Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs

Copp, Janine N.; Williams, Elizabeth; Rich, Michelle H; Patterson, Adam V.; Smaill, Jeff B.; Ackerley, David F.

doi:10.1093/protein/gzu025

Cited by 38 publications

(66 citation statements)

References 31 publications

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“…) meant an enrichment of 4.4 × 10 6 fold in only one round of selection and screening. Other systems were just tested with initial ratios up to 1:10 6 and required at least two FACS rounds or one selection round to get to a more than 10 5 fold enrichment (van Sint Fiet et al ., ; Copp et al ., ; Jha et al ., ). Thus, the system described here is able to obtain a very good enrichment, and it is relatively easy, short and cheap, compared with, for example, FACS.…”

Section: Resultsmentioning

confidence: 99%

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

Rossum

Muras

Baur

et al. 2017

Microbial Biotechnology

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SummaryThe use of bioreporters in high‐throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants. Of four developed systems, the best system was the medium copy system with KmR as selection reporter. As a proof of principle, the system was tested for the selection of cells expressing an l‐arabinose isomerase derived from mesophilic Escherichia coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with l‐arabinose isomerase activity was demonstrated by selection and exclusion of false positives by screening. This dual selection/screening system is an important step towards an improved detection method for small molecules, and thereby for finding novel biocatalysts.

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Section: Resultsmentioning

confidence: 99%

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

Rossum

Muras

Baur

et al. 2017

Microbial Biotechnology

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“…Compounds PR-104A, EF5, HX4, and Metronidazole A diverse panel of 58 native oxygen-independent nitroreductases, extended from our previous studies (Prosser et al, 2013;Copp et al, 2014), was screened to select an optimal starting gene for subsequent engineering. To quantify DNA damage caused by specific activation of the target compounds, we measured the E. coli SOS response at sub-lethal PR-104A concentrations, or growth inhibition (half maximal inhibitory concentration, IC 50 ) of nitroreductase-expressing cells at lethal EF5, HX4, or metronidazole concentrations.…”

Section: E Coli Nfsa Efficiently Activates the Key Clinicalmentioning

confidence: 99%

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

Copp

Mowday

Williams

et al. 2017

Cell Chemical Biology

115

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Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the "black box" of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates.

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“… E. coli BW25113 strains bearing individual gene deletions were obtained from the Keio knockout collection ( 51 ). Δ7NR and Δ7NR tolC mutants were generated via sequential knockout as previously described ( 52 ). New Zealand clinical isolates used in this study were A. baumannii NZRM3289, P. aeruginosa NZRM4034, K. pneumoniae NZRM4387, and E. coli NZRM4403 (obtained from the New Zealand Reference Culture Collection, Environmental Science and Research Ltd.).…”

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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Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs

Cited by 38 publications

References 31 publications

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

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

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

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