Overcoming fluoroquinolone resistance: Mechanistic basis of non-quinolone antibacterials targeting type II topoisomerases

Abstract

Fluoroquinolones are among the most widely prescribed antibacterials worldwide. However, their usefulness is being diminished by the rise in drug resistance. The most common and detrimental form of fluoroquinolone resistance results from specific mutations in gyrase and topoisomerase IV, which are the targets for these drugs. Understanding how fluoroquinolones interact with their targets and how mutations alter drug-enzyme interactions has opened the door to overcoming resistance by the development of new drug classes that utilize different interactions than fluoroquinolones.

The first part of this dissertation describes the mechanism of action of a novel class of gyrase poisons: Mycobacterium tuberculosis gyrase inhibitors (MGIs), a subclass of novel bacterial topoisomerase inhibitors (NBTIs). In contrast to fluoroquinolones, the MGIs induced only single-stranded DNA breaks and suppressed gyrase-mediated double-stranded breaks. Furthermore, they maintained activity against fluoroquinolone resistance mutations in M. tuberculosis gyrase and displayed no activity against human topoisomerase II.

The second part of this dissertation examined the actions of GSK126, a naphthyridone/ aminopiperidine-based NBTI in the parent class of MGIs. Results indicate that GSK126 has a broader spectrum of activity against bacterial type II enzymes than MGIs, but displays similar mechanistic characteristics, including the induction of only single-stranded breaks and the suppression of double-stranded DNA scission. GSK126 also maintained activity against fluoroquinolone resistant enzymes.

The last part of this dissertation describes the mechanistic and structural basis for the actions of gepotidacin, the most clinically relevant NBTI. Gepotidacin has successfully completed phase II clinical trials for skin/skin structure infections (including those from Staphylococcus aureus) and uncomplicated gonorrhea. Despite this clinical promise, nothing has been reported regarding gepotidacin-enzyme interactions. Therefore, I characterized the actions of gepotidacin against S. aureus gyrase. The compound was a potent inhibitor of gyrase catalytic activity. Furthermore, it induced high levels of enzyme-mediated single-stranded DNA breaks and suppressed double-stranded breaks. Finally, as determined by a 2.31Å resolution crystal structure of gepotidacin with a S. aureus GyrB27-A56/Y123F fusion truncate and DNA, a single molecule of gepotidacin was situated midway between the two DNA scissile bonds cleaved by the enzyme.