Discovery of a novel polymyxin adjuvant against multidrug-resistant Gram-negative bacteria through oxidative stress modulation

Antibiotic adjuvants offer a promising strategy for restoring antibiotic sensitivity, expanding antibacterial spectra, and reducing required dosages. Previously, compound 15 was identified as a potential adjuvant for Polymyxin B (PB) against multidrug-resistant (MDR) Pseudomonas aeruginosa DK2; however, its clinical utility was hindered by high cytotoxicity, uncertain in vivo efficacy, and an unclear synergetic mechanism. To address these challenges, we synthesized and evaluated a series of novel benzamide derivatives, with A22 emerging as a particularly promising candidate. A22 demonstrated potent synergistic activity to PB, minimal cytotoxicity, improved water solubility, and broad-spectrum synergism of polymyxins against various clinically isolated MDR Gram-negative strains. In vivo studies using Caenorhabditis elegans and mouse models further confirmed the efficacy of A22. Moreover, A22 effectively suppressed the development of PB resistance in Pseudomonas aeruginosa DK2. Mechanistic investigations revealed that A22 enhances polymyxins activity by inducing reactive oxygen species production, reducing ATP levels, increasing NOX activity, and inhibiting biofilm formation, leading to bacterial death. These findings position A22 as a highly promising candidate for the development of polymyxin adjuvants, offering a robust approach to combating MDR Gram-negative bacterial infections.