A novel penicillin-binding protein inhibitor with unprecedented intracellular activity eradicates multiple pathogenic bacteria.

The escalating crisis of antibiotics against Gram negative bacteria resistance demands innovative strategies to combat recalcitrant intracellular pathogens. We reposition RS 17053, an α1A-adrenoceptor antagonist with unexplored antibacterial function, as a mechanistically distinct broad-spectrum antibiotic. We proved RS 17053 has strong binding ability with Class A penicillin binding proteins (PBPs), which overcomes the β-lactam barrier by penetrating host cells and disrupts the biosynthesis of peptidoglycan by uniquely targeting MrcA/MecA. RS 17053 eradicated Gram-negative (Brucella, Salmonella) and Gram-positive (Methicillin resistant Staphylococcus aureus, MRSA) pathogens in vitro (MICs 0.25-4 μg/mL) and eliminated intracellular Brucella within macrophages at 8 μg/mL. Crucially, upon serial passaging under drug pressure, RS 17053 showed a slower progression to high-level resistance. In murine zoonotic infection models, 10 mg/kg/day RS 17053 achieved higher survival rate by caused a 2-log CFU/mL reduction in organs and normalizing pro-inflammatory cytokines. Multi-omics and phenotypic analyses confirmed selective PBP targeting, causing peptidoglycan collapse and bacterial lysis. This work has provided a promising therapeutic against intractable intracellular infections.