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

Gonorrhea, a sexually transmitted infection caused by <i>Neisseria gonorrhoeae</i>, 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 <i>N. gonorrhoeae</i> gyrase and topoisomerase IV have not been reported. Consequently, we determined the targeting of gepotidacin in <i>N. gonorrhoeae</i> cells and its effects on purified <i>N. gonorrhoeae</i> 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 <i>N. gonorrhoeae</i> gyrase (GyrA^D90) and topoisomerase IV (ParC^D86) 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.