Synthesis, molecular dynamics, and antimicrobial evaluation of peptide-antibiotic conjugates designed for dual-mechanism pathogen inhibition.

Introduction: In the context of rapid rise of resistant pathogen strains, antimicrobial peptides (AMPs) represent promising scaffolds that complement conventional antibiotics, especially given the specific toxicities that limit classic drugs like fluoroquinolones and nitroimidazoles. AMP monotherapy faces major drawbacks, primarily due to inherent host-cell toxicity and a short half-life. Methods: To overcome these limitations, this study details the design and synthesis of dual-mechanism prodrugs by conjugating the membrane-active AMPs scaffolds of urechistachykinin I and decoralin to norfloxacin and metronidazole via a pathogen-cleavable ester linker. To minimize off-target toxicity, both AMP sequences were synthesized with a C-terminal carboxyl group, as these variants exhibit negligible hemolytic and neurotoxic activity compared to their amidated analogues. The designed antibiotic-peptide conjugates were synthesized on solid support starting from the C-terminus, followed by N-terminal chain elongation to attach the antibiotics. Specific derivatization methods ensured the covalent ester linkage of norfloxacin and metronidazole to the N-termini of urechistachykinin I (resulting in conjugates Results: All conjugates exhibited increased flexibility and solvent exposure relative to their parent peptides, as evidenced by increased RMSD, radius of gyration, H-bonding with water and higher solvent accessible surface area. In vitro screening identified Discussion: These in vitro assays demonstrate retained peptide membrane-disrupting properties and synergistic effects with the antibiotics. The dual-mechanism design represents a promising strategy to reduce toxicity and overcome antimicrobial resistance.