Discovery and antibacterial mechanism of a novel antibacterial agent based on target screening and membrane permeability optimization.

The escalating crisis of multidrug-resistant (MDR) bacterial infections has rendered many conventional antibiotics ineffective, necessitating more agents with rapid action and low resistance potential. Here, we integrated consensus-based target identification with permeability-driven molecular optimization. Through comparative genomics and off-target exclusion, we identified highly conserved essential bacterial proteins as promising targets. High-throughput virtual screening of a natural product library (n = 582,719) followed by structure-guided modification yielded a series of oxadiazole benzoate derivatives. Umbrella sampling simulations guided membrane permeability optimization, identifying the brominated derivative Com8 with the lowest free energy barrier for transmembrane transport (ΔG = 24 kcal mol-1). Com8 exhibited potent bactericidal activity against both Pseudomonas aeruginosa and Staphylococcus aureus, with no resistance development after 20 passages. Mechanistic studies revealed that Com8 disrupts membrane integrity, causes depolarization, increases membrane permeability, and eradicates mature biofilms. In a murine S. aureus-infected wound model, Com8 significantly accelerated wound healing, reduced bacterial load, and promoted tissue regeneration without systemic toxicity. Collectively, this work establishes a rational, generalizable framework for broad-spectrum antibacterial discovery and identifies Com8 as a promising lead candidate against MDR infections.