Liu Tianqi, Zhu Ningyi, Zhong Chao, Zhu Yuewen, Gou Sanhu, Chang Linlin, Bao Hexin, Liu Hui, Zhang Yun, Ni Jingman
School of Pharmacy, Lanzhou University, Lanzhou 730000, China.
School of Pharmacy, Lanzhou University, Lanzhou 730000, China; Key Laboratory of Preclinical Study for New Drugs of Gansu Province School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China.
Eur J Pharm Sci. 2020 Sep 1;152:105453. doi: 10.1016/j.ejps.2020.105453. Epub 2020 Jul 7.
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 10-10 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 kanamycin. 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 bactericidal 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.
随着耐药菌的增加以及新型抗生素研发缓慢，抗菌肽作为一种潜在的抗生素替代品受到了越来越多的关注。它们不仅具有广谱抗菌活性，而且由于其独特的膜裂解活性，难以诱导耐药性的产生。在此，为了改善Anoplin的局限性，首先使用N-甲基氨基酸替换Anoplin在敏感酶切位点（亮氨酸、异亮氨酸、赖氨酸和精氨酸）的氨基酸。随后，筛选出具有高稳定性的N-甲基化类似物M3.6/M4.7/M5.7，并通过N端脂肪酸偶联进行进一步修饰，以开发具有强大抗菌活性和高蛋白水解稳定性的新型抗菌肽类似物，设计并合成了12种新的Anoplin类似物Cn-M3.6/M4.7/M5.7（n = 8、10、12、14）。我们的结果表明，与天然Anoplin相比，这些N-甲基化脂肽对胰蛋白酶和糜蛋白酶降解的稳定性提高了10 - 100倍。此外，它们在生理盐溶液和血清环境下仍具有强大的抗菌活性。其中，新设计的类似物C12-M3.6/M4.7/M5.7表现出最佳抗菌活性，当它们与传统抗生素多粘菌素B、利福平和卡那霉素联合使用时，还观察到协同和相加作用。此外，它们可以有效抑制铜绿假单胞菌和金黄色葡萄球菌生物膜的形成。对抗菌机制的研究表明，这些N-甲基化脂肽可以通过破坏细菌细胞膜发挥快速杀菌作用。值得注意的是，与大肠杆菌连续培养20代后，这些新设计的肽未检测到耐药性。综上所述，我们通过N-甲基氨基酸取代和N端脂肪酸偶联设计了一类具有强大抗菌活性和高蛋白水解稳定性的新型抗菌肽类似物。本研究也为未来抗菌肽的修饰提供了新的思路和方法。
