Discovery of isoquinoline sulfonamides as allosteric gyrase inhibitors with activity against fluoroquinolone-resistant bacteria

Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of DNA gyrase with antibacterial activity against fluoroquinolone-resistant clinical isolates of Escherichia coli. Screening of a small-molecule library revealed an initial isoquinoline sulfonamide hit, which was optimized via medicinal chemistry efforts to afford the more potent antibacterial LEI-800. Target identification studies, including whole-genome sequencing of in vitro selected mutants with resistance to isoquinoline sulfonamides, unanimously pointed to the DNA gyrase complex, an essential bacterial topoisomerase and an established antibacterial target. Using single-particle cryogenic electron microscopy, we determined the structure of the gyrase–LEI-800–DNA complex. The compound occupies an allosteric, hydrophobic pocket in the GyrA subunit and has a mode of action that is distinct from the clinically used fluoroquinolones or any other gyrase inhibitor reported to date. LEI-800 provides a chemotype suitable for development to counter the increasingly widespread bacterial resistance to fluoroquinolones. Global antibiotic scarcity looms owing to bacterial resistance. Now the discovery of a class of allosteric inhibitors targeting DNA gyrase—essential for bacteria—yields a compound LEI-800 that exhibits activity against fluoroquinolone-resistant E. coli. The compound’s unique mode of action, revealed through cryo-EM, makes it a promising candidate for countering bacterial resistance.