Mechanism of Quinolone Resistance

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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 Anti- microbials,” and these drugs are the most heavily prescribed antibacterials at Veterans Administration hospitals. 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. Both gyrase and topoisomerase IV are physiological targets for fluoro- quinolones, but 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 and their use to treat US Veterans. For example, fluoroquinolones were used routinely to treat gonorrhea (which is caused by Neisseria gonorrhoeae), a sexually transmitted disease that is prevalent in the military and elevated in Veteran populations, starting in 1993. However, their use as front-line therapy was discontinued in 2006 due to the high incidence of resistance. 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 fluoroquinolones and gyrase/topoisomerase IV. By characterizing fluoroquinolone-enzyme interactions, the PI has designed novel drugs that overcome resistance mutations in Mycobacterium tuberculosis gyrase and Bacillus anthracis gyrase and topoisomerase IV. The identification and characterization of novel agents that act against these well-validated topoisomerase targets and overcome fluoroquinolone resistance could have important ramifications for the health of Veterans. 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 their basis of resistance. There is an urgent need to develop more effective drugs that display activity against fluoroquinolone-resistant bacteria. The premise underlying the proposed research is that understanding how drugs interact with their enzyme targets places us in a far better position to develop agents that overcome resistance. Thus, the specific aims of this proposal are to 1) determine the mechanistic basis for fluoroquinolone action and resistance with gyrase and topoisomerase IV across species; and 2) determine the mechanistic basis for the actions of NBTIs and SPTs against gyrase and topoisomerase IV across species. Proposed experiments will build upon previous studies from the Osheroff laboratory and preliminary data on the mechanism of bacterial type II topoisomerases and their interactions with fluoroquinolones, NBTI, and SPTs. Research will benefit greatly from the broad library of wild-type and drug-resistant gyrase/topoisomerase IV that the Osheroff laboratory has established. This library includes enzymes from B. anthracis, E. coli, Staphylococcus aureus, M. tuberculosis, Neisseria gonorrhoeae, Francisella tularensis, and Acinetobacter baumannii. Many of these pathogens routinely affect the health of US Veterans. Initial studies will focus on N. gonorrhoeae, M. tuberculosis, and E. coli as the model systems.