High-throughput LacZ/CPRG assay for identifying Gram-negative envelope-targeting antibiotics.

Background: Objectives: We aimed to develop a liquid-based high-throughput screening (HTS) assay to identify small molecules that target GN bacterial envelope integrity and to assess their antibacterial potential, distinct from traditional viability-based screens. Methods: Envelope disruption in Results: The assay demonstrated robust, reproducible performance across independent optimization runs, as evidenced by high Z'-factor and signal-to-noise (S/N) ratios. Screening identified 72 active compounds, including β-lactams and four non-antibiotic molecules (suloctidil, isorotenone, alexidine, and thiram) that showed concentration-dependent increases in CPRG reporter signal, consistent with envelope permeabilization. Thiram was excluded due to its fungicidal use, and the remaining three compounds were further characterized. Alexidine showed the most potent activity, with the lowest K Conclusion: The LacZ/CPRG reporter assay enables rapid identification of compounds targeting bacterial envelopes, including novel molecules active against GN pathogens, providing a high-throughput functional platform that complements established permeability assays and accelerates discovery of new therapeutics.