Overcoming fluoroquinolone resistance in Neisseria gonorrhoeae

项目总结

PROJECT SUMMARY Fluoroquinolone-based drugs are the most widely used oral antibacterials worldwide. However, the rise of resistance to this class of antibacterials has impacted their usage. The fluoroquinolones were a first-line therapy for the 78 million annual cases of gonorrheal infections, caused by Neisseria gonorrhoeae, until 2007. At that time, the Centers for Disease Control altered recommendations in response to growing resistance rates in the bacterium. The most important fluoroquinolone resistance mutations are target mediated and develop in the bacterial type II topoisomerases, gyrase and topoisomerase IV. Finding new targets for antibacterials is a difficult task. However, two alternative approaches to overcoming fluoroquinolone resistance are 1) altering fluoroquinolone structure to identify derivatives with increased potency and/or efficacy and 2) developing new classes of compounds that target the validated bacterial type II topoisomerases, but interact with different residues in the enzymes. Efforts in the latter approach have resulted in a new class of compounds called novel bacterial topoisomerase inhibitors (NBTIs). The enzyme targets of fluoroquinolones and NBTIs, gyrase and topoisomerase IV, are essential for regulating DNA topology in bacterial cells. They modulate levels of DNA supercoiling and untangle and unknot the genome by passing a segment of DNA through a transient double-stranded break made in a separate segment. Both drug classes target the covalent topoisomerase-cleaved DNA complex (cleavage complex) and inhibit ligation of the broken strands. If replication or transcription machinery encounters the covalent topoisomerase-DNA cleavage complex, the genome can be fragmented, which leads to SOS responses and cell death in the bacteria. The primary goal of this project is to overcome fluoroquinolone resistance in N. gonorrhoeae by increasing our understanding of fluoroquinolone and NBTI interactions with their targets and target-mediated resistance development to aid drug design. This goal will be addressed by three specific aims: In Specific Aim 1, I will determine if and how a water-metal ion bridge, shown to facilitate topoisomerase- fluoroquinolone interactions in other bacterial species, functions in N. gonorrhoeae. To this end, I will use assays that measure DNA cleavage, supercoiling, and relaxation with wild-type and resistant enzymes. I also intend to generate fluoroquinolone binding data to further quantify the effects of the bridge using fluorescence anisotropy. In Specific Aim 2, I will focus on the NBTI class to determine how NBTIs function against N. gonorrhoeae gyrase and topoisomerase IV, and how resistance mutations in gyrase and topoisomerase IV abrogate drug action. This will require similar enzymological activity assays as in Specific Aim 1. Finally, in Specific Aim 3, I will assess the cellular effects of fluoroquinolones and NBTIs as they correlate with topoisomerase-DNA cleavage complex formation using an in vivo complex of enzyme bioassay. These studies have the potential to aid in the development of drugs to overcome antibacterial resistance.