. The first screening on RmGST identified 30 compounds with the ability to modify the enzymatic activity of this enzyme. These compounds included different chemical families, like chalcones, diarylideneketones, flavone, thiazoles, thiourea, steroids, thiadiazines, indazoles, and hydrazine. The most potent compounds against RmGST belong to the diarylideneketones family with an inhibition concentration of 50% of activity (IC50) between 7-50 μM. Interestingly, one of the most potent compounds was also an inhibitor of the GST from other tick species. Experiments with R. microplus adults and larvae showed toxicity at 150 μM, suggesting a potential acaricidal effect of these molecules