Discovery of an N,N'-diarylurea with potent activity against multidrug-resistant Staphylococcus aureus via perturbation of membrane homeostasis.

The global prevalence of multidrug-resistant (MDR) Staphylococcus aureus highlights the urgent need for the development of new therapeutic strategies. Recent studies have revealed that selected N,N'-diarylurea derivatives display potent antibacterial activity without inducing resistance, highlighting the potential of this chemotype for anti-staphylococcal agents development. In this study, we designed and synthesized a library of 45 N,N'-diarylurea derivatives and systematically evaluated their antibacterial activities and cytotoxicities. Primary screening identified six compounds with a selectivity index (ratio of cytotoxicity to antibacterial activity) greater than 95. Among these, compound 30 was selected as a lead candidate based on its superior efficacy in the Galleria mellonella larval infection model. Compound 30 exhibited potent activity against MDR S. aureus in multiple physiological states, including planktonic cells, persister cells, and biofilms. In addition, it significantly improved survival outcome in the murine peritonitis infection model, further supporting its in vivo efficacy. Mechanistic investigations revealed that compound 30 disrupts bacterial membrane function by altering membrane fluidity, dissipating membrane potential, and compromising membrane integrity, leading to rapid bacterial killing. Extended serial passage experiments over 31 generations did not result in detectable resistance, and no cross-resistance was observed across a panel of MDR S. aureus strains with diverse resistance patterns. Collectively, these results establish N,N'-diarylurea as a valuable scaffold for the development of membrane-targeting antibacterial agents and highlight compound 30 as a promising lead for further medicinal chemistry optimization against MDR S. aureus infections.