Effects of DNA Supercoil Handedness on Catalytic Inhibition of Mycobacterial Gyrase by Gepotidacin

Abstract Gepotidacin, the first-in-class triazaacenaphthylene antibacterial, kills bacteria by stabilizing DNA cleavage complexes and inhibiting the catalytic activities of the type II topoisomerases, gyrase and topoisomerase IV. Mycobacterial species encode gyrase as their sole type II topoisomerase, meaning that inhibition of this enzyme blocks several essential cellular functions through a single target. Unlike fluoroquinolones and spiropyrimidinetriones, which inhibit gyrase by inserting two drug molecules, one at each of the cleaved scissile bonds, gepotidacin intercalates a single molecule into the intact double helix between the two scissile bonds within the enzyme active site. To determine how the distinct binding mechanism of gepotidacin affects its inhibitory profile, we examined the ability of the drug to inhibit decatenation, supercoiling of relaxed DNA, and relaxation of positive supercoils catalyzed by Mycobacterium tuberculosis and Mycobacteroides abscessus gyrase. Gepotidacin inhibited decatenation and supercoiling at comparable concentrations but required higher concentrations to inhibit relaxation of positively supercoiled DNA, a pattern distinct from fluoroquinolones and spiropyrimidetriones. Similar results were observed with GSK000, a Mycobacterium tuberculosis gyrase inhibitor that also intercalates into intact DNA in the gyrase active site. Using mutant gyrase enzymes capable of relaxing both positively and negatively supercoiled DNA, we found that gepotidacin and GSK000 inhibited relaxation of negative supercoils at substantially lower concentrations (∼13-fold on average) than relaxation of positive supercoils. This sensitivity to supercoil handedness was markedly higher than observed for nonintercalative drugs (moxifloxacin, ciprofloxacin, zoliflodacin, and novobiocin). These findings suggest that the intercalative binding mechanism of gepotidacin confers heightened sensitivity to DNA supercoil handedness.