Design and Evaluation of β-Amino-Acid-Containing Autoinducing Peptide Analogues as Inhibitors of the Staphylococcus aureus Agr Quorum Sensing System

Antimicrobial resistance in Staphylococcus aureus (S. aureus), particularly methicillin-resistant S. aureus, has created an urgent need for alternative therapeutic strategies. Targeting bacterial virulence rather than viability has emerged as a promising approach to reduce selective pressure for resistance development. The accessory gene regulator (agr) quorum-sensing system plays a central role in controlling the expression of numerous virulence factors in S. aureus and therefore represents an attractive anti-virulence target. This study investigated the design and characterisation of β-amino-acid-containing peptide inhibitors targeting the AgrC receptor. Guided by structure-based design and molecular modelling, three groups of analogues were designed and synthesised using Fmoc-based solid-phase peptide synthesis and evaluated using an agrP3-lux reporter assay. Two lead compounds derived from the AIP-I scaffold exhibited potent inhibitory activity, with nanomolar IC50 values and no detectable effects on bacterial growth. Incorporation of β-amino acids and replacement of the native thiolactone with a lactam ring substantially improved resistance to hydrolysis and proteolytic degradation compared with the native autoinducing peptide. Molecular docking, pharmacophore modelling and molecular dynamics simulations were employed to investigate ligand recognition and receptor activation. The results supported a model in which agonists and antagonists occupy distinct regions of the AgrC binding pocket and suggested a structural mechanism underlying AgrC-mediated signal transduction. Preliminary mammalian cell compatibility studies demonstrated no obvious cytotoxicity towards J774 macrophages. Furthermore, agr inhibition significantly reduced intracellular and extracellular S. aureus burdens in a macrophage infection model, suggesting reduced bacterial-mediated host cell damage and enhanced macrophage-mediated bacterial clearance. Overall, this work demonstrates that β-amino-acid-containing agr inhibitors can combine potent quorum-sensing inhibition with improved stability and favourable biological compatibility, supporting their further development as anti-virulence agents against S. aureus infections.