Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase

Abstract

Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 μM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 μM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or with intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound.

Cryo-EM structure of the completeE. coliDNA Gyrase nucleoprotein complex

“…The high resolution features of the DNA binding domain structure now makes it possible to detect the density of small molecules and possibly catalytic ions in the cryo-EM map. We were able to observe the Gepotidacin molecule inserted in the DNA a on the two-fold axis at the GyrA dimer interface, as previously observed in crystal structures of the S. aureus DNA Gyrase in complex with the NBTI 21,24,37,38 . Gepotidacin is currently in clinical trials for the treatment of N. gonorrhoeae infections 22,23 , a species that is phylogenetically closer to E. coli and also harbors an Isoleucine at this position in the Gyrase sequence ( Supplementary Figure 11b-c).…”

“…Although the overall resolution of the pre-opening state is lower, a density for the Gepotidacin molecule could be identified at a lower rmsd level, suggesting that the Gepotidacin binding site is compatinble with conformational fluctuations at the DNA gate. Such flexibility of the DNA binding and cleavage domain in presence of Gepotidacin has also been observed in the S. aureus DNA crystal structures of DNA binding domains with an intact or doubly nicked DNA 24 .…”

“…7a-b and Supplementary Figure 4). The molecule intercalates in the DNA duplex between positions +2 and +3 forming hydrophobic interactions and pi-stacks with the protein and the DNA, at the same location as previously observed for the NBTIs and crystallized with S. aureus DNA Gyrase 21,24,37,38 (Supplemental Figure 11a). The main difference in the binding pocket of S. aureus and E. coli gyrase lies in the subunit Methionine residue (M75) that is replaced by an Isoleucine residue in (I74) (Supplementary Figure S11a).…”

“…The comparison of the cryo-EM map with the electron density map of crystal structures 24 shows that the cryo-EM map information is of comparable quality in this region for the identification of small compounds ( Supplementary Figure 14).…”

“…More recently, novel bacterial topoisomerase inhibitors (NBTIs), have been developed that also target the DNA cleavage activity of Gyrase, but with a mechanism and target site different from the quinolones 21 . These molecules and in particular Gepotidacin, represent a promising alternative and are currently in clinical trial for the treatment of bacterial infections [22][23][24] . However, the use of any antibiotics may lead to the appearance of mutations and the development of resistant strains 25 .…”