Verbenol acts as a low-entropy pan-inhibitor of quorum sensing to attenuate Pseudomonas aeruginosa virulence and resolve pulmonary inflammation.

The rise of multidrug-resistant Pseudomonas aeruginosa necessitates urgent anti-virulence strategies designed to disarm pathogens without driving selective resistance. Here, we present an integrated in vitro, in vivo, and in silico investigations establishing the plant-derived monoterpenoid verbenol as a potent, multi-target quorum sensing (QS) inhibitor, targeting bacterial virulence. In vitro, while exhibiting limited direct bactericidal pressure (MIC = 6.25 mg/mL), sub-inhibitory concentrations of verbenol profoundly suppressed key QS-regulated virulence traits. It effectively disrupted initial bacterial adhesion, eradicated pre-formed biofilm, paralyzed flagellar and pili-mediated motilities, and drastically reduced the production of pyocyanin and rhamnolipids. Translating these findings to an acute murine lung infection model, verbenol treatment significantly reduced bacterial virulence and mitigated pneumonia-associated tissue damage without detectable systemic toxicity. Crucially, it exerted dual host-protective effects by restoring pulmonary redox homeostasis and dampening the pro-inflammatory cytokine cascade (IL-6 and TNF-α). To elucidate the molecular basis of this broad anti-virulence efficacy, deep-learning-driven molecular docking and extended explicit-solvent molecular dynamics (200 ns) were performed. CNN-guided pose validation (RMSD ≤2.0 Å) and computational thermodynamic profiling revealed that verbenol functions as a highly efficient, low-entropy pan-inhibitor. By maintaining exceptional Ligand Efficiency, it anchors simultaneously into the hydrophobic binding pockets of the LasR, RhlR, and PqsR master regulators, completely bypassing the entropic penalties that hinder flexible native autoinducers. Collectively, this study highlights verbenol as a dual-action therapeutic adjuvant capable of silencing bacterial communication and shielding host lung tissues from inflammatory injury, paving the way for sustainable therapies against persistent P. aeruginosa infections.