Design, synthesis and biological evaluation of 3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazole derivatives as AcrB inhibitors with potent antibiofilm effect for reversing bacterial multidrug resistance

A series of novel 3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazole derivatives were rationally designed, synthesized and evaluated for their biological activity as AcrB inhibitors. The compounds were assessed for their Antibiotic potentiating effects, followed by evaluation of Nile Red efflux inhibition, and off-target effects including activity on the outer and inner Bacterial membranes. Ten compounds potentiated Antibiotic activity at sub-inhibitory concentrations, reducing the minimum inhibitory concentrations (MICs) of at least one of the tested Antibiotics by at least 8-fold, with three derivatives (7c, 11g, and 11i) achieving 32-fold MIC reductions at 128 μg/mL. The identified compounds were also able to inhibit Nile Red efflux at concentrations between 50 μM and 200 μM. The compounds did not perturb Bacterial membrane integrity or dissipate the proton motive force (p.m.f), and acted specifically on AcrB. All active derivatives exhibited low hemolytic activity in murine erythrocytes. Compound 11i demonstrated superior therapeutic potential, both extending the post-antibiotic effects of azithromycin and linezolid by 2 and 1.5 h, respectively, and reducing significantly preformed biofilm biomass in combination therapy. Cytotoxicity testing assay suggested that 11i was relatively safe for the human embryonic kidney (HEK) 293 cell line. Moreover, the pharmacokinetic characteristics of 11i were also tested to guide pharmacodynamic studies in vivo. Subsequent in vivo assessments using Galleria mellonella and murine models revealed favorable biosafety and efficacy. Molecular docking studies elucidated critical hydrogen-bonding and hydrophobic interactions between 11i and distal binding pocket of AcrB, with molecular dynamics (MD) simulations further demonstrating the stability of the compound within this binding pocket. The 3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazole core represents a structurally promising chemical scaffold for the development of novel AcrB inhibitors with potent antibiofilm effect.

3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazole derivatives; AcrB; Antibacterial sensitizing activity; Efflux pump inhibitor.