PT - JOURNAL ARTICLE AU - Harry Morgan AU - Magdalena Lipka-Lloyd AU - Anna J. Warren AU - Naomi Hughes AU - John Holmes AU - Nicolas P. Burton AU - Eshwar Mahenthiralingam AU - Ben D. Bax TI - A 2.8 Å structure of zoliflodacin in a DNA-cleavage complex with <em>Staphylococcus aureus</em> DNA gyrase AID - 10.1101/2022.11.30.518515 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.11.30.518515 4099 - http://biorxiv.org/content/early/2022/11/30/2022.11.30.518515.short 4100 - http://biorxiv.org/content/early/2022/11/30/2022.11.30.518515.full AB - Since 2000 some thirteen quinolones/fluoroquinolones have been developed and come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA-cleavage complexes with DNA gyrase and topo IV, the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here we report a 2.8 Å X-ray crystal structure which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA-cleavage site(s) as quinolones sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA) suggesting it may be more difficult for bacteria to develop target mediated resistance. We found that zoliflodacin had an MIC of 4 µg/mL against Acinetobacter baumannii, an improvement of 4-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, are likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.Competing Interest StatementThe authors have declared no competing interest.