A carregar...
Publicação
Identificação in silico e validação experimental de novos fármacos inibidores de efluxo ou biofilmes em Staphylococcus aureus
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 2.54 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
Staphylococcus aureus é agente de uma vasta gama de infeções, de fraca severidade até
potencialmente fatais. A emergência de estirpes resistentes aos antibióticos, particularmente
S. aureus resistente à meticilina (MRSA), é preocupante devido às limitadas opções
terapêuticas.
Neste trabalho, foi utilizada uma estratégia de reposicionamento de fármacos baseada em
quimiogenómica para a identificação de novos fármacos que possam atuar em dois
mecanismos associados à resistência aos antimicrobianos em S. aureus, o efluxo e a
formação de biofilmes. Esta estratégia consiste na pesquisa in silico de potenciais inibidores
de efluxo (IE) e/ou agentes antibiofilmes que são depois sujeitos a um algoritmo
experimental para validar a sua atividade biológica.
Compilou-se informação sobre transportadores membranares de S. aureus (na base de dados
“TransportDB”) e proteínas associadas à formação de biofilmes (no repositório “UniProt”),
gerando uma lista de potenciais alvos. Estes alvos foram usados para interrogar a base de
dados “DrugBank”, que compila informação sobre fármacos aprovados pela FDA, fármacos
investigacionais e experimentais, e os seus respetivos alvos. Este processo gerou uma lista
de alvos homólogos aos de S. aureus e uma lista de fármacos (n = 826) associados. Aplicando
vários critérios de exclusão, obteve-se uma lista curada de fármacos candidatos (n = 243)
com potencial atividade no efluxo e/ou biofilmes em S. aureus. Foram escolhidos sete
fármacos, pertencentes às duas superclasses químicas mais representativas para validação
experimental: desipramina, cloroquina, atovaquone, topiramato, amlodipina, tariquidar e
sulpirida.
O modelo experimental incluiu as estirpes MRSA COL, S. aureus ATCC25923 e sua
derivada ATCC25923_EtBr adaptada ao brometo de etídio, que sobreexpressa o gene norA
que codifica para NorA, uma das principais bombas de efluxo em S. aureus. Os fármacos
candidatos foram comparados com os IE controlo tioridazina, clorpromazina, verapamil e
reserpina. A concentração mínima inibitória (CMI) dos fármacos/IE controlo foram
determinadas por microdiluição. Depois, cada fármaco/IE controlo foi avaliado quanto à sua
capacidade de, em concentração subinibitória, reduzir significativamente (≥4x) a CMI de
um painel de compostos antimicrobianos, que incluem substratos e não substratos de NorA.
Os fármacos que apresentaram efeito significativo foram avaliados quanto ao sinergismo
com diferentes antimicrobianos por ensaios de checkerboard e quanto ao seu potencial de
inibição da formação de biofilme pelo método de adesão em placa com violeta de cristal.
Todos os fármacos candidatos testados mostraram uma CMI elevada (>64 mg/L) contra as
estirpes de S. aureus. Os fármacos tariquidar, amlodipina, desipramina e cloroquina
(ordenados pela maior atividade) reduziram a CMI de todos ou alguns substratos de NorA
em ATCC25923_EtBr, sugerindo que possuem atividade inibitória de efluxo. Os ensaios de
checkerboard mostraram que o tariquidar a 1/32x CMI (8 mg/L) e a amlodipina a 1/16x CMI
(4 mg/L) apresentam sinergismo com substratos de NorA. Testes adicionais mostraram que
apenas a amlodipina é capaz de abolir a formação de biofilme a ½ CMI (64 mg/L), um efeito
menor do que o observado para verapamil e tioridazina.
Os nossos resultados evidenciam a amlodipina como o único fármaco candidato que
apresentou atividade quer a nível de inibição do efluxo quer na formação de biofilmes. No entanto, o tariquidar foi o que apresentou maior atividade inibitória de efluxo, quando
comparado com IEs controlo e outros fármacos candidatos. Estudos adicionais poderão
elucidar os mecanismos de ação destes fármacos, particularmente a associação efluxo biofilme e amlodipina.
Em suma, este trabalho destaca a utilidade do reposicionamento de fármacos para identificar
novos fármacos que visam o efluxo e/ou produção de biofilme em S. aureus, revelando a
amlodipina como um novo potencial fármaco de duplo alvo e o tariquidar como um potente
IE, os quais podem no futuro ser explorados no combate às infeções por S. aureus resistentes
a antimicrobianos.
Staphylococcus aureus is a pathogen that causes mild to life-threatening infections. Emergence of antimicrobial-resistant strains, particularly methicillin-resistant S. aureus (MRSA), is worrisome due to the limited therapeutic options. In this work, a drug repurposing strategy based on chemogenomics was used to identify new drugs that can target two mechanisms associated with resistance in S. aureus, drug efflux and biofilm formation. The strategy consists of an in silico search for new potential efflux inhibitors (EI) and/or antibiofilm agents that are then subjected to an experimental algorithm to validate their biological activity. Information was compiled on S. aureus membrane transporters (TransportDB database) and proteins associated with biofilm formation (UniProt repository), generating a list of S. aureus targets. These targets were used to interrogate the DrugBank database, which compiles information on FDA-approved drugs, drugs in R&D, and their targets. This process generated a list of targets homologous to the S. aureus targets and a list of drugs (n = 826) that act upon them. Applying several exclusion criteria, we obtained a final curated list of candidate drugs (n = 243) with potential activity upon efflux and/or biofilms in S. aureus. Seven candidate drugs of the two most represented chemical superclasses were chosen for experimental validation: desipramine, chloroquine, atovaquone, topiramate, amlodipine, tariquidar and sulpiride. The experimental model included strains MRSA COL, S. aureus ATCC25923 and its ethidium-bromide adapted counterpart ATCC25923_EtBr that overexpresses the norA gene encoding NorA, one of the main S. aureus efflux pumps. The candidate drugs were compared to the control EIs, thioridazine, chlorpromazine, verapamil and reserpine. The minimum inhibitory concentrations (MIC) of the drugs and control EIs were determined by broth microdilution. Then, each drug/control EI (at a subinhibitory concentration) was evaluated for their ability to significantly reduce (≥four-fold) the MIC of a panel of antimicrobials that included NorA substrates and non-substrates. The ones with significant effect were then further tested for their synergism with antimicrobials by checkerboard assays and potential to inhibit biofilm formation by the crystal violet microplate adhesion method. All candidate drugs showed a high MIC value (> 64 mg/L) against all S. aureus strains tested. The drugs tariquidar, amlodipine, desipramine and chloroquine (from highest to lowest activity) were able to reduce the MICs of all or some NorA substrates in ATCC25923_EtBr, suggesting that they possess efflux inhibitory activity. Checkerboard assays showed that tariquidar at 1/32x MIC (4 mg/L) and amlodipine at 1/16x MIC (8 mg/L) present synergism with NorA substrates. Further testing showed that only amlodipine was able to abolish biofilm formation at ½ MIC (64 mg/L), a lower effect than the one observed for verapamil and thioridazine. Our data showed that amlodipine was the only candidate drug presenting both efflux inhibitory and antibiofilm formation activities. However, tariquidar showed the highest efflux inhibitory activity when compared with control EIs and other candidate drugs. Additional studies should be carried out to elucidate the mechanisms of action, particularly regarding efflux-biofilm association and amlodipine. vi In sum, this work highlights the value of drug repurposing to identify new drugs that target efflux and/or biofilm formation in S. aureus, revealing amlodipine as a new potential dual target drug and tariquidar as a potent EI, both of which may be explored as new drugs to fight antimicrobial-resistant S. aureus infectionIn sum, this work highlights the value of drug repurposing to identify new drugs that target efflux and/or biofilm formation in S. aureus, revealing amlodipine as a new potential dual target drug and tariquidar as a potent EI, both of which may be explored as new drugs to fight antimicrobial-resistant S. aureus infections.
Staphylococcus aureus is a pathogen that causes mild to life-threatening infections. Emergence of antimicrobial-resistant strains, particularly methicillin-resistant S. aureus (MRSA), is worrisome due to the limited therapeutic options. In this work, a drug repurposing strategy based on chemogenomics was used to identify new drugs that can target two mechanisms associated with resistance in S. aureus, drug efflux and biofilm formation. The strategy consists of an in silico search for new potential efflux inhibitors (EI) and/or antibiofilm agents that are then subjected to an experimental algorithm to validate their biological activity. Information was compiled on S. aureus membrane transporters (TransportDB database) and proteins associated with biofilm formation (UniProt repository), generating a list of S. aureus targets. These targets were used to interrogate the DrugBank database, which compiles information on FDA-approved drugs, drugs in R&D, and their targets. This process generated a list of targets homologous to the S. aureus targets and a list of drugs (n = 826) that act upon them. Applying several exclusion criteria, we obtained a final curated list of candidate drugs (n = 243) with potential activity upon efflux and/or biofilms in S. aureus. Seven candidate drugs of the two most represented chemical superclasses were chosen for experimental validation: desipramine, chloroquine, atovaquone, topiramate, amlodipine, tariquidar and sulpiride. The experimental model included strains MRSA COL, S. aureus ATCC25923 and its ethidium-bromide adapted counterpart ATCC25923_EtBr that overexpresses the norA gene encoding NorA, one of the main S. aureus efflux pumps. The candidate drugs were compared to the control EIs, thioridazine, chlorpromazine, verapamil and reserpine. The minimum inhibitory concentrations (MIC) of the drugs and control EIs were determined by broth microdilution. Then, each drug/control EI (at a subinhibitory concentration) was evaluated for their ability to significantly reduce (≥four-fold) the MIC of a panel of antimicrobials that included NorA substrates and non-substrates. The ones with significant effect were then further tested for their synergism with antimicrobials by checkerboard assays and potential to inhibit biofilm formation by the crystal violet microplate adhesion method. All candidate drugs showed a high MIC value (> 64 mg/L) against all S. aureus strains tested. The drugs tariquidar, amlodipine, desipramine and chloroquine (from highest to lowest activity) were able to reduce the MICs of all or some NorA substrates in ATCC25923_EtBr, suggesting that they possess efflux inhibitory activity. Checkerboard assays showed that tariquidar at 1/32x MIC (4 mg/L) and amlodipine at 1/16x MIC (8 mg/L) present synergism with NorA substrates. Further testing showed that only amlodipine was able to abolish biofilm formation at ½ MIC (64 mg/L), a lower effect than the one observed for verapamil and thioridazine. Our data showed that amlodipine was the only candidate drug presenting both efflux inhibitory and antibiofilm formation activities. However, tariquidar showed the highest efflux inhibitory activity when compared with control EIs and other candidate drugs. Additional studies should be carried out to elucidate the mechanisms of action, particularly regarding efflux-biofilm association and amlodipine. vi In sum, this work highlights the value of drug repurposing to identify new drugs that target efflux and/or biofilm formation in S. aureus, revealing amlodipine as a new potential dual target drug and tariquidar as a potent EI, both of which may be explored as new drugs to fight antimicrobial-resistant S. aureus infectionIn sum, this work highlights the value of drug repurposing to identify new drugs that target efflux and/or biofilm formation in S. aureus, revealing amlodipine as a new potential dual target drug and tariquidar as a potent EI, both of which may be explored as new drugs to fight antimicrobial-resistant S. aureus infections.
Descrição
Palavras-chave
Microbiologia médica Staphylococcus aureus Resistência a antibióticos Efluxo Biofilmes Reposicionamento de fármacos Biologia molecular Saúde tropical e internacional-saúde pública