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

Abstract

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 1a and 1b) and decoralin (resulting in conjugates 2a and 2b). Structural behavior was assessed by 200 ns molecular dynamics simulations in water. Antimicrobial activity was evaluated by broth microdilution against Gram-negative, Gram-positive bacteria and fungi, and compared to parent peptides and reference antibiotics as positive controls. 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 1a, 2a and 2b as the most active conjugates. Norfloxacin conjugate 1a exhibited potent activity against Escherichia coli ATCC 8739 (MIC 0.56 μM) and Candida parapsilosis (MIC 2.26 μM). Norfloxacin conjugate 2a showed broad-spectrum inhibition, including Escherichia coli ATCC 8739 (MIC 8.64 μM), Salmonella enterica ATCC BAA-2162 (MIC 8.64 μM) and Listeria monocytogenes DSMZ 115292 (MIC 4.32 μM), with improved activity over unmodified decoralin. Metronidazole conjugate 2b was active against Listeria monocytogenes DSMZ 115292 (MIC 11.86 μM) and Enterococcus faecalis ATCC 29212 (MIC 23.12 μM). 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.