A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

To combat antibiotic-resistant priority pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium, novel antibiotics are urgently needed1-3. DNA polymerases, essential for DNA replication but structurally distinct between bacteria and eukaryotes, represent promising yet underexplored targets4,5. The replicative polymerase in Gram-positive bacteria, PolC, is specifically inhibited by ibezapolstat, a guanine nucleobase analogue with an aromatic modification at the N2-position6,7. Following the success of ibezapolstat against Clostridioides difficile infections (CDI) in Phase 2 studies8,9, next-generation compounds with distinct pharmacokinetic properties are being designed to target other Gram-positive pathogens10. However, mechanistic insight into the mode of action of PolC inhibitors is lacking. Here, using cryo-electron microscopy, we show that ibezapolstat and next-generation inhibitor ACX-801 use the nucleobase analogue to pair with DNA within the active site of E. faecium PolC, similar to dGTP. The N2-linked aromatic group is positioned perpendicularly to the nucleobase and inserted into an induced binding pocket. Our results explain reduced susceptibility due to mutation of F1276, that plays a pivotal role in the creation of this pocket and engages in interactions with both the aromatic and nucleobase moieties. This work lays the foundation for rational development of an innovative class of antimicrobials against Gram-positive priority pathogens.