Design,Synthesis And Activity Evaluation Of New Analogs Of Natural Antimicrobial Peptide Anoplin By Non-natural D-amino Acid Modification And Side-chain Link Strategy And The Synergia Antibacterial Effectiveness Study Of Ursolic Acid Combined With Antimic

Antimicrobial peptides participated in the formation of internal immune system and have attracted more and more attention due to small molecular weight and the unique mechanism of action,which makes it difficult to induce drug resistance.However,natural antimicrobial peptides having some defects,such as poor stability,low bioavailability and cytotoxicity,restricted the development and application of antimicrobial peptides.In the first part of this study,non-natural D-amino acids（D-Leu,D-Lys,D-Arg）were used to partially replace in polar and nonpolar surface of natural antimicrobial peptide Anoplin.Ten new analogs of Anoplin with partial D-amino acid substitutions were designed and obtained.In order to screen out the most sensitive site of Anoplin,a systematic structure-activity relationship studies were undertaken.It was found that the substitution of D-amino acids at different sites had different effects on the antimicrobial activity,stability and secondary structure.The results of antimicrobial activity showed that the analogs of Anoplin-D9,10 and Anoplin-D9,10,3 retained the antimicrobial activity.What’s more,the antimicrobial activity of Anoplin-D9,10,3 was increased one-fold against E.coli（ATCC 25922）,when compared with parent peptide Anoplin;However,the rest of other analogs lost antimicrobial activity.The stability of all these D-amino acids substitution analogs was investigated,and we found that the ability of the non-polar surface D-amino acid substitution analog Anoplin-D9,10,3 to resist chymotrypsin degradation was 10 times higher than that of the parent peptide Anoplin,while the resistance to trypsin degradation was not obviously improved;The polar surface D-amino acid substitution analogs had better tolerance against trypsin and chymotrypsin degradation.In particular,the analogs of Anoplin-D4,7 and Anoplin-D5,7 had increased tolerance against trypsin and chymotrypsin degradation by 10⁴ and 10³ times,respectively.The secondary structures of all D-amino acids substitution analogs were studied.It was found that the parent peptide Anoplin and the D-amino acids substitution analogs of Anoplin-D9,10 and Anoplin-D9,10,3 could formαhelical structure,but the secondary structure of the other analogs were destroyed,which could be an important factor to influence the antibacterial activity of peptides.In addition,all the D-amino acids substitution analogs had very low hemolytic activity.Therefore,we successfully found the fourth,seventh or the fifth,seventh amino acids of the Anoplin were the most sensitive enzymatic degradation site.In the second part of the study,in order to obtain new antimicrobial peptide analogs with long-term effect and enhance antibacterial activity,the peptide analogs of Anoplin-D9,10,3;Anoplin-D4,7;Anoplin-D5,7 which have the enhanced stability in the first part,were chosen to design and synthesize the new dimer analogs.New homodimer and heterodimer analogs of J-AA-（D4,7）₂;J-AA-（D5,7）₂;J-AA-（D9,10,3+D5,7）and J-AA-（D4,7+D5,7）,were designed and synthesized by using the reaction of 1,3-dipolar cycloaddition.The result of antimicrobial activity showed that the antimicrobial activity of all these antimicrobial peptide dimer analogs containing D-amino acids was significantly enhanced,especially against the Gram-negative bacteria,which was increased 4-16 fold in comparison with the parent peptide Anoplin and the corresponding of D-amino acid substitution monomer peptide.The time-killing kinetics displayed that all these dimer analogs had rapid antibacterial effect and dose-dependent characteristics against Escherichia coli（ATCC 25922）and Klebsiella pneumonia（ATCC 700603）.And the dimer analogs of J-AA-（D9,10,3+D5,7）and J-AA-（D5,7）₂ had synergy and additivity effects against Klebsiella pneumonia when in combination with rifampicin or kanamycin,respectively.What’s more,all these dimer analogs had stronger activity to inhibit the formation of biofilm than the antibiotic of Rifampin.Additionally,the stability against enzyme degradation showed that the four dimer analogs had the significantly enhanced stability to resist the degradation of trypsin and chymotrypsin as the same as the D-amino acid substition template peptides Anoplin-D9,10,3;Anoplin-D4,7;Anoplin-D5,7.All dimer analogs had low hemolysis（hemolysis rate of them was less than 1%,at 300μM）.At the same time,all these dimer analogs were incubated with normal mice fibroblast cell NIH3T3 for 24 h.And we found that in the range of active concentration,cell survival rate was not affected.WeselectedthreedimeranalogsofJ-AA-（D4,7）₂;J-AA-（D4,7+D5,7）;J-AA-（D9,10,3+D5,7）with relatively high yield to evaluate their availability in vivo.In the treatment of acute peritonitis in mice,the therapeutic effects of dimer analogues showed a dose-dependent characteristic.In the high dose group of 40 mg/kg,the therapeutic effect of dimer analogs of J-AA-（D9,10,3+D5,7）was significantly increased,and the survival rate of mice was reached to 50%.In addition,in the combinational treatment of acute peritonitis in mice,the group of 20mg/kg of J-AA-（D9,10,3+D5,7）combined with 10mg/kg of rifampicin had greater effect than that of the individual group.And the survival rate of the mice in the the group of 20mg/kg of J-AA-（D9,10,3+D5,7）combined with 10mg/kg of rifampicin was 83%.Finally,the ability of dimer analogs to protect the mice from bacterial infection was also investigated.Rifampicin was used as control drug and normal saline group as negative control.The three antimicrobial peptide dimer analogs could reduced the risk of acute peritonitis death of the mice with low immunity,especially the protective effect of 10 mg/kg dimer analogs J-AA-（D9,10,3+D5,7）was the most significant and the survival rate of infected mice increased to 67%,while the saline negative control group was 0%.The mechanism study showed that all these dimer analogs had the classical function of destroy the bacterial cell membrane,and we also found that they could induce a large amount of reactive oxygen species（Ros）produced inside the bacteria to cause molecular damage.In addition,circular dichroism analysis showed that all these dimer analogs could formα-helical structure in the environment of bacteria membrane.In the third part of this study,the combined antimicrobial effect of ursolic acid and a dimer analog J-AA-（D9,10,3+D5,7）were discussed.The results showed that ursolic acid was sensitive to Gram-positive bacteria and had no antimicrobial activity against Gram-negative bacteria.The antibacterial mechanism showed that ursolic acid had the same ability to destroy bacterial cell membrane as antimicrobial peptides.The results showed that the ursolic acid combined with dimer analog J-AA-（D9,10,3+D5,7）had additivity effect against Staphylococcus aureus（ATCC25923）,but had no effect against Bacillus subtilis（ATCC 23857）.The combined drug group had rapid bactericidal characteristics.The PI flow cytometry was used to quantitatively analyze the fluorescence intensity of the combinational group,which proved that the fluorescence intensity of the combinational group was the strongest,compared with each group alone.The results were consistent with the additivity effect of the combinational group against Staphylococcus aureus.The aim of this paper is to design and synthesize new antimicrobial peptide analogs with new strategies.A novel group of antimicrobial peptide dimer analogs containing non-natural D-amino acid modification was designed and the antibacterial activity of them was significantly enhanced.It is hoped that this study will provide new ideas and methods for structural modification of natural antimicrobial peptides in the future.