Structure-based virtual screening identifies naturally derived histidine kinase VbrK inhibitors as potential...

The issue of antibacterial resistance (AMR) mediated by β-lactamases raises an urgent need for the development of novel anti-infective strategies. In V. parahaemolyticus, the histidine kinase VbrK directly detects β-lactam antibiotics and initiates resistance signaling. The absence of a crystal structure for its intracellular kinase domain has impeded drug discovery. In this research, we have built a protein model of VbrK by grafting the essential ligand and metal cofactor onto AlphaFold predictions. Based on this model, a virtual screening was performed against a compound library composed of 7 665 natural products. The top 35 hits were screened using a competitive fluorescence polarization assay to verify their binding affinity to VbrK. Phlorizin, diprophylline and baicalin were identified as novel VbrK inhibitors with binding affinities in the micromolar range. Minimum inhibitory concentration testing confirmed that phlorizin and baicalin enhanced the susceptibility to carbenicillin in AMR strains of V. parahaemolyticus. Furthermore, nitrocefin hydrolysis assays demonstrated that phlorizin effectively repressed the carbenicillin-induced production of functional β-lactamases at the phenotypic level. Flexible docking and molecular simulation indicated stable interactions with residues Arg326 and Gly445. This study provided novel hit compounds for VbrK, with the expectation of being optimized as mechanistically distinct β-lactam adjuvants complementary to the existing β-lactamase inhibitor therapy.