High-throughput LacZ/CPRG screen identifies novel potential antibiotics targeting Gram-negative bacterial envelopes to combat resistance
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Background
Bacterial resistance, exacerbated by multidrug-resistant Gram-negative (GN) pathogens, poses a public health threat due to their impermeable envelopes, which block many antibiotics.
Objectives
We aimed to develop a high-throughput screening (HTS) method to identify small molecules targeting GN bacterial envelopes and assess their antibacterial potential.
Methods
Envelope disruption in Escherichia coli and Pseudomonas aeruginosa was assessed using a β -galactosidase (LacZ)/CPRG reporter assay in LB at 37°C. The assay was validated through screening the LOPAC 1280 and KD2 4761 compound libraries. Concentration–response relationships, permeabilisation constants (K 50 ), co-permeabilisation assays, minimal inhibitory concentration (MIC) measurements, and bacterial microscopy post-MICs were performed.
Results
The assay demonstrated robust performance, evidenced by high Z’-factor and signal-to-noise (S/N ratios. Screening identified 57 active compounds (1.2% of the library), including β -lactams and three non-antibiotic molecules—suloctidil, isorotenone, and alexidine—that exhibited concentration-dependent antibacterial activity. Alexidine showed the most potent activity, with the lowest K 50 (2.7×10 −3 mM) and MICs of 0.004 mM for E. coli and 0.015 mM for P. aeruginosa . Suloctidil and isorotenone induced spherical cell morphology, while alexidine induced a filamentous phenotype, indicative of envelope disruption. The assay also identified antibiotics for monotherapy and combination therapy, with ampicillin, alexidine, and suloctidil enhancing chloramphenicol’s efficacy against E. coli MG1655.
Conclusions
The LacZ/CPRG reporter assay effectively identified compounds targeting bacterial envelopes, including novel molecules with antibacterial activity against GN pathogens, making it a promising tool for antibiotic discovery or combination therapy.
