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Avaliação da atividade antimicrobiana de compostos inibidores do metabolismo energético em Escherichia coli
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Ao longo dos anos, as bactérias desenvolveram vários mecanismos de resistência aos
antimicrobianos, nomeadamente bombas de efluxo. Uma estratégia promissora para
combater e prevenir a resistência mediada por sistemas de efluxo em bactérias Gram negativas, incluindo Escherichia coli, é a utilização de fármacos não antibióticos que,
quando utilizados em combinação com antibióticos, promovem ou restauram a atividade
destes últimos. Vários estudos sugerem que os inibidores de efluxo mais eficazes atuam
a nível do metabolismo energético, nomeadamente na fosforilação oxidativa.
Uma estratégia para a identificação de inibidores de efluxo consiste no reposicionamento
de fármacos, uma abordagem menos dispendiosa e mais curta do que o processo
convencional de desenvolvimento de novos medicamentos.
Nesta Dissertação, avaliou-se a atividade in vitro de fármacos aprovados, identificados
previamente por um método de reposicionamento in silico como potenciais inibidores de
proteínas envolvidas no metabolismo energético, em E. coli.
Os compostos candidatos a reposicionamento testados neste trabalho foram:
doxorubicina, acetazolamida, tiabendazol, atovaquone, benztiazida, topiramato,
amlodipina, sulpirida e metformina. Para estudar o mecanismo de ação dos compostos
candidatos, utilizou-se um sistema modelo de expressão de bombas de efluxo em E. coli,
que inclui as estirpes seguintes: AG100, com o sistema de efluxo AcrAB-TolC funcional;
AG100A, com o sistema AcrAB-TolC inativo; AG100TET, que apresenta sobre-expressão
de bombas de efluxo, incluindo AcrAB-TolC. Estes compostos foram também avaliados
quanto ao seu efeito adjuvante da atividade de antibióticos selecionados em E. coli,
através da determinação da concentração mínima inibitória (CMI) de antibióticos na
presença e na ausência do composto candidato, e quanto à sua capacidade de inibição de
efluxo, através de um método fluorométrico semiautomático. Os resultados obtidos
demonstraram que a doxorrubicina, a amlodipina e o atovaquone apresentam maior
atividade antimicrobiana na estirpe AG100, do que os restantes compostos. No entanto,
a doxorrubicina demonstrou ser substrato do sistema de efluxo AcrAB-TolC.
Por outro lado, verificou-se efeito significativo dos compostos na redução da CMI da
gentamicina e do brometo de etídio na estirpe AG100A, que apresenta o sistema AcrAB TolC inativo. Este estudo também demonstrou que a amlodipina foi o composto com
atividade inibitória de efluxo mais promissora nos ensaios de fluorometria em tempo real.
Este composto também promoveu a redução significativa da CMI da tetraciclina,
gentamicina e brometo de etídio em isolados clínicos de E. coli resistentes à tetraciclina.
Concluindo, os resultados obtidos neste trabalho permitiram identificar a amlodipina
como um composto promissor, com potencial aplicação no desenvolvimento de
estratégias terapêuticas futuras para combater a emergência de resistência em E. coli e
outras bactérias Gram-negativas.
Over the years, bacteria developed several drug resistance mechanisms, namely efflux pumps. A promising strategy to fight and prevent efflux systems-mediated resistance in Gram-negative bacteria, including Escherichia coli, is the use of non-antibiotics that, when used in combination with antibiotics, promote or restore the latter’s activity. Several studies have suggested that the most effective efflux inhibitors act at the level of energy metabolism, namely oxidative phosphorylation. A strategy for the identification of efflux inhibitors consists in drug repositioning, a more inexpensive and shorter approach than the conventional drug development process. In this Dissertation, we evaluated the in vitro activity of approved drugs, previously identified by an in silico repositioning method as potential inhibitors of proteins involved in energy metabolism, in E. coli. The repositioning candidate compounds tested in this work were: doxorubicin, acetazolamide, thiabendazole, atovaquone, benzthiazide, topiramate, amlodipine, sulpiride and metformin. To study the mechanism of action of the candidate compounds, we used an efflux pump expression model system in E. coli that includes the following strains: AG100, with a functional AcrAB-TolC efflux system; AG100A, with an inactive AcrAB-TolC system; AG100TET that presents overexpression of efflux pumps, including AcrAB-TolC. These compounds were also evaluated for their adjuvant effect of the activity of selected antibiotics in E. coli, by the determination of the minimum inhibitory concentration (MIC) of antibiotics in the presence and absence of compound, and for their efflux inhibition capacity, by a semiautomatic fluorometric method. The obtained results demonstrated that doxorubicin, amlodipine and atovaquone present higher antimicrobial activity in the AG100 strain, than the remaining compounds. However, doxorubicin was shown to be a substrate of the AcrAB-TolC efflux system. On the other hand, there was a significant effect of the compounds in the reduction of the MIC of gentamycin and ethidium bromide in the strain AG100A, which presents an inactive AcrAB-TolC system. This study also demonstrated that amlodipine was the compound with the most promising efflux inhibition activity in the real time fluorometric assays. This compound also promoted significant reduction of the MIC of tetracycline, gentamycin, and ethidium bromide in E. coli clinical isolates resistant to tetracycline. In conclusion, the results obtained in this work allowed the identification of amlodipine as a promising compound, with potential application in the development of future therapeutic options to fight the emergence of resistance in E. coli and other Gram negative bacteria
Over the years, bacteria developed several drug resistance mechanisms, namely efflux pumps. A promising strategy to fight and prevent efflux systems-mediated resistance in Gram-negative bacteria, including Escherichia coli, is the use of non-antibiotics that, when used in combination with antibiotics, promote or restore the latter’s activity. Several studies have suggested that the most effective efflux inhibitors act at the level of energy metabolism, namely oxidative phosphorylation. A strategy for the identification of efflux inhibitors consists in drug repositioning, a more inexpensive and shorter approach than the conventional drug development process. In this Dissertation, we evaluated the in vitro activity of approved drugs, previously identified by an in silico repositioning method as potential inhibitors of proteins involved in energy metabolism, in E. coli. The repositioning candidate compounds tested in this work were: doxorubicin, acetazolamide, thiabendazole, atovaquone, benzthiazide, topiramate, amlodipine, sulpiride and metformin. To study the mechanism of action of the candidate compounds, we used an efflux pump expression model system in E. coli that includes the following strains: AG100, with a functional AcrAB-TolC efflux system; AG100A, with an inactive AcrAB-TolC system; AG100TET that presents overexpression of efflux pumps, including AcrAB-TolC. These compounds were also evaluated for their adjuvant effect of the activity of selected antibiotics in E. coli, by the determination of the minimum inhibitory concentration (MIC) of antibiotics in the presence and absence of compound, and for their efflux inhibition capacity, by a semiautomatic fluorometric method. The obtained results demonstrated that doxorubicin, amlodipine and atovaquone present higher antimicrobial activity in the AG100 strain, than the remaining compounds. However, doxorubicin was shown to be a substrate of the AcrAB-TolC efflux system. On the other hand, there was a significant effect of the compounds in the reduction of the MIC of gentamycin and ethidium bromide in the strain AG100A, which presents an inactive AcrAB-TolC system. This study also demonstrated that amlodipine was the compound with the most promising efflux inhibition activity in the real time fluorometric assays. This compound also promoted significant reduction of the MIC of tetracycline, gentamycin, and ethidium bromide in E. coli clinical isolates resistant to tetracycline. In conclusion, the results obtained in this work allowed the identification of amlodipine as a promising compound, with potential application in the development of future therapeutic options to fight the emergence of resistance in E. coli and other Gram negative bacteria
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Microbiologia médica Bactérias Resistência aos atibióticos Bombas de efluxo Inibidor de efluxo Reposicionamento de fármacos Escherichia coli Biologia molecular Saúde pública - internacional