Fluoroquinolones, such as ciprofloxacin, are among the most efficacious and broad-spectrum oral antibacterials in clinical use. The World Health Organization lists them in their five “Highest Priority Critically Important Antimicrobials,” and these drugs are the most heavily prescribed antibacterials worldwide. The cellular targets of fluoroquinolones are the bacterial type II topoisomerases, gyrase and topoisomerase IV. These essential enzymes regulate DNA under- and overwinding and remove knots and tangles from the genome by generating transient double-stranded breaks in the genetic material. Fluoroquinolones act by increasing levels of double-stranded DNA breaks generated by gyrase and topoisomerase IV, which converts these enzymes into cellular toxins that fragment the genome. Although gyrase and topoisomerase IV are both physiological targets for fluoroquinolones, their relative importance to drug action appears to be species- and drug-dependent. There is a growing crisis in antibacterial resistance and fluoroquinolone resistance is becoming prevalent. This resistance is threatening the clinical efficacy of fluoroquinolones. Initial fluoroquinolone resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV that occur at a serine residue (originally described as Ser83 in the GyrA subunit of Escherichia coli gyrase) and a glutamic/aspartic acid residue 4 amino acids downstream. Based on a published structure and functional studies from the Osheroff laboratory, these residues are proposed to anchor a water-metal ion bridge that serves as the primary conduit between fluoro- quinolones and gyrase/topoisomerase IV. The identification and characterization of novel agents that act against these well-validated enzyme targets and overcome fluoroquinolone resistance could have important health ramifications. Recently, two new classes of gyrase/topoisomerase IV-targeted agents have been described that appear to overcome this resistance, Novel Bacterial Topoisomerase Inhibitors (NBTIs) and Spiropyrimidinetriones (SPTs). Members of these classes, gepotidacin (NBTI) and zoliflodacin (SPT), have advanced to Phase 3 clinical trials. NBTIs are unique, as they induce single- rather than double-stranded enzyme-generated DNA breaks. However, little is known about the actions of NBTIs and SPTs against gyrase/topoisomerase IV or the mechanism of drug resistance. There is an urgent need to identify drugs that display activity against fluoroquinolone-resistant bacteria. Thus, the goals of this project are to further define the mechanism of action of fluoroquinolones, NBTIs, and SPTs against gyrase and topoisomerase IV in vivo and in cells, to characterize the basis of target-mediated drug resistance, and to identify novel compounds that overcome resistance. Research will benefit from the broad library of wild- type and drug-resistant gyrase/topoisomerase IV available in the Osheroff laboratory, which includes enzymes from Bacil...