Stapled Modification And Activity Study Of Antimicrobial Peptides Feleucin-K3 And CPF-C1

The long-term and large-scale abuse of antibiotics leads to widespread bacterial drug resistance.At present,bacterial drug resistance has become a serious public health problem,threatening human life and health and bringing huge social and economic burden.However,the research and development of antibiotics is obviously lagging behind,and the infection caused by drug-resistant bacteria lacks effective means of treatment,so it is urgent to develop new antibacterial drugs.Antimicrobial peptides not only have the advantages of peptide drugs,but also become a hot research and development in the field of antibacterial because of their unique mechanism of action,good antibacterial activity,rapid bactericidal action and low drug resistance tendency,and have a good application prospect.Feleucin-K3（FLKLLKLL-NH₂）and CPF-C1（GFGSLLGKALRLGANVL-NH₂）are two natural antibacterial peptides isolated from amphibian skin secretions.Both of them are amphiphilic cationic polypeptides withα-helical conformation,and have broad-spectrum antibacterial activity.It also shows good antibacterial activity to clinically isolated drug-resistant strains,and is not easy to develop drug resistance,which is a good research template with the potential of patent medicine.However,as natural linear peptides,Feleucin-K3 and CPF-C1 both showed poor stability and low bioavailability.In the previous study,the two antibacterial peptides were modified by substitution of natural amino acids,modification of non-natural amino acids and introduction of peptide-like small molecules respectively,and analogizes with improved antibacterial activity and stability were screened through biological activity detection.However,in order to develop these two antimicrobial peptides as lead antibacterial compounds,they still need to be optimized for medicinal properties,especially their stability needs to be further improved.Stapled peptide is a modification technique that forms a covalent bridge between the side chains of the peptide to stabilize theα-helical active conformation and protect the peptide chain from enzymatic hydrolysis.It has been proved to be an effective method to improve the stability of the peptide and improve the physiological activity of the peptide.Based on the purpose of improving the stability and activity of antibacterial peptides Feleucin-K3 and CPF-C1,this project applied the modification scheme of hydrocarbon stapling,introduced pentenyl amino acids and formed covalent Bridges by olefin complex decomposition reaction（RCM）to connect olefin chains outside the two polypeptide main chains,and scanned and bound the points of parent peptides.Two series of stapling analogs were synthesized,including 8Feleucin-K3 series analogs and 17 CPF-C1 series analogs.The physicochemical properties,antibacterial activity,hemolysis activity and stability of the analogs were tested after mass spectrometry characterization and purification analysis,and the structure-activity relationship was studied.The experimental results showed that after stapling modification,the content ofα-helix in the active conformation of the two analogs was significantly increased compared with the parent peptide,for example,the content of helix in Feleucin-K3analogue FK3-S4 was 95.19%（the content of helix in the parent peptide Feleucin-K3was 64.97%）.The helix content of CPF-S4 was 95.23%（the helix content of CPF-C1was 71.35%）.In the short Feleucin-K3 series,the hydrophobicity of most analogs decreased,while in the long CPF-C1 series,the hydrophobicity partly increased and partly decreased,which was related to different stapling sites.At the same time,the change of amphiphilia was also related to the stapling site,but there was no obvious rule.In terms of antibacterial activity,the antibacterial activity of most compounds in Feleucin-K3 stapling analogues was greatly improved,and the MIC of the four tested bacteria was 4μg/m L（the MIC of the parent peptide Feleucin-K3 was 8-32μg/m L）,but the antibacterial activity of a few analogues was decreased or lost.The antibacterial activity of CPF-C1 stapling analogs was not significantly changed compared with that of parent peptide.In terms of the stability of this subject,the experimental results showed that the stability of the two series of analogues was significantly improved after the stapling modification,and they still maintained their original antibacterial activity in physiological salt environment,and their half-life in serum was significantly higher than that of their parent peptides.The half-life of Feleucin-K3 analogue FK3-S2 is 40.28 h(T_1/2Feleucin-K3=3.53 h).The half-life of CPF-S9 was 49.75 h(T_1/2CPF-C1=1.43 h).The hemolytic activity of the two analogues increased to a certain extent.The structure-activity relationship analysis of the compounds confirmed that the Feleucin-K3 analogs could maintain good antibacterial activity by maintaining 4 positive charge numbers,and the stapling site could not cross the amphiphilic interface,otherwise the hydrophilic and hydrophobic balance would be broken and the activity would be lost.Because the sequences of the Feleucin-K3 series were short,the hemolytic activity is mainly affected by the significantly increased spiral degree after stapling,and the subsequent stapling should focus on adjusting the spiral degree to reduce the hemolytic activity.For CPF-C1series analotes with long sequences,it is speculated that the 9th and 13th sites may be active sites according to the scanning results of stapling sites,which will affect the antibacterial activity.At the same time,since hydrophobic and neutral amino acid residues account for the majority of their sequence composition,the polypeptide is more hydrophobic as a whole.Therefore,the decrease of charge number,the increase of helical degree and the extension of hydrophobic interface have little influence on the antibacterial activity,but relatively great influence on the hemolytic activity.The subsequent stapling needs to introduce hydrophilic amino acid moderately to adjust the amphiphilic ratio and increase the charge number to improve the antibacterial activity,and then affect the change of helical degree and hydrophobic interface to reduce the hemolytic activity.Due to the introduction of unnatural amino acids and stable helical structure,the stability of stapling analogues in both series was significantly improved.In summary,by scanning and stapling at various points of Feleucin-K3 and CPF-C1,a total of 25 analogs in two series were synthesized in this study.Moreover,physical and chemical properties,antibacterial activity,hemolytic activity and stability were detected and structure-activity relationship analysis was conducted.Further in-depth and accurate stapling modification provided theoretical and experimental references.Access to new antimicrobial drugs provides a research basis.