title: A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria.
abstract: Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.
authors.affiliation: Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
authors.affiliation: Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Boudewijn L M
authors.initials: BL
authors.lastname: de Jonge
authors.affiliation: Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Kathy
authors.initials: K
authors.lastname: MacCormack
authors.affiliation: Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Shubha
authors.initials: S
authors.lastname: Sriram
authors.affiliation: Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: McLaughlin
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Plant
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom.
authors.forename: Marian
authors.initials: M
authors.lastname: Preston
authors.affiliation: Infection Chemistry, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Fleming
authors.affiliation: Infection Chemistry, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Albert
authors.affiliation: Infection Chemistry, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.
authors.forename: Melinda
authors.initials: M
authors.lastname: Foulk
authors.affiliation: Infection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA scott.mills@astrazeneca.com.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Mills
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D015374:Biosensing Techniques; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D020128:Inhibitory Concentration 50; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013014:SOS Response, Genetics / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; C073126:tolC protein, E coli; D049452:Green Fluorescent Proteins; D011938:Rec A Recombinases; D027081:DNA Gyrase