Inhibitory Effect of Amentoflavone on the Virulence of <scp>MRSA</scp> by Targeting <scp>ClpP</scp>

Methicillin-resistant Staphylococcus aureus (MRSA) poses significant therapeutic challenges due to its global spread and virulence. Targeting the critical virulence regulator ClpP presents a promising antivirulence strategy. This study investigated AMF's mechanism against MRSA through molecular dynamics simulations, FRET and TSA. Phenotypic analyses revealed AMF's inhibition of MRSA haemolytic activity (72% reduction) and biofilm formation (58% decrease) without affecting bacterial growth. Molecular docking identified key AMF-ClpP interaction sites (ARG-171, ASP-170, ASP-172), validated via CETSA. AMF reduced transcription of critical virulence genes (hla, psmα) by 3.8-fold and inhibited ClpP enzymatic activity by 65%. Cellular studies demonstrated AMF's protection of A549 lung cells from MRSA infection (82% viability vs. 43% control). In murine pneumonia models, AMF treatment enhanced survival rates from 20% to 75% while reducing proinflammatory cytokines (IL-6, TNF-α) by 60%-70%. Histopathological analysis showed significant mitigation of lung tissue damage. These findings establish AMF as a potent ClpP inhibitor that attenuates MRSA virulence through dual mechanisms: suppression of toxin production and biofilm formation. The compound's therapeutic potential stems from its ability to disarm pathogenic mechanisms while maintaining commensal microbiota integrity. This study provides proof-of-concept for antivirulence strategies targeting ClpP, offering a promising alternative to traditional antibiotics against MRSA infections.