novobiocin and dihydronovobiocin

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.

Topics: Anti-Bacterial Agents; Binding Sites; Biotinylation; Coumarins; DNA Gyrase; Drug Evaluation, Preclinical; Enzyme Inhibitors; Escherichia coli; Novobiocin; Peptides, Cyclic; Radioligand Assay; Topoisomerase II Inhibitors

DNA gyrase is the target of the coumarin group of antibacterial agents. The drugs are known to inhibit the ATPase activity of gyrase and bind to the 24-kDa N-terminal subdomain of gyrase B protein. Supercoiling assays with intact DNA gyrase and ATPase assays with a 43-kDa N-terminal fragment of the B protein suggest that the drugs bind tightly, with Kd values <10(-7) M. In addition, the ATPase data suggest that 1 coumermycin molecule interacts with 2 molecules of the 43-kDa protein while the other coumarins form a 1:1 complex. This result is confirmed by cross-linking experiments. Rapid gel-filtration experiments show that the binding of ADPNP(5'-adneylyl beta,gamm-imidodiphosphate) and coumarins to the 43-kDa protein is mutally exclusive, consistent with a competitive mode of action for the drugs. Rapid gel-filtration binding experiments using both the 24-and 43-kDa proteins also show that the drugs bind with association rate constants of >10(5) M-1.s-1, and dissociation rate constants of approximately 3x10(-3)s-1 and approximately 4x10(-3)s-1 for the 43-and 24-kDa proteins, respectively. Titration calorimetry shows that the Kd values for coumarins binding to both proteins are approximately 10-8M and that binding is enthalpy driven.

Topics: Adenylyl Imidodiphosphate; Aminocoumarins; Anti-Bacterial Agents; Binding Sites; Coumarins; Cross-Linking Reagents; DNA Gyrase; DNA Topoisomerases, Type II; Kinetics; Novobiocin; Protein Folding; Topoisomerase II Inhibitors

Topics: Adolescent; Anti-Bacterial Agents; Biomedical Research; Bronchitis; Child; Drug Therapy; Geriatrics; Infant; Infant, Newborn; Inositol; Novobiocin; Pharyngitis; Phytic Acid; Tetracycline; Toxicology

Topics: Asthma; Bronchiectasis; Bronchitis; Humans; Novobiocin; Pneumonia; Respiratory Tract Infections; Tetracycline

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Humans; Infections; Lung Diseases; Novobiocin; Respiratory Tract Infections; Urinary Tract Infections

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Humans; Infections; Lung Diseases; Novobiocin; Respiratory Tract Infections; Urinary Tract Infections