Structural basis for the mode of action of inhibitors of the DNA polymerase PolC in Gram-positive priority pathogens

To combat antibiotic-resistant priority pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE), novel antibiotics are urgently needed. DNA polymerases, essential for DNA replication and structurally distinct between bacteria and eukaryotes, represent a promising yet underexplored target. The replicative polymerase in Gram-positive bacteria, PolC, is specifically inhibited by N2-substituted nucleobase analogues like Ibezapolstat. Following the success of Ibezapolstat against Clostridioides difficile infections, next-generation compounds with distinct pharmacokinetic properties are being designed to target other pathogens. However, mechanistic insight into the mode of action is lacking. Here, we show using cryo-electron microscopy that ACX-801 and Ibezapolstat contain a distinctive >90 kink at the Cx position to orients an aromatic group perpendicularly to the nucleobase, thereby inducing a conformational change in PolC that is stabilized by a quartet of aromatic residues. Our results explain reduced susceptibility due to mutations of F1276, which is part of the residues that form the opening of the induced pocket and additionally engages in pi-pi interactions with the nucleobase moiety, and A1281, that is also part of the binding pocket. This work lays the foundation for the rational development of an innovative class of antimicrobials against Gram-positive priority pathogens.