Effect of N-methylated and fatty acid conjugation on analogs of antimicrobial peptide Anoplin

With the increment of drug-resistant bacteria and the slow development of novel antibiotics, antimicrobial peptides have gained increasing attention as a potential antibiotic alternative. They not only displayed a broad-spectrum antimicrobial activity but also were difficult to induce resistance development because of their unique membrane-lytic activity. Herein, 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). Afterward, the N -methylated analogs M3.6/M4.7/M5.7 with high stability were screened out and further modified by N-terminal fatty acid conjugation to develop new antimicrobial peptide analogs with both potent antimicrobial activity and high proteolytic stability, and 12 new Anoplin analogs Cn-M3.6/M4.7/M5.7 (n = 8,10,12,14) were designed and synthesized. Our results showed that compared with native Anoplin, the stability of these N-methylated lipopeptides against trypsin and chymotrypsin degradation were increased by 10(4)-10(6) times. Besides, they still possessed potent antimicrobial activity under physiological salts and serum environment. Among them, the new designed analogs C12-M3.6/M4.7/M5.7 showed the optimal antimicrobial activity, synergy and additive effects were also observed when they were combined with traditional antibiotics polymyxin B, rifampin, and kana-mycin. Moreover, they could effectively inhibit the formation of biofilms by P. aeruginosa and S. aureus. The antimicrobial mechanism studied revealed that these N-methylated lipopeptides could display a rapid bacter-icidal effect by destroying the bacterial cell membrane. Notably, no detectable resistance of these new designed peptides was developed after continuous cultured with E. coli for 20 passages. 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 conjugation. This study also provides new ideas and methods for the modification of antimicrobial peptides in the future.