Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

Mardle, Charlotte E.; Goddard, Layla R.; Spelman, Bailei C.; Atkins, Helen S.; Butt, Louise E.; Cox, Paul A.; Gowers, Darren M.; Vincent, Helen A.; Callaghan, Anastasia J.

doi:10.1016/j.bbrep.2020.100773

Cited by 8 publications

(9 citation statements)

References 35 publications

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“…A fourth inhibitor, glutathione, is a metabolic tripeptide involved in regulating cellular redox state and detection of reactive-oxygen species in E. coli [ 46 , 47 ]. There is growing evidence that enzymes involved in nucleic acid metabolism are regulated/inhibited by metabolites [ 33 , 48 , 49 ]. It would be interesting to investigate if this is also the case for DNA ligases.…”

Section: Discussionmentioning

confidence: 99%

“…The strategy of combining structure-based in silico screening with in vitro validation provides a high-throughput method for identifying small molecule inhibitors, provided that structural and functional characterisation of the target protein is available. Previously, we have successfully applied this approach to identify inhibitors of RNase E [ 32 , 33 ]. We have now applied the strategy to identify novel inhibitors of EC-LigA and potentially novel inhibitor target sites within EC-LigA.…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we aimed to investigate the potential of targeting EC-LigA more broadly as an inhibitory/antibacterial strategy. We applied a structure-based in silico screening and in vitro validation strategy, which we have used previously to rapidly identify inhibitory lead compounds of antibacterial targets [ 32 , 33 ], to identify new inhibitors that target NAD + ligases. The strategy involved first identifying potential inhibitor binding sites within an NAD + ligase in silico.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Novel Inhibitors of Escherichia coli DNA Ligase (LigA)

et al. 2021

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Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3′ hydroxyl and a 5′ phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11–2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of Novel Inhibitors of Escherichia coli DNA Ligase (LigA)

et al. 2021

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“…Some progress has been made in validating RNase E/ rne as an antibacterial target through the identification of small molecule inhibitors of RNase E, using structure-based virtual high-throughput screening, and the characterisation of their inhibitory activity in vitro. Through this approach, a number of small molecules have been identified that inhibit RNase E from multiple bacterial pathogens in vitro [ 12 , 13 ]. However, the half maximal inhibitory concentration (IC 50 ) for each of these inhibitors was in the low millimolar range, much higher than would be desired for an effective antibiotic [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…Through this approach, a number of small molecules have been identified that inhibit RNase E from multiple bacterial pathogens in vitro [ 12 , 13 ]. However, the half maximal inhibitory concentration (IC 50 ) for each of these inhibitors was in the low millimolar range, much higher than would be desired for an effective antibiotic [ 12 , 13 ]. Even enhanced inhibition, obtained using a combination of inhibitory small molecules, required millimolar concentrations of inhibitors [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

et al. 2021

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The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

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Engineering an Escherichia coli based in vivo mRNA manufacturing platform

Curry,

Muir,

et al. 2024

Biotech & Bioengineering

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Synthetic mRNA is currently produced in standardized in vitro transcription systems. However, this one‐size‐fits‐all approach has associated drawbacks in supply chain shortages, high reagent costs, complex product‐related impurity profiles, and limited design options for molecule‐specific optimization of product yield and quality. Herein, we describe for the first time development of an in vivo mRNA manufacturing platform, utilizing an Escherichia coli cell chassis. Coordinated mRNA, DNA, cell and media engineering, primarily focussed on disrupting interactions between synthetic mRNA molecules and host cell RNA degradation machinery, increased product yields >40‐fold compared to standard “unengineered” E. coli expression systems. Mechanistic dissection of cell factory performance showed that product mRNA accumulation levels approached theoretical limits, accounting for ~30% of intracellular total RNA mass, and that this was achieved via host‐cell's reallocating biosynthetic capacity away from endogenous RNA and cell biomass generation activities. We demonstrate that varying sized functional mRNA molecules can be produced in this system and subsequently purified. Accordingly, this study introduces a new mRNA production technology, expanding the solution space available for mRNA manufacturing.

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Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

Cited by 8 publications

References 35 publications

Identification of Novel Inhibitors of Escherichia coli DNA Ligase (LigA)

Identification of Novel Inhibitors of Escherichia coli DNA Ligase (LigA)

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

Engineering an Escherichia coli based in vivo mRNA manufacturing platform

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