Optimized peptide inhibitor Aqs1C targets LasR to disrupt quorum sensing and biofilm formation in Pseudomonas aeruginosa: Insights from MD simulations and in vitro studies

Pseudomonas aeruginosa (PA) is a critical pathogen, and its antibiotic resistance is largely driven by the quorum-sensing regulator LasR. Herein, we report the design, synthesis, and characterization of Aqs1C, a mutated peptide derivative of Aqs1, optimized to inhibit LasR and its quorum-sensing pathway. By introducing a targeted mutation, Aqs1C exhibited enhanced stability and binding affinity for LasR protein compared to its predecessor, Aqs1B. Using molecular dynamics simulations (MDS), the Aqs1C-LasR complex demonstrated a marked increase in structural stability, reflected in reduced root mean square deviation (RMSD) values and lower binding free energy. Electrostatic complementarity analysis showed stronger and more favorable interactions between Aqs1C and LasR. Further, GaMD experiments were able to reproduce the binding state between Aqs1C and LasR, indicating the binding mechanism between them. These molecular insights correlated with functional in vitro assays. Aqs1C effectively inhibited quorum-sensing-associated virulence factors in PA, involving biofilm formation (77.6 % inhibition), pyocyanin production (75.7 % inhibition), protease secretion (61.1 % inhibition), and rhamnolipid production (74.1 % inhibition), at a 100 μg/mL concentration, in a comparable or superior pattern to azithromycin (AZM). Molecular modelling, MDS, and GaMD insights and in vitro assays established Aqs1C as a promising candidate for therapeutic development to mitigate PA infections through targeted quorum-sensing disruption.