Rational Design, Computational Analysis and Antibacterial Activities of Synthesized Peptide-Based Molecules Targeting...

Background/Objective: The rise in bacterial resistance necessitates novel therapeutic strategies beyond conventional antibiotics. Antimicrobial peptides represent promising candidates but face challenges such as instability, enzymatic degradation, and host toxicity. To overcome these limitations, conjugation and structural modifications are being explored. This study focuses on designing peptide-based inhibitors of the quorum-sensing (QS) regulator LasR in Pseudomonas aeruginosa, a key mediator of biofilm formation and antibiotic resistance. Methods: Rationally designed tripeptides and dipeptides conjugated with coumarin-3-carboxylic acid and dihydro-3-amino-2-(3H)-furanone were evaluated using molecular docking. The most promising ligand-protein complexes were further analyzed using molecular dynamics (MD) simulations conducted with the CHARMM-GUI and AMBER tools to assess the stability of the ligand-protein complex systems, and the binding affinities were evaluated using Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculations. Pharmacokinetic and toxicity profiles were predicted using ADMETLab 3.0. Selected compounds were synthesized via solid-phase peptide synthesis, structurally confirmed by ¹H NMR and ESI-MS, and tested for antibacterial and antibiofilm activity against P. aeruginosa ATCC 27853. Results: Computational analyses identified several promising inhibitors with stronger binding affinities than the native autoinducer OdDHL. Coumarin conjugates C004 and C006 showed superior docking scores, while MM-PBSA indicated P004 and C004 had the most favorable binding energies. MD simulations confirmed stable ligand-protein complexes. ADMET predictions highlighted C004 and C006 as having excellent pharmacokinetic properties. Experimental assays showed moderate antibacterial activity (MIC 512-1024 µg/mL) and strong antibiofilm inhibition, particularly for C004 (83% inhibition at ½ MIC). Conclusions: The study demonstrates that peptide-coumarin conjugates, especially C004, are promising tools for disrupting QS and biofilm formation in P. aeruginosa. Further optimization and in vivo validation are needed to advance these compounds toward therapeutic application.