Study On Structure-activity Relationship,Antibacterial Mechanism And Stability Of Antimicrobial Peptide Chensinin-1

Antimicrobial peptides are widely existed in the bodies,and show strong cationicity,good thermal stability,no resistance,and no cytotoxicity against eukaryotic cells,which make them be a good candidate for new antibiotics with novel mode of action.In this paper,a series of analogues were designed and synthesized by using the natural antimicrobial peptide chensinin-1,which was isolated and purified from the skin secretions of Chinese brown frog Rana chensinensis,the structural parameters such as hydrophobicity andα-helix content of chensinin-1 peptide were varied to investigate the structure-activity relationship of antimicrobial peptide chensinin-1 and the antibacterial mechanism,including the following aspects:1)The substitution of Gly in chensinin-1 sequence with Trp resulted in an increase in the hydrophobicity of MC1-1 and the antimicrobial activity of MC1-1 was increased accordingly.In order to examine the function of His,three His were removed from the sequence of MC1-1 or replaced by Arg to obtain MC1-2 and MC1-3,respectively.The results showed that the and antibacterial activity of MC1-2 were increased compared with MC1-1 which could be due to the enhancement of hydrophobicity.However,the hydrophobic values of MC1-3 and MC1-1 are unchanged,after the addition of three positive charges in MC1-3,the antibacterial activity was not improved.The depolarization of cell membrane and the release of calcein in the mimetic cell membrane showed that all three analogues of chensinin-1 could increase the permeability of cell membrane which proved that the three peptide targets were cell membrane.Isothermal thermal titration（ITC）and dynamic light scattering（DLS）experiments indicated that three analogues could bind with LPS,which helped chensinin-1 analogues insert into the cell membrane,and then kiled the bacteria.2)Chensinin-1b showed potent,broad-spectrum bactericidal activity with no hemolytic activity.The results of structural analysis of nuclear magnetic resonance spectroscopy showed that theα-helix region of chensinin-1b falled in the interval of 8-13 amino acid residues in the hydrophobic environment of the simulated membrane.In this paper,a series of analogues are designed to study the structure-activity relationship of chensinin-1b analogues.The experimental results showed that the amino acid residues at the carboxy terminus played an important role in the antibacterial activity of chensinin-1b than the N-terminal amino acid residues.Theα-helix content and cationicity have different effects on the antibacterial activity.The increase in the number of positive charges could improve the antibacterial activity,but theα-helix and antibacterial activity was not linearly related.3)The instability of antimicrobial peptides in the body limits its clinical application.In this paper,a series of chensinin-1b lipopeptides were designed and synthesized by coupling CH₃-（CH₂）₆-COOH,CH₃-（CH₂）₁₀-COOH and CH₃-（CH₂）₁₄-COOH at the amino carboxyl to try to improve their stability both in vivo and in vitro,the antibacterial activity and hemolytic activity were also investigated.The results showed that chensinin-1b lipopeptide retained the original antibacterial activity,and the secondary structure did not change significantly.Endometrial and extracellular membrane permeability experiments showed that the target of chensinin-1b lipopeptides are the cell membrane.which is same with chensinin-1b.The experiment proved that the stability of lipopeptide was improved in the existance of both trypsin and in mice,indicating that increased fat chain could increase the stability of antimicrobial peptides.