Electrostatic Trojan Horse: Charge-Anchored Pleuromutilin Breaches Anionic Barriers to Hijack Ribosomes in Antibiotic-Resistant Bacteria.

Antimicrobial resistance threatens health, and new agents are needed. Lefamulin is the only approved antibiotic of a new class in two decades. It targets the 50S peptidyl transferase center (PTC). Its efficacy against multidrug-resistant pathogens is limited by anionic envelopes, limiting penetration. We report a charge-anchored pleuromutilin exploiting an electrostatic Trojan horse to breach barriers and engage the ribosome. An N-pyridinium provides a cationic localizer. A two-step mechanism operates. Long-range electrostatics enrich ligands at anionic interfaces and the PTC. Short-range interactions secure high-affinity placement of the tricyclic core. Computer simulations support the occupancy of the activity pocket and the field-guided orientation of the cationic side chain for PNY-6b. Proteomics highlights ribosomal proteins as dominant targets. Cellular assays show biofilm eradication and membrane depolarization. In murine infections, PNY-6b lowers burden and improves survival. Electrostatic complementarity offers a generalizable design principle for targets with electrostatic fields and for pathogens with poor envelope permeability.