title: A scintillation proximity assay amenable for screening and characterization of DNA gyrase B subunit inhibitors.
abstract: DNA gyrase is the target of coumarin and cyclothialidine antibacterials, which bind to the B subunit of the enzyme (GyrB). Currently available GyrB inhibitors have not been clinically successful, but their high in vitro potency against DNA gyrase has raised interest in the development of novel noncoumarin antibacterials acting at the same site. We report the development of a simple scintillation proximity assay (SPA) for the study of binding interactions between coumarin or noncoumarin antibacterials and GyrB, which prevents the needs of separation steps and can be run in microtiter plate formats. The assay is based on the detection of the binding of a radioligand, [3H]dihydronovobiocin, to a biotin-labeled 43-kDa fragment of GyrB (biotin-GyrB43), which is captured by streptavidin-coated SPA beads. The typical assay was conducted in 96-well microtiter plates, with final concentration of 10 nM for biotin-GyrB43, 20 nM for [3H]dihydronovobiocin, and 33 microg of SPA beads/well. From saturation experiments, an equilibrium dissociation constant (K(d)) for dihydronovobiocin of 8.10 nM was found. Displacement studies gave 50% inhibitory concentrations (IC(50)) of 42, 64, and 11 nM for novobiocin, dihydronovobiocin, and the cyclothialidine analogue GR122222X, respectively, consistent with previous findings. The assay was found to be robust to dimethyl sulfoxide up to 5% (v/v) and can be used for high-throughput screens of large chemical collections in the search of novel DNA gyrase inhibitors.
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Via Fleming, 4, Verona 37135, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Gevi
authors.forename: Enrico
authors.initials: E
authors.lastname: Domenici
mesh_terms: D000900:Anti-Bacterial Agents / Q000032:analysis / Q000494:pharmacology; D001665:Binding Sites; D019921:Biotinylation / Q000379:methods; D003374:Coumarins / Q000032:analysis / Q000494:pharmacology; D027081:DNA Gyrase / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology*; D004926:Escherichia coli; D009675:Novobiocin / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000032:analysis / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods*; D059005:Topoisomerase II Inhibitors
chemical_list: D000900:Anti-Bacterial Agents; D003374:Coumarins; D004791:Enzyme Inhibitors; D010456:Peptides, Cyclic; D059005:Topoisomerase II Inhibitors; C088717:GR 122222X; D009675:Novobiocin; C099094:dihydronovobiocin; C030123:coumarin; D027081:DNA Gyrase