Discovery and Characterization of QPT-1, the Progenitor of a New Class of Bacterial Topoisomerase Inhibitors (cid:1) †

QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the ( (cid:1) )-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic collection. Generation of resistant strains. Either ethyl methanesulfonate-mutagenized Staphylococcus aureus cultures prepared as described previously (16) or untreated S. aureus cultures (for spontaneous resistance) were grown at 37°C to an optical density at 600 nm of 1.0 (cid:2) 10 10 , and 100 (cid:3) l was plated on Mueller-Hinton agar plates which contained 4 (cid:3) g/ml PNU-286607 or ciprofloxacin. Individual resistant colonies were confirmed by restreaking the colonies and subsequent MIC analysis (see Tables 2 and 4). The gyrAB and parCE topoisomerase genes from these and other resistant strains were amplified by PCR and sequenced at the Pfizer (formerly Pharmacia) sequencing core facility. Molecular biology. Genomic DNA was isolated from overnight cultures of S. aureus grown in brain heart infusion medium with a FastDNA kit (Qbiogene, 20, 30, 60, 120, 240, 360, and 480 min. The vehicle for the oral formulation was pH 4.5 acetate-buffered, 20% DMSO-methylcellulose suspension. The vehicle for the intravenous formulation was 20% hydroxypropyl beta-cyclodextrin (pH 7 phosphate buffer). The extent of binding of PNU-286607 to mouse plasma was determined by ultracentrifugation. PNU-286607 demonstrated moderate binding (70%), with the ( (cid:5) )- and ( (cid:1) )-enantiomers showing similar levels of protein binding in mouse plasma. Phar- macokinetic analysis was performed by noncompartmental analysis using Watson Laboratory Information Management System (Thermo, Inc., Philadelphia, PA). The mean concentration-time data (three mice per time point) were used for calculation of the values of the pharmacokinetic parameters.