Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence.

Motoharu Hirano,Ryuji Kawano,Chihiro Goto,Takashi Misawa,Hidetomo Yokoo,Yosuke Demizu,Chihiro Saito

doi:10.3390/molecules26020444

Abstract

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

Highlights

Magainin 2 (Mag2), a representative antimicrobial peptide (AMP), plays an important role in the natural immune system and has recently received attention as a next-generation drug for multi-drug resistance bacteria [1,2]
The peptides were cleaved from the resin and precipitated using cold ether, and the resulting residues were purified using reverse-phase high-performance liquid chromatography (HPLC); from this, the target peptide was identified with liquid phase chromatography–mass spectrometry (LC-MS)
We investigated the antimicrobial activity of the synthesized peptides 2–9 against gram-positive Staphylococcus aureus and gram-negative Escherichia coli DH5α, Pseudomonas aeruginosa, and multiple-drug resistant P. aeruginosa (MDRP)

Summary

Introduction

Magainin 2 (Mag2), a representative antimicrobial peptide (AMP), plays an important role in the natural immune system and has recently received attention as a next-generation drug for multi-drug resistance bacteria [1,2]. Mag comprises cationic and hydrophobic amino acid residues and employs a helical structure in aqueous solution These properties are required for interaction with microbial membranes, and subsequent Mag insertions into membranes induce bacterial pore formation and ion efflux to exert their antimicrobial activity. Peptide 1 showed resistance against digestive enzymes due to the unnatural amino acid residues These data suggest that further structural developments are required to stabilize the helical structure of the essential fragment of Mag; this may be achieved by using non-proteinogenic amino acids as a promising strategy for increasing antimicrobial activity. We designed several peptides bearing the stapling side chain and synthesized them in the hydrophobic regions and the N-terminal fatty acyl group and subsequently evaluated their preferred secondary structures, antimicrobial activity against gram-positive and gram-negative bacteria, and hemolytic activity against human RBCs

Methods

Results

Conclusion