# Inhibitory Effect of Amentoflavone on the Virulence of MRSA by Targeting ClpP

## TL;DR

Amentoflavone (AMF) reduces MRSA virulence by inhibiting ClpP, a key protein, without killing bacteria, offering a new approach to combat antibiotic-resistant infections.

## Contribution

AMF is shown to inhibit MRSA virulence via ClpP targeting, offering a novel antivirulence strategy over traditional antibiotics.

## Key findings

- AMF inhibits MRSA haemolytic activity by 72% and biofilm formation by 58% without affecting bacterial growth.
- AMF reduces transcription of virulence genes hla and psmα by 3.8-fold and ClpP enzymatic activity by 65%.
- AMF protects lung cells from MRSA infection and increases murine survival rates from 20% to 75% in pneumonia models.

## Abstract

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.