Neochlorogenic acid targets caseinolytic protease P to Suppress MRSA pathogenicity and Unlocks a novel therapeutic avenue for osteomyelitis.

Osteomyelitis is a complex and challenging infectious disease in orthopedics that is caused predominantly by methicillin-resistant Staphylococcus aureus (MRSA). The development of novel anti-infective strategies to overcome antibiotic resistance is urgently needed. Caseinolytic protease (ClpP), a key regulator of MRSA virulence, represents a promising target. Using fluorescence resonance energy transfer screening, this study identified neochlorogenic acid (NCA) as an effective ClpP inhibitor, with an IC50 of 17.96 ± 1.25 μg/mL (50.7 ± 3.5 μM) and excellent safety. Fluorescence quenching, a thermal shift assay, a cellular thermal shift assay (CETSA), and molecular docking confirmed strong interactions between NCA and ClpP. In vitro experiments demonstrated that NCA effectively suppressed the transcription and expression of key MRSA virulence factors, including α-hemolysin and Panton-Valentine leukocidin (PVL). It also significantly reduced MRSA adhesion to fibrinogen, increased urease activity, and increased sensitivity to hydrogen peroxide, thereby attenuating virulence without inhibiting bacterial growth. Furthermore, NCA alleviated MRSA invasion of MC3T3-E1 cells and mitigated associated cellular damage. In vivo, NCA exhibited significant therapeutic efficacy in a rat osteomyelitis model and effectively suppressed MRSA virulence in a Galleria mellonella systemic infection model. These findings suggest that the use of NCA, a potent ClpP inhibitor and a promising antivirulence lead compound, provides a scientific basis for the development of novel therapeutics against MRSA infections.