Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections

ABSTRACT One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of an...

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Autores principales:	Janine N. Copp, Daniel Pletzer, Alistair S. Brown, Joris Van der Heijden, Charlotte M. Miton, Rebecca J. Edgar, Michelle H. Rich, Rory F. Little, Elsie M. Williams, Robert E. W. Hancock, Nobuhiko Tokuriki, David F. Ackerley
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	synergy repurposing niclosamide colistin efflux nitroreductase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0e72123ce7a44e038133dd43455c6ea7
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spelling	oai:doaj.org-article:0e72123ce7a44e038133dd43455c6ea72021-11-15T16:19:09ZMechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections10.1128/mBio.02068-202150-7511https://doaj.org/article/0e72123ce7a44e038133dd43455c6ea72020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02068-20https://doaj.org/toc/2150-7511ABSTRACT One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies. IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.Janine N. CoppDaniel PletzerAlistair S. BrownJoris Van der HeijdenCharlotte M. MitonRebecca J. EdgarMichelle H. RichRory F. LittleElsie M. WilliamsRobert E. W. HancockNobuhiko TokurikiDavid F. AckerleyAmerican Society for MicrobiologyarticlesynergyrepurposingniclosamidecolistineffluxnitroreductaseMicrobiologyQR1-502ENmBio, Vol 11, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	synergy repurposing niclosamide colistin efflux nitroreductase Microbiology QR1-502
spellingShingle	synergy repurposing niclosamide colistin efflux nitroreductase Microbiology QR1-502 Janine N. Copp Daniel Pletzer Alistair S. Brown Joris Van der Heijden Charlotte M. Miton Rebecca J. Edgar Michelle H. Rich Rory F. Little Elsie M. Williams Robert E. W. Hancock Nobuhiko Tokuriki David F. Ackerley Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections
description	ABSTRACT One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies. IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.
format	article
author	Janine N. Copp Daniel Pletzer Alistair S. Brown Joris Van der Heijden Charlotte M. Miton Rebecca J. Edgar Michelle H. Rich Rory F. Little Elsie M. Williams Robert E. W. Hancock Nobuhiko Tokuriki David F. Ackerley
author_facet	Janine N. Copp Daniel Pletzer Alistair S. Brown Joris Van der Heijden Charlotte M. Miton Rebecca J. Edgar Michelle H. Rich Rory F. Little Elsie M. Williams Robert E. W. Hancock Nobuhiko Tokuriki David F. Ackerley
author_sort	Janine N. Copp
title	Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections
title_short	Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections
title_full	Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections
title_fullStr	Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections
title_full_unstemmed	Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections
title_sort	mechanistic understanding enables the rational design of salicylanilide combination therapies for gram-negative infections
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/0e72123ce7a44e038133dd43455c6ea7
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Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections

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