In this study, through virtual screening and in vitro bioactivity assays, we discovered that (−)-epicatechin gallate (ECG), a polyphenol compound extracted from green tea, demonstrated marked anti-Ser/Thr phosphatase (Stp1) activity towards Staphylococcus aureus (S. aureus) with an IC50 value of 8.35 μM. By targeting S. aureus Stp1, ECG prevented the up-regulation of virulence gene and the formation of antibody membrane and protected the mice from S. aureus infection. Through MD simulation, the allosteric inhibitory mechanism of ECG on Stp1 was determined. The Stp1-ECG complex model underwent a significant change in conformation; its flap subdomain changed from opening to closing, whereas Stp1 activity was lost when bound to ECG. In addition, the MD simulation results of Stp1 and several tea polyphenol compounds showed that gallate groups and fewer adjacent phenolic hydroxyl groups contributed to the binding of Stp1 and inhibitors. As an inhibitor targeting S. aureus Stp1, ECG reduced the pathogenicity of S. aureus without inhibiting S. aureus, which largely reduced the possibility of drug resistance. Our findings demonstrated a novel molecular mechanism of green tea as the usual drink against S. aureus infection and elucidated the future design of allosteric inhibitors targeting Stp1.
