Design And The Structure-activity Relationship Study Of Analogs Of Anoplin By N-methyl Amino Acids Substitution And N-terminal Fatty Acid Modification

The emergence and growth of drug-resistant bacteria caused by the abuse of antibiotics has become a serious public health problem that threatens human health.As an important part of the innate immune system,antimicrobial peptides can effectively combat infections caused by multiple pathogens,including multi-drug resistant bacteria,through non-specific membrane disruption.And it is not easy for antimicrobial peptides to induce bacteria to develop drug resistance.Therefore,antimicrobial peptides are considered as a new type of antibacterial agent with great development potential.Anoplin?GLLKRIKTLL-NH₂?is an amphipathic?-helix antimicrobial peptide which was isolated from the venom of the solitary wasp.It has been shown to have broad-spectrum antimicrobial activity towards bacterial membrane and neglectable hemolysis activity towards mammalian membrane.However,Anoplin is susceptible to protease and its antibacterial effect is not ideal.In this study,in order to improve the limitations of Anoplin,the N-methyl amino acids were first used to replace the amino acids of Anoplin at sensitive enzymatic cleave sites?Leu,Ile,Lys and Arg?.And 14 new analogs of Anoplin with N-methyl amino acids substitution were designed and synthesized.The results of proteolytic stability study showed that the stability of the analogs after N-methyl amino acids substitution was significantly improved.Among them,compared with the parent peptide Anoplin,the resistance of analog M4.7 towards trypsin and chymotrypsin degradation was increased by 10⁶ and 10⁵ times.And the analog M5.7had increased resistance to trypsin and chymotrypsin degradation by 10⁶ and 10⁴times.Similarly,the stability of analog M3.6 to both proteases was increased 10⁵times.However,the results of antimicrobial activity study showed that the introduction of N-methyl amino acids have an adversely effect on the antimicrobial activity of antimicrobial peptides.And the antimicrobial activity of most of these new designed analogs against Gram-positive and Gram-negative bacteria was decreaed to different degrees.Studies on the secondary structure of all N-methyl amino acids substitution analogs showed that in the cell membrane mimic environment,the parent peptide Anoplin and the analog M2 could form?-helix structure,but other analogs were more inclined to form?-turn structure.In order to develop new antimicrobial peptide analogs with both potent antimicrobial activity and high proteolytic stability,we performed N-terminal fatty acid modification on the three N-methylated analogs M3.6,M4.7 and M5.7 screened in the above studies.And 12 new Anoplin analogs Cn-M3.6/M4.7/M5.7?n=8,10,12,14?with N-methyl amino acids substitution and N-terminal fatty acid modification were designed and synthesized.The results of antimicrobial activity study showed that the N-terminal fatty acid modification significantly improved the antimicrobial activity of the corresponding N-methyl amino acids replacement analogs.And the antimicrobial activity of the analogs had fatty acid chain length-dependent characteristics.Among them,the new analogs C12-M3.6/M4.7/M5.7 showed the optimal antimicrobial activity.Moreover,these analogs also exhibited good antimicrobial activity in physiological salt conditions and serum environment.The proteolytic stability study showed that all new analogs with N-methyl amino acids substitution and N-terminal fatty acid modification still retained high stability against proteolytic degradation.In addition,the results of the safety study showed that as the fatty acid chain length increased,the hemolytic activity of the analogs gradually increased.Circular dichroism study showed that these new analogs with N-methyl amino acids substitution and N-terminal fatty acid modification had secondary structures similar to those of N-methyl amino acids replacement template peptides.According to the results of antimicrobial activity and hemolytic activity studies,we selected the analogs C12-M3.6/M4.7/M5.7 for more in-depth activity and mechanism research.When these three analogs were used in combination with the traditional antibiotics rifampin,vancomycin,kanamycin and polymyxin B against S.aureus and E.coli,they showed a significant synergy and additive antibacterial effect.Besides,these new analogs could also effectively inhibit the formation of biofilms by S.aureus and P.aeruginosa.The antimicrobial mechanism study showed that the analogs C12-M3.6/M4.7/M5.7 could play a rapid bactericidal effect by destroying the bacterial cell membrane.In addition,the results of the induced resistance study showed that the analogs C12-M3.6/M4.7/M5.7 could hardly develop resistance after continuous cultured with E.coli for 20 passages compared with antibiotic rifampin.In summary,we have designed a new class of antimicrobial peptide analogs with potent antimicrobial activity and high proteolytic stability through N-methyl amino acids substitution and N-terminal fatty acid modification.This study also provides new ideas and methods for the modification of antimicrobial peptides in the future.