Insight into the Dual inhibitory Mechanism of verbascoside targeting serine/threonine phosphatase Stp1 against Staphylococcus aureus.

Abstract The eukaryotic-like serine/threonine phosphatase (Stp1) is an enzyme-dependent protein phosphatase involved in regulating various virulence factors of Staphylococcus aureus. Owing to its role in S. aureus infections, Stp1 has become a potential target for antibiotic development. Unfortunately, there are very few reports describing Stp1 inhibitors. Using virtual screening, we have identified a potent and effective Stp1 inhibitor, verbascoside (VBS). Interestingly, the kinetics of the enzymatic reaction revealed that this natural inhibitor acts via both competitive and allosteric mechanisms. To explore the mechanism of interaction between VBS and Stp1, standard molecular dynamics (MD) simulations were performed for the Stp1-VBS complex. Consistent with the experimental results, competitive and allosteric binding sites for VBS were identified in Stp1. Met39, Gly41, His42, Arg161, and Asn162 residues were involved in the competitive binding of VBS, while Arg122, Ser136, Asp137, Asn142, and Val145 residues were associated with the allosteric binding of VBS. The contributions of these residues were confirmed by amino acid site-directed mutagenesis and fluorescence quenching experiments. This work demonstrates that VBS is a potent ant-virulence compound against S. aureus infection, laying the foundation for the further development of novel ant-virulence agents.