Rational hybrid design and computational characterization of PA-Hyb1: a novel peptide inhibitor targeting the MexB efflux pump in Pseudomonas aeruginosa.

CONTEXT: The MexAB-OprM efflux pump is a primary driver of multidrug resistance in Pseudomonas aeruginosa. While antimicrobial peptides (AMPs) offer a promising alternative to small-molecule inhibitors, understanding their dynamic interactions with large, multi-domain membrane transporters like MexB requires rigorous computational modeling. Rational hybrid peptide design combined with physics-based Molecular Dynamics simulations provides a strategic pathway to optimize AMP efficacy and predict binding stability prior to experimental synthesis. Ten hybrid peptides were designed and screened in silico for toxicity and physicochemical properties. The lead candidate, PA-Hyb1, exhibited a stable α-helical conformation and favorable safety profiles. During extended MD simulations, PA-Hyb1 displayed structural flexibility, anchoring its hydrophobic N-terminus via persistent π-π stacking with Phe178/Phe610, while its cationic C-terminus dynamically scanned acidic vestibule residues. MM/PBSA calculations over the equilibrated trajectory (from 300 to 500 ns) yielded a binding enthalpy of -28.5 ± 2.4 kcal/mol. This interaction energy was significantly superior to levofloxacin (-14.2 ± 1.8 kcal/mol; p < 0.0001) and outperformed scrambled and parent fragment controls, closely approaching the rigid small-molecule inhibitor D13-9001 (-30.1 ± 1.8 kcal/mol), despite the inherent entropic penalty of peptide flexibility. The computational pipeline successfully identified PA-Hyb1 as a high-affinity, sequence-specific candidate for MexB inhibition, establishing a strong rationale for its in vitro synthesis and validation. These binding-mode findings were obtained in a DPPC/POPE bilayer used as a simplified membrane-mimetic system rather than the native Pseudomonas aeruginosa inner-membrane lipid composition (predominantly phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin), and outer-membrane penetration by PA-Hyb1 was not evaluated; these limitations, together with a preliminary host-safety-only ADMET screen, temper the translational claims below and are discussed further in Sect. 4.1. METHODS: Ten hybrid peptides were designed by fusing functional motifs from Cecropin A, Magainin 2, and LL-37. Candidates were screened in silico for toxicity and physicochemical properties. The lead candidate, PA-Hyb1, was structurally modeled using PEP-FOLD4 and energy minimized under the CHARMM36m force field. Molecular docking (AutoDock Vina) targeted the distal binding pocket (Cavity C3) of MexB (PDB ID: 6IIA). To evaluate dynamic stability and binding thermodynamics, 500-ns all-atom Molecular Dynamics simulations were conducted in an explicit asymmetric DPPC/POPE lipid bilayer. Binding affinities were quantified using MM/PBSA free energy calculations and rigorously compared against a scrambled sequence control, parent fragments, levofloxacin, and the D13-9001 reference inhibitor.