Dual-targeting sulfamethoxazole derivatives overcome multidrug resistance in ESKAPE pathogens by inhibiting quorum sensing and efflux pumps.

The rapid emergence of pan-drug-resistant pathogens necessitates innovative antimicrobial strategies that overcome conventional resistance. This study reports the structure-guided design of sulfamethoxazole derivatives as dual inhibitors of quorum sensing (LasR) and efflux pumps (AcrB), alongside the classic dihydropteroate synthase (DHPS) target. High-throughput virtual screening of 54 derivatives, followed by MM-GBSA analysis, prioritized five novel compounds for synthesis. Their structures were confirmed by 1H/13C NMR, FT-IR, and mass spectrometry. All compounds demonstrated potent growth inhibition (MICs 15.625-125 µg mL-1) against Proteus mirabilis, Salmonella typhi, and Escherichia coli, though activity against the more resistant Pseudomonas aeruginosa was reduced. Critically, lead compounds SMX033 and SMX015 achieved >99% biofilm inhibition against P. mirabilis, and SMX033 showed significant efflux pump inhibition, directly validating our in silico predictions of a multi-target mechanism. In particular, compound SMX033 exhibited the best antimicrobial activity and lowest cytotoxicity of all the sulfamethoxazole derivatives with a CCC50 value of 286.20 µM against Vero cells. Despite their predicted in silico genotoxicity, these derivatives provide promising chemical scaffolds for combating multidrug-resistant infections by concurrently attenuating virulence and restoring antibiotic susceptibility.