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.

细菌已经进化出对几乎所有已知抗菌药物的耐药性，这强调需要识别通过新机制发挥作用的抗生素。在这里，我们报道了一类 DNA 旋转酶变构抑制剂，对氟喹诺酮耐药临床大肠杆菌分离株具有抗菌活性。对小分子文库的筛选显示出最初的异喹啉磺酰胺效果，通过药物化学工作对其进行优化，以提供更有效的抗菌 LEI-800。靶点识别研究，包括对体外选定的异喹啉磺酰胺耐药突变体进行全基因组测序，一致指出DNA旋转酶复合物、一种重要的细菌拓扑异构酶和已确定的抗菌靶点。使用单颗粒低温电子显微镜，我们确定了旋转酶-LEI-800-DNA 复合物的结构。该化合物占据 GyrA 亚基中的变构疏水口袋，其作用模式不同于临床上使用的氟喹诺酮类药物或迄今为止报道的任何其他旋转酶抑制剂。 LEI-800 提供了一种适合开发的化学型，以对抗日益广泛的细菌对氟喹诺酮类药物的耐药性。