Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy

Copp, Janine N.; Mowday, Alexandra M.; Williams, Elizabeth; Guise, Christopher P.; Ashoorzadeh, Amir; Sharrock, Abigail V.; Flanagan, Jack U.; Smaill, Jeff B.; Patterson, Adam V.; Ackerley, David F.

doi:10.1016/j.chembiol.2017.02.005

Cited by 68 publications

(132 citation statements)

References 45 publications

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“…Although NTR 2.0 exhibited improved MTZ activity across all model systems, some of the other NTR variants tested over the course of this study showed less correlation across species. In addition to the lack of activity presented here for NTR 1.1 in HEK-293 cells, we have previously encountered expression instability issues for the NfsB_Ec F70A/F108Y double mutant and other native or engineered NTR variants in HCT-116 cells 23,[35][36][37] . Although not reported here, we have also been unable to generate stable transgenic zebrafish lines for several NTR variants that expressed well in both bacteria and HEK-293 cells.…”

Section: Discussionmentioning

confidence: 97%

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

Sharrock

Mulligan

Hall

et al. 2020

Preprint

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Heterologously-expressed bacterial nitroreductase (NTR) enzymes sensitize eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrate systems. However, first-generation NTRs require confoundingly toxic prodrug treatments (e.g. 10 mM MTZ) and some cell types have proven resistant. We used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy. Toxicity tests in zebrafish showed no deleterious effects of prolonged MTZ treatments of ≤1 mM. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and enable modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic resources to facilitate dissemination of NTR 2.0 to the research community.

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Section: Discussionmentioning

confidence: 97%

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

Sharrock

Mulligan

Hall

et al. 2020

Preprint

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“…To analyze the activity of NfsA variants, library subsets were selected on agar plates using 0, 0.2, and 2 μg · ml −1 of niclosamide. Ninety colonies from each subset were subsequently screened via growth assays with niclosamide, metronidazole, and nitrofurantoin at 2, 10, and 5 μg · ml −1 , respectively; growth was measured via OD 600 following 4 h of incubation as previously described ( 56 ).…”

Section: Methodsmentioning

confidence: 99%

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

Copp

Pletzer

Brown

et al. 2020

mBio

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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.

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“…To analyze the activity of NfsA variants, library subsets were selected on agar plates using 0, 0.2 and 2 μg.mL −1 of niclosamide. Ninety colonies from each subset were subsequently screened via growth assays with niclosamide, metronidazole, and nitrofurantoin at 2, 10, and 5 μg.mL −1 respectively; growth was measured via OD 600 following 4 h incubation as previously described (53).…”

Section: Methodsmentioning

confidence: 99%

Mechanistic understanding enables the rational design of salicylanilide combination therapies for Gram-negative infections

Copp

Pletzer

Brown³

et al. 2020

Preprint

Self Cite

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21One avenue to combat multidrug-resistant Gram-negative bacteria is the co-administration 22 of multiple drugs (combination therapy), which can be particularly promising if drugs 23 synergize. The identification of synergistic drug combinations, however, is challenging. 24 Detailed understanding of antibiotic mechanisms can address this issue by facilitating the 25 rational design of improved combination therapies. Here, using diverse biochemical and 26 genetic assays, we reveal the molecular mechanisms of niclosamide, a clinically-approved 27 salicylanilide compound, and demonstrate its potential for Gram-negative combination 28 therapies. We discovered that Gram-negative bacteria possess two innate resistance 29 mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated 30 by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was 31 compromised, niclosamide became a potent antibiotic, dissipating the proton motive force 32 (PMF), increasing oxidative stress and reducing ATP production to cause cell death. These 33 insights guided the identification of diverse compounds that synergized with salicylanilides 34 when co-administered (efflux inhibitors, membrane permeabilizers, and antibiotics that are 35 expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may 36 have broad utility in combination therapies. We validate these findings in vivo using a 37 murine abscess model, where we show that niclosamide synergizes with the membrane 38 permeabilizing antibiotic colistin against high-density infections of multidrug-resistant 39 Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase 40 activity is a potential route to adaptive niclosamide resistance but show that this causes 41 collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how 42 mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can 43 rationally guide the discovery, development and stewardship of novel combination 44 therapies. 45 46 Importance 47There is a critical need for more effective treatments to combat multidrug-resistant Gram-48 negative infections. Combination therapies are a promising strategy, especially when these 49 enable existing clinical drugs to be repurposed as antibiotics. We reveal the mechanisms 50 of action and basis of innate Gram-negative resistance for the anthelmintic drug 51 niclosamide, and subsequently exploit this information to demonstrate that niclosamide 52 and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug 53 efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in 54 vitro against a diverse range of recalcitrant Gram-negative clinical isolates, and in vivo in 55 a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to 56 niclosamide, but show that evolution of these enzymes for enhanced niclosamide resistance 57 confers a collateral s...

show abstract

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy

Cited by 68 publications

References 45 publications

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

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

Mechanistic understanding enables the rational design of salicylanilide combination therapies for Gram-negative infections

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