Safety And Activity Evaluation In Vivo Of Antimicrobial Peptides J-AA?J-AR And J-RR And Design,Synthesis And Activity Study Of Their New Analogs

As novel potential antimicrobial agents,antimicrobial peptides?AMPs?caused widespread concern at the growing problem of drug resistance.While in vitro study of antimicrobial peptides deepening,studies in vivo has become the focus.The first part of this study,in vivo safety and antibacterial activity of antimicrobial peptide J-AA,J-AR and J-RR were evaluated,which had been studies in vitro by our group.In vivo safety was evaluated by acute toxicity with the method of median lethal dose(LD₅₀),the results show that J-AR and J-RR have very low toxicity,and the LD₅₀ of J-AA is 53.6mg/kg.In vivo Antimicrobial activity was evaluated by the cure rate and blood content in the mouse model of pathogenic bacteria.J-AA,J-AR and J-RR have all shown significant antibacterial effect in the bacteremia model caused by E.coli,but no treatment in the mice peritonitis model of infection caused by E.coli.In a mouse model of infection caused by MRSA,J-RR shows a significant treatment,and the mice cure rates at the doses of 10,5,2.5mg/kg were 100%,66.7%and 33.3%,respectively.But J-AA and J-AR have no treatment effect.Antibacterial activity in vivo studies showed that the combinational antimicrobial peptides J-AA,J-AR and J-RR have a clear and selective bactericidal activity in vivo.The second part of this study,to improve the enzymatic stability of J-AA,J-AR and J-RR,new analoges were designed and synthesized.The method was that The 7,8 sequences of parent peptide Anoplin,and the 1,5 sequence of parent peptide RW were first substituded by D-amino acid,then new analogues J-AA-1,J-AR-1 and J-RR-1 were synthesized through 1,3-dipolar cycloaddition at side chain.The influences of new analoges on multiple aspects,such as antibacterial activity,anti-enzymatic ability,antibacterial mechanism,and hemolytic toxicity after simultaneously applying D-amino acid substitutions and the side connection strategy on structural transformation of antimicrobial peptide Anoplin and RW were discussed.Compared with the parent peptides,the antibacterial activities of new analogues in vitro were significantly increased by 2 to 32 times and have no resistance.Surprisely,the antibacterial activity of J-AA-1 increased 2 to 8 times on gram-negative bacteria,but lost activity on the gram-positive bacteria.Sterilization kinetics study found J-RR-1 had rapid bactericidal capacity.In the enzymatic stability experiment,the new analogues were incubated 6h with different concentrations of trypsin,it shows that the stable trypsin concentrations of J-AA-1 and J-AR-1 is up to 10 times,J-RR-1 is up to 10⁴-fold in comparing with parent peptides.Plating the maximum of J-RR-1 and stable concentration trypsin which is 0.2mg/mL onto MH agar after incubated 4h,the pictures showed it can still kill bacteria completely.At the same time HPLC analysis showed that the degradation rates of Anoplin and RW are 82.3%and 70.9%,while the degradation rates of J-AA-1 and J-AR-1 are 23.8%and 17.1%,respectively,J-RR-1 is not degraded after the peptides incubated with trypsin whose concentration is 0.2mg/mL for 1h.With a total incubation time is extended,the parent peptides are completely degraded in 2h,but the degradation rates of J-AA-1and J-AR-1 are less than 50%,J-RR-1 has still not been degraded.The outer and inner membranes permeability experiments and PI uptake experiments showed that the mechanism of new analogs is rapidly to destroy bacterial membrane which is the same as their mother peptides.When the concentration of new analogues is 150?M,the hemolysis rate is less than 1.5%,who indicates they are hemolysis safety.In addition,the result of circular dichroism certified the three new analogs are?helix structure in the biofilm environment.It is shown that the strategy of application simultaneously of D-amino acid substitutions and side chain modification strategy with 1,3-dipolar cycloaddition for Structural modification of antimicrobial peptides could improve antimicrobial activity and the hydrolysis stability,and reduce hemolysis.But J-AA-1 has no antimicrobial activity to S.aureus,it suggesting that we have to further explore the better sites of D-amino acid substitution of Anoplin,at the same time concern about the antimicrobial mechanism of S.aureus.The study gives a good basis to make the structural modification of the natural antimicrobial peptides more successful,and screens antimicrobial peptide J-RR with a broad spectrum of activity and low toxicity in vivo,and new antimicrobial peptide J-AR-1 and J-RR-1 with a broad spectrum of activity and low toxicity and strong stable in vitro.Our research established foundation for clinical application of new antimicrobial peptides.