Characterization of a novel topoisomerase inhibitor (NBTI) with broad-spectrum antibacterial activity, including mycobacterial pathogens

Abstract Resistance to fluoroquinolone antibiotics is rising across bacterial pathogens, including the important pathogen Mycobacterium tuberculosis ( Mtb ), posing a major threat to global health. Novel bacterial topoisomerase inhibitors (NBTIs) can overcome fluoroquinolone resistance. However, the only FDA-approved NBTI, the triazaacenaphthylene compound gepotidacin, is not indicated for mycobacterial infections. Here we characterize compound B1 , a previously identified NBTI with broad-spectrum antibacterial activity, including against Gram-negative clinical isolates and the mycobacterium Mtb . B1 selectively inhibited prokaryotic topoisomerases, including Mtb gyrase, with greater potency than the FDA-approved NBTI gepotidacin. Consistent with the NBTI mode of action, B1 primarily induced single-strand DNA breaks, and structural studies revealed binding within the NBTI pocket of gyrase. B1 showed enhanced efficacy against Mtb in broth culture and in an ex-vivo Mtb macrophage infection model compared with gepotidacin. In zebrafish embryos, B1 cleared infections with clinical isolates of Klebsiella pneumoniae and Acinetobacter baumannii , but—like gepotidacin—was inactive against Mycobacterium marinum . Although well tolerated in vivo, B1 strongly inhibited the human ether-à-go-go (hERG) channel, indicating a cardiotoxicity risk. Together, our findings position B1 as a promising NBTI scaffold for anti(myco)bacterial drug development, requiring optimization to overcome hERG liability and enable in vivo studies in an appropriate Mtb infection model.