A high-throughput, homogeneous, bioluminescent assay for Pseudomonas aeruginosa gyrase inhibitors and other DNA-damaging agents.
A homogeneous, sensitive, cellular bioluminescent high-throughput screen was developed for inhibitors of gyrase and other DNA-damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as one-fourth minimum inhibitory concentration (MIC) with Z' scores greater than 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole-cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2000 known bioactive compounds, 13 compounds generated reproducible signals >50% of that of the control (ciprofloxacin at one-half MIC) using bioluminescence readings after 7 h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other 3 are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis but by a variety of different DNA-damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA-damaging agents, which may lead to potent new antibacterials.
Moir
Donald T
DT
Microbiotix, Inc., Worcester, Massachusetts 01605, USA. dmoir@microbiotix.com
Ming Di
Opperman
Timothy
T
Schweizer
Herbert P
HP
Bowlin
Terry L
TL
D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000494:pharmacology*; D004249:DNA Damage; D004269:DNA, Bacterial / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000032:analysis; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008954:Models, Biological; D030781:Organisms, Genetically Modified; D010865:Pilot Projects; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D059005:Topoisomerase II Inhibitors
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D059005:Topoisomerase II Inhibitors