Targeting quorum sensing in Pseudomonas aeruginosa: Novel benzo[d]imidazole-based LasR inhibitors with tunable head, α,β-unsaturated linker, and tail segments.

Targeting quorum sensing is a promising anti-virulence strategy to combat Pseudomonas aeruginosa pathogenicity and antimicrobial resistance. Based on this, a series of benzo[d]imidazoles derivatives were rationally designed, synthesized and structurally elucidated. Two α,β-unsaturated linkers-based scaffolds were investigated: acrylonitrile-linked (7a-g, 9a-c) and prop-2-enone-linked (13a-f, 15a-d) derivatives encompassing both NH-unsubstituted and N-methylated benzo[d]imidazole analogues. Antimicrobial evaluation of all the target compounds against P. aeruginosa PAO1 showed inhibition zones of 18-26 mm and MIC values ranging from 15.6 to 125 μg/mL. Among these, compounds 7d, 9a, 13b, and 15b exhibited the highest antibacterial activity. At sub-inhibitory concentrations (½ MIC), the tested compounds significantly suppressed key virulence factors, including protease production, biofilm formation, and motility phenotypes (twitching and swarming) (7d; 70.4, 79.4, 57.9, 74.7%), (9a;58.0, 55.4, 75.0, 62.7%), (13b;72.6, 86.0, 65.8, 65.3%), (15b;83.3, 81.8, 65.8, 66.7%), respectively. In vitro LasR antagonism assays confirmed effective quorum sensing inhibition (IC₅₀ values: 7d= 0.261, 9a= 0.94, 13b= 4.32, 15b= 3.04 μM). Molecular docking and molecular dynamics simulations validated the potency of 7d, revealing stable binding within the LasR active site, characterized by favorable hydrophobic interactions and disruption of receptor dimerization. Collectively, these findings identify 7d as a promising lead benzo[d]imidazole analogue possessing dual antibacterial and antivirulence activities, through quorum sensing interference via LasR inhibition.