Design and synthesis of novel thiazolotriazole-sulfonamide hybrids tackling antimicrobial resistance: mechanistic insights into efflux pump inhibitor, gene downregulation, and machine-aided results.

The escalating global threat of antimicrobial resistance necessitates innovative therapeutic strategies. In this study, a novel series of thiazolotriazole-sulfonamide hybrids was designed using a molecular hybridization strategy. Computational potency was predicted via pharmacophore modelling, identifying biologically active scaffolds against resistant pathogens. The designed molecules(7a-j) were synthesized andevaluated for their in vitro antibacterial and antifungal efficacy. Compounds 7a, 7b, 7j emerged as potentantimicrobial agentswith favorable safety margin. 7j showed robust antibacterial activity (MIC: 15.625 μg/ml) against B. cereus and S. typhimurium. 7a displayed dual-action potency, matching this MIC against B. cereus and inhibiting C. albicans (MIC: 31.25 μg/ml). Also,7bshowed antifungal efficacy (31.25 μg/ml). P. aeruginosa antibiofilm assays revealed dose-dependent attenuation; 7b achieved 44% inhibition (1/2 MIC) against the standard strain and clinical isolate 1, whereas 7a excelled against isolate 2. Notably, 7j displayed a dual mechanism against MDR pathogens, inhibiting the efflux pump protein and achieving exceptional downregulation of the MexA, MexC, and MexE genes, as confirmed by qRT-PCR. This multi-level approach achieved a 16-fold potentiation of Ciprofloxacin's activity.Mechanistic elucidation integrated experimental data with docking and MD simulations targeting FabI, DNA gyrase A, and dihydrofolate reductase (DHFR).Molecular dynamics simulations further predicted the stabilityof these compounds within the catalytic domain ofthe targeted enzymes.In silico ADMET analyses indicated theirfavorable pharmacokinetic profiles.In vitro assays laterconfirmed their low toxicity in normal HEK-293 and WI-38 cell lines. In conclusion,these hybridsserve as promising candidates to overcome antimicrobial resistance.