Fluorescence Polarization-Based High-Throughput Screening Enables Identification of Vibrio β-Lactam Resistance Sensor Kinase Inhibitors to Counteract Antibiotic Resistance

Combination therapies of β-lactam Antibiotics with β-lactamase inhibitors are widely used, yet resistance to these regimens continues to emerge, highlighting the need for alternative strategies to control β-lactam resistance. Our previous work showed that bacteria can sense β-lactam Antibiotics prior to overt cell wall damage. The activation of histidine kinase VbrK by β-lactams initiating transcription of β-lactamase genes, thereby promoting β-lactam resistance. Using an optimized fluorescence polarization-based screening assay followed by hit-to-lead optimization, we identified LPZ-51 as an effective inhibitor of VbrK. LPZ-51 repressed blaA transcription without affecting Bacterial growth but markedly potentiated the activity of β-lactam Antibiotics. In a bacteria-challenged zebrafish model, LPZ-51 synergized with carbenicillin to reduce Bacterial burden, alleviate intestinal inflammation, and improve host survival. Together, these findings demonstrate that targeting VbrK attenuates β-lactam resistance by blocking β-lactamase induction at the transcriptional level, providing a mechanistically distinct antiresistance strategy complementary to existing β-lactam therapies.