Rigidity vs Activity: Design of Gramicidin S Analogs against Multidrug-Resistant Bacteria Based on Molecular Engineering

Abstract Antimicrobial peptides are a promising class of therapeutics to address antibiotic resistance; yet, their clinical use is limited by toxicity and narrow-spectrum activity. To better understand how conformational rigidity influences efficacy and safety, a series of β-sheet antimicrobial peptide analogs based on gramicidin S were designed and synthesized. Two stapled derivatives (GSC-FB and GSC-SS) and a flexible linear analog (GS-L) were prepared and evaluated. GSC-FB retained potent activity against Gram-positive bacteria with a significantly reduced cytotoxicity. GS-L, characterized by increased conformational flexibility, showed broader-spectrum activity, including activity against Gram-negative strains, and similarly improved safety. Circular dichroism spectroscopy revealed that all analogs displayed structural perturbations relative to native gramicidin S. Molecular dynamics simulations indicated that only flexible or moderately rigid analogs effectively interact with membrane models. These findings demonstrate that conformational rigidity is a key parameter in the design of antimicrobial peptides, enabling the optimization of antimicrobial potency while mitigating toxicity.