Study On Biological Characteristics And Half-life Determination Of Cyclic Antimicrobial Peptide C-LR18

With the increasing drug resistance of pathogenic bacteria,the research and development of new antibiotics and their analogues is imminent.Antimicrobial peptides（AMPs）have unique antibacterial mechanism and have become one of the most potential antibiotic substitutes.However,most antimicrobial peptides have the weaknesses of low stability and short half-life,which limits their clinical application.Therefore,improving the stability of antimicrobial peptides and prolonging the half-life of antimicrobial peptides have become the focus of the research and development of new antimicrobial peptides.Peptide cyclization refers to a modification method that applies chemical methods to cyclize or partially cyclize the head and tail of a linear peptide to make it into a more stable ring structure,so as to improve the stability of the peptide and prolong its half-life.Compared with linear peptides,cyclized peptides have stronger biological activity,protease stability（including exopeptidase and endopeptidase）,and cell selectivity.Therefore,cyclization of linear antimicrobial peptides is expected to be an effective method to improve the stability of antimicrobial peptides,extend the half-life of antimicrobial peptides,and then improve the absorption and utilization of antimicrobial peptides in vivo.LR18（LRRLLRLPRRPLRLLRRL-NH₂）is an antimicrobial peptide composed of18 amino acid residues obtained from our laboratory.It is two mirror symmetrical heptapeptide sequences connected with PRRP as the central axis of symmetryα-secondary spiral structure.The previous research of the research group shows that LR18has broad-spectrum antibacterial activity and good stability in different salt ions,temperatures and p H environments,but its protease stability and serum stability are poor and its half-life is short,which affects its further application.Therefore,in order to prevent the carboxyl end of lysine at the head and tail of LR18 from being hydrolyzed by sulfhydryl protease,this study cyclized LR18 through disulfide bond by adding two cysteines.Designed and synthesized a new cyclized antimicrobial peptide with stable three-dimensional structure,named C-LR18.The biological activity,half-life and therapeutic effect of C-LR18 on mouse model of E.coli infection were studied.The following are the main contents and results of our research:1.Design and bioactivity determination of C-LR18The antimicrobial activity of cyclized peptide C-LR18 was higher than linear peptide LR18.C-LR18 had higher antimicrobial activity against both gram-positive and gram-negative bacteria,and its antimicrobial activity against gram-negative bacteria was obviously better than gram-positive bacteria.The cyclized peptide C-LR18 retained the advantages of low cytotoxic solubility and low hemolytic activity compared with the original peptide LR18,and showed no hemolytic activity and cytotoxicity at the inhibitory concentration（1-128μg/m L）.In order to study the stability of C-LR18,the environmental sensitivity of C-LR18 was tested.Compared with the original peptide LR18,the papain stability and serum stability of the cyclized peptide C-LR18 were significantly improved.After treatment with papain for 1h,the antimicrobial activity of C-LR18 had no significant change,and the MIC against E.coli ATCC25922 was still4μM.After treatment with 50%mouse serum and fetal bovine serum,the MIC of cyclized peptide C-LR18 to E.coli ATCC25922 increased only 4 times,while the MIC of protopeptide LR18 increased 16 times.2.Determination of LR18 and C-LR18 half-lifes in vivo and in vitro in SD ratsIn order to investigate the change of cyclized antibacterial peptide half-life,the half-life of C-LR18 and LR18 in vitro and in vivo were determined by HPLC.The half-lives of C-LR18 and LR18 in rats were 12.73±0.79min and 2.73±0.74min in vivo,209.45±20.21min and 62.12±5.66min in plasma,respectively.After cyclization,the half-life of the cyclized peptides in SD rats was extended to 4.66 times and 3.37 times respectively.3.In vivo therapeutic effect of C-LR18 on mice infected with Escherichia coliIn order to study the therapeutic effect of C-LR18 on mice infected with E.coli,the safety of C-LR18 was evaluated by acute toxicity test,and the LD₅₀ of C-LR18 on mice was 37.8mg/kg,which was lower than the toxicity of many antibacterial peptides reported at present.The mice model of pathogenic E.coli infection and the treatment model were established.It was found that high dose（7.5 mg/kg）C-LR18 could improve the survival rate of mice and inhibit the pathogenic bacteria in target organs.In conclusion,in this study,theα-helix antibacterial peptide LR18 was used as the propeptide and cycled to obtain a new cyclized antibacterial peptide C-LR18 with better antibacterial activity,higher stability and effectively extended half-life,and C-LR18showed certain therapeutic effect on mice infected with E.coli.They had laid the groundwork for the research and development of new antibacterial peptide drugs.