Study On The Relationship Between Structure And Function Of Lipopeptide Antibacterial Agents

The increasing resistance of bacteria and fungi against antibiotics is a major concern worldwide,which has led to tremendous efforts to develop new antibiotics.The structure of natural lipopeptides usually consists of a linear or cyclic hydrophobic alkyl chain covalently linked to 4-12 amino acid peptides.Natural lipopeptides have the advantages of broad-spectrum antibacterial properties,wide applications,not easy to make bacteria resistance.So,they are expected to substitute for current antibiotics.However,the relationship between the antibacterial activity of lipopeptides and their structural features is currently unclear.Based on the previous research,this article used antibacterial peptides G（VVKK）₂V-NH₂ and G（IIKK）₂I-NH₂ as templates,we modified the N-terminus of templates peptide with different alkyl chain lengths.We explored the influence of the addition of alkyl chains on the physical and chemical properties of lipopeptides,such as assembly structure,hydrophobicity,surface activity,helicity,etc.Base on this experiments,we studied the effects of lipopeptides on the permeability of bacterial membranes by fluorescent microscope staining and flow cytometry detection,and clarified the antibacterial mechanism of lipopeptides.This study not only explored the relationship between the structure of lipopeptides and its antibacterial function,but also provided theoretical basis and data support for the development of antibacterial drugs with high efficiency and low toxicity.The main contents of this paper are list as follows:Synthesis and structure-activity relationship of C_n（VVKK）₂V-NH₂（n=8/12/16）series lipopeptides study:（1）The alkyl chain length of synthetic lipopeptides affected its hydrophobicity,surface activity and secondary structure,and further influenced the interaction between lipopeptides and bacterial cell membranes.With the extension of the alkyl chain,the hydrophobicity and surface activity of synthetic lipopeptides increased.Lipopeptides wereβ-sheets in the membrane environment of DPPC/DPPG（7:3,w/w）SUVs,which means that the interaction between lipopeptides and bacteria tends to adoptβ-sheet secondary conformation.（2）The alkyl chain length of the synthetic lipopeptide affected the assembly and disassembly ability of the lipopeptide.The lipopeptide C₁₂（VVKK）₂V-NH₂ with stronger disassembly ability can exert better antibacterial activity.With the extension of the alkyl chain,the CAC value of lipopeptides decreased and the assembly ability increased.C₁₆（VVKK）₂V-NH₂ lipopeptides tend to form long nanofibers through hydrophobic interaction and intramolecular hydrogen bond interactions.The lipopeptide molecules bound to bacteria through electrostatic interactions and could not destroy the bacterial cell membrane.（3）Alkyl chain modification can compensate for the hydrophobicity of the parent peptide and improve the membrane breaking ability of synthetic lipopeptides.However,the alkylation modification also increased the cytotoxicity of synthetic lipopeptides to human red blood cells.The lipopeptide C₁₂（VVKK）₂V-NH₂ modified with the appropriate alkyl chain length has suitable hydrophobicity and assembly ability,thus exhibiting the best selectivity index.C_nG₃（VVKK）₂V-NH₂（n=8/12/16）series lipopeptides study:（1）The existence of"linker"affected the basic physical and chemical properties of synthetic lipopeptides.Compared with lipopeptides without"linker",the hydrophobicity of lipopeptides was slightly decreased but the surface activity was increased.The antibacterial activity was slightly reduced,so the hydrophobicity played a dominant factor in the VVKK system.The existence of the flexible segment"linker"promoted the formation ofα-helix secondary structure of lipopeptides in the membrane environment of DPPC/DPPG（7:3,w/w）SUVs.（2）The lipopeptide with the best antibacterial effect in this system was still the lipopeptide C₁₂G₃（VVKK）₂V-NH₂ with moderate alkyl chain length.The existence of"linker"did not affect the antibacterial activity to a large extent,so it is still the alkyl chain of lipopeptides that affects its antibacterial activity.C_nG_x（IIKK）₂I-NH₂（n=8/12/16 x=0,3）series lipopeptides study:（1）The antibacterial activity and hemolytic activity of lipopeptides increased overall the replacement of hydrophobic amino acid.Among them,C₈G₃（IIKK）₂I-NH₂ had strong antibacterial activity of both Gram-positive and Gram-negative bacteria and exhibited higher selectivity index.（2）The basic physical and chemical properties of synthetic lipopeptides were changed after amino acid replacement,and the hydrophobicity and surface activity were improved overall.Compared with the hydrophobicity of lipopeptides of VVKK,it was found that lipopeptides had the best hydrophobicity threshold.Lipopeptides wereα-helices in the membrane environment of DPPC/DPPG（7:3,w/w）SUVs,which means that the interaction between lipopeptides and bacteria tends to adopt aα-helix secondary conformation.The antibacterial activity of lipopeptides was closely related toα-helicity and rate constant（K）.（3）Amino acid substitutions affected the cut-off effect point of lipopeptides.In this series,the cut-off point of lipopeptides was changed to lipopeptides with an alkyl chain length of C₈.