Mechanism of Action of Gepotidacin: Well-Balanced Dual-Targeting against Neisseria gonorrhoeae Gyrase and Topoisomerase IV in Cells and In Vitro.

Gonorrhea, a sexually transmitted infection caused by Neisseria gonorrhoeae, remains a major public health concern. Fluoroquinolones, which target gyrase and topoisomerase IV, once served as first-line therapy for gonorrhea. However, rising target-mediated resistance led to their removal from treatment guidelines. In response to growing antibacterial resistance, gepotidacin, a first-in-class triazaacenaphthylene, offers a promising new treatment strategy. Gepotidacin targets gyrase and topoisomerase IV but is structurally and mechanistically different from fluoroquinolones. A phase III clinical trial of gepotidacin in the treatment of uncomplicated urogenital gonorrhea demonstrated a positive outcome. However, interactions of the drug with N. gonorrhoeae gyrase and topoisomerase IV have not been reported. Consequently, we determined the targeting of gepotidacin in N. gonorrhoeae cells and its effects on purified N. gonorrhoeae gyrase and topoisomerase IV. Although fluoroquinolones primarily target gyrase in Gram-negative bacteria, gepotidacin displayed well-balanced dual-targeting of both gyrase and topoisomerase IV in cultured cells. Reduced gepotidacin susceptibility required concurrent target-specific mutations in both enzymes, predicting a low propensity for developing target-mediated resistance. Consistent with this cellular dual-targeting, gepotidacin inhibited gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed DNA decatenation at similar low micromolar concentrations. Gepotidacin also induced primarily single-stranded DNA breaks mediated by both enzymes at comparable concentrations. Finally, mutations in aspartic acid residues predicted to mediate important gepotidacin-protein interactions in N. gonorrhoeae gyrase (GyrAD90) and topoisomerase IV (ParCD86) markedly diminished the activity of gepotidacin against the respective enzymes. Our findings differentiate gepotidacin targeting and mechanism from those of fluoroquinolones and highlight its potential to combat drug-resistant gonorrhea.