Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump

ABSTRACT Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their m...

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Autores principales:	Elizabeth M. Grimsey, Chiara Fais, Robert L. Marshall, Vito Ricci, Maria Laura Ciusa, Jack W. Stone, Alasdair Ivens, Giuliano Malloci, Paolo Ruggerone, Attilio V. Vargiu, Laura J. V. Piddock
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	antibiotic resistance efflux pump inhibitors AcrB antipsychotic drugs efflux pumps Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c0b86e8c528248f1b64fee868c1d93ae
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spelling	oai:doaj.org-article:c0b86e8c528248f1b64fee868c1d93ae2021-11-15T15:56:47ZChlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump10.1128/mBio.00465-202150-7511https://doaj.org/article/c0b86e8c528248f1b64fee868c1d93ae2020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00465-20https://doaj.org/toc/2150-7511ABSTRACT Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors. IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.Elizabeth M. GrimseyChiara FaisRobert L. MarshallVito RicciMaria Laura CiusaJack W. StoneAlasdair IvensGiuliano MallociPaolo RuggeroneAttilio V. VargiuLaura J. V. PiddockAmerican Society for Microbiologyarticleantibiotic resistanceefflux pump inhibitorsAcrBantipsychotic drugsefflux pumpsMicrobiologyQR1-502ENmBio, Vol 11, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	antibiotic resistance efflux pump inhibitors AcrB antipsychotic drugs efflux pumps Microbiology QR1-502
spellingShingle	antibiotic resistance efflux pump inhibitors AcrB antipsychotic drugs efflux pumps Microbiology QR1-502 Elizabeth M. Grimsey Chiara Fais Robert L. Marshall Vito Ricci Maria Laura Ciusa Jack W. Stone Alasdair Ivens Giuliano Malloci Paolo Ruggerone Attilio V. Vargiu Laura J. V. Piddock Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump
description	ABSTRACT Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors. IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.
format	article
author	Elizabeth M. Grimsey Chiara Fais Robert L. Marshall Vito Ricci Maria Laura Ciusa Jack W. Stone Alasdair Ivens Giuliano Malloci Paolo Ruggerone Attilio V. Vargiu Laura J. V. Piddock
author_facet	Elizabeth M. Grimsey Chiara Fais Robert L. Marshall Vito Ricci Maria Laura Ciusa Jack W. Stone Alasdair Ivens Giuliano Malloci Paolo Ruggerone Attilio V. Vargiu Laura J. V. Piddock
author_sort	Elizabeth M. Grimsey
title	Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump
title_short	Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump
title_full	Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump
title_fullStr	Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump
title_full_unstemmed	Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump
title_sort	chlorpromazine and amitriptyline are substrates and inhibitors of the acrb multidrug efflux pump
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/c0b86e8c528248f1b64fee868c1d93ae
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Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump

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