Design,Synthesis,and Antimicrobial Activities Of Stapled Peptides For Cecropin A

Objectives:In this study,we investigated whether the "staple" chemical modification of Cecropin A could positively affect the drug properties of Cecropin A by using an all-carbon conformation-locking strategy.The purity and secondary structure of the peptides were determined by high-performance liquid chromatography and circular dichroism,and the toxicity of the stapled peptides was tested by fluorescence detection.Sequencing of the biological prokaryotic transcriptome by explore the antimicrobial mechanism of the preferred stapled peptide CEC-2-9.Methods:(1)Design and synthesis: The peptides were synthesized by the traditional solid phase synthesis(SPPS)method,and the purity of the peptides was determined by Waters high-performance liquid chromatography,and the molecular weight composition of the peptides was analyzed by Q-TOF mass spectrometry.(2)In vitro antibacterial activity test: The minimum inhibitory concentration of the stapled peptides was determined by micro-broth two-fold dilution method to explore whether the stapled peptides could exert a broader and more potent antibacterial effect.(3)Helicity test: The helicity of the Cecropin A series stapled peptide was tested by using Jasco-815 circular dichroism chromatograph.(4)Protein stability test: Protein stability assay using chymotrypsin and trypsin co-incubated with peptides to test the degradation of Cecropin A series stapled peptides in protease.(5)Cytotoxicity experiment: The experiment uses a multifunctional enzyme standardization instrument to detect the degree of hemolysis of the peptide with human blood red blood cells and the size of cytotoxicity of the peptides to RAW264.7 after incubation by fluorescence analysis,and the HC50 and IC50 values of each peptide are calculated.(6)In vivo antibacterial experiment: The experiment was conducted to establish a mouse sepsis model with E.coli ATCC25922 strain,and the colony counting method was used as the antibacterial results of in vivo treatment with Cecropin A and its stapled peptides.(7)Antibacterial mechanism: The prokaryotic transcriptome sequencing technology was used for Cecropin A preferential stapled peptide,and after GO pathway and KEGG pathway analysis of significantly different genes,the biological information related to the antibacterial mechanism of Cecropin A preferential stapled peptide was screened and identified.Results:(1)In vitro antimicrobial activity test: As analyzed by the minimum inhibitory concentration value results,the antibacterial activity of stapled Cecropin A was significantly higher than that of Cecropin A against Gram-positive and Gram-negative bacteria,as well as the antibacterial activity of CEC-2 series among the stapled peptides were significantly higher than that of CEC-1 series.(2)Peptide helicity: The ɑ-helicity of the original peptide CEC-0 was 18.99%,theɑ-helicity of the preferred stapled peptide CEC-2-9 was 20.51%,and the helicity of most other stapled peptides was higher than the original peptide by the analysis of CD value calculation.(3)Protein stability test: In the degradation test of trypsin and chymotrypsin,it can be found that the remaining content of the original peptide and stapled peptide at 4hours is similar,so the protein stability of both of them is the same.(4)Cytotoxicity assay: In the hemolysis assay,the HC50 value of stapled peptide CEC-2-9 was more than 400 μM,while the HC50 value of CEC-0 was 174.2 μM,which showed that the stapling modification strategy could reduce the hemolytic activity of Cecropin A;in the RAW264.7 cytotoxicity assay,the IC50 value of CEC-2-9 was lower than that of the original peptide CEC-0,which proved that the stapling modification had lower toxicity to RAW264.7 cells.(5)In vivo antibacterial activity test: The results of the colony counting assay showed that the growth of E.coli was significantly reduced,the antibacterial effect was significantly enhanced.(6)Antibacterial mechanism: Transcriptome sequencing further revealed that the mechanism by which CEC-2-9 can exert antibacterial effects is that it affects the cellular pentose phosphate metabolism to the extent that it negatively interferes with the synthesis of nucleic acids in bacterial cells,play a powerful broad-spectrum antibacterial effect.Conclusions:The broad chemical diversity of antimicrobial peptides makes them an important source of new compounds for future drug development,and the stapling modification strategy can effectively enhance the α-helicity of peptides and improve protein stability.The preferred stapled peptide CEC-2-9 provided in this study has the potential to become a new broad-spectrum antibacterial drug for clinical research and is expected to be developed into a new drug,which is important for reducing current antibiotic resistance levels and preventing infectious disease.That has profound importance for the development process of pharmaceutical research,the livestock industry and science and technology for people’s livelihood.