Design Modification And Activity Study Of Feleucin-k3 Antimicrobial Peptide By Introducing α-（4-pentenyl）-alanine At Different Sites

Antibiotics are considered to be one of the greatest achievements in the history of human medicine.However,the long-term unregulated use or even abuse of antibiotics has caused an irreversible bacterial resistance crisis,especially the emergence and widespread spread of"ESKAPE"superbugs,making the problem of bacterial drug resistance increasingly serious.Currently,almost all antibiotics are ineffective against these superbugs,making it the biggest problem in the treatment of clinical bacterial infections,and the most effective breakthrough to solve this problem is the development of new antibacterial drugs.Antimicrobial peptides have opened up a new field for people to search for ideal antimicrobial drugs because of their broad-spectrum antimicrobial activity,unique mechanism of action and low drug resistance tendency.Antimicrobial peptides come from a wide range of sources,and amphibian skin secretions are an important resource pool for them.Feleucin-K3（FLKLLKKLL-NH₂）is a short amphiphilic cationic peptide secreted from the skin of the Bombina Orientali with simple sequence composition and broad-spectrum antimicrobial activity,which is an ideal template for antimicrobial peptide drug development.The preliminary study of our group found that the chemically synthesized Feleucin-K3 peptide showed good antibacterial activity against standard and resistant strains of common clinical bacteria,but it has the defects of poor enzymatic stability and systemic toxicity common to natural antibacterial peptides.In order to improve the druggability of Feleucin-K3 antimicrobial peptide,the group introducedα-（4-pentenyl）-alanine,an unnatural amino acid,into the 4th site of the peptide chain in a previous study modification,which significantly improved the antimicrobial activity and stability of the parental peptide,and reduced its hemolytic activity in the range of antimicrobial concentrations.In view of the effectiveness of the introduction ofα-（4-pentenyl）-alanine in enhancing the biological activity of Feleucin-K3 peptide,the present work investigated the effect of deletion or replacement of amino acid residues in the original site on the antibacterial activity,toxicity and stability of Feleucin-K3 peptide through the strategy of amino acid deletion and replacement into different sites of the peptide chain.In vitro activity screening showed that the analogues obtained byα-（4-pentenyl）-alanine substitution showed good antibacterial activity（MIC=4-8μg/m L）against both standard and resistant strains,while those synthesized by amino acid deletion strategy showed significantly lower antibacterial activity.Then,we evaluated the hemolytic activity and stability of the substituted analogues and found that the N-terminal substituted analogue K65 and the C-terminal substituted analogue K70 had low hemolytic activity（The hemolysis rates of K65 and K70 at 128μg/m L were 31.6%and 0.99%,respectively）,and both showed significantly higher enzymatic resistance in serum（with K65 and K70residues were 56.36%and 40.6%,respectively,after 8 h incubation with serum）.Based on the results of antibacterial and hemolytic activity assays,two analogues K65 and K70 with good antibacterial activity and low hemolytic activity were screened out in this study,and both were investigated for induction of resistance and in vitro antibiofilm activity.The results of the induced resistance assay showed that the K65 and K70analogs presented a significantly lower incidence of resistance than the control antibiotics.The anti-biofilm activity results showed that K65 and K70 analogues had significant inhibitory effects on bacterial biofilm formation in vitro;in vivo mouse dorsal catheter biofilm infection model,the two analogues also showed strong inhibition of bacterial biofilm formation and the inhibition of methicillin-resistant Staphylococcus aureus（MRSA）biofilm formation was stronger than the control drug amoxicillin at high concentration.Finally,a preliminary exploration of the antibacterial mechanisms of the two analogues was conducted,and the results showed that the K65and K70 analogues killed bacteria mainly by interacting with bacterial cell membranes,disrupting the integrity of the cell membrane and causing the leakage of contents.In summary,a series of analogues were designed by introducingα-（4-pentenyl）-alanine into different sites of Feleucin-K3 antimicrobial peptide through amino acid deletion and unnatural amino acid substitution strategy,and the antibacterial activity,hemolytic activity and stability of the analogues were evaluated.Both of them are less susceptible to drug resistance and showed good antibiofilm activity in vitro and in vivo,and the antibacterial mechanism was investigated.This study provides a theoretical and experimental basis for further modification of Feleucin-K3 peptides and the eventual development of peptide antibacterial lead compounds for the treatment of drug-resistant bacterial infections.