由於抗生素具有容易讓細菌產生抗藥性的缺點，應此具有化學和結構豐富性的抗菌胜肽 (AMP) 便成為下一代抗菌化合物的重要研究目標。然而當我們要從頭設計和優化新的 AMP 所面臨的關鍵挑戰之一便是對抗菌活性和細胞毒性的分子機制知之甚少。在這裡，我們利用分子動力學 (MD) 模擬以及傘型採樣 (US)的方式來模擬兩種具有廣譜體外活性的 AMP (arenicin-3 和 AA139) 在穿膜過程的反應，並提出了一種可設計的 AMP 的作用機制來提升其抗菌活性和降低其溶血性。
當AMP在靠近帶較多陰離子的細菌細胞膜膜時，AMP便會 “開啟”它們的抗菌活性模式：AMP 的陽離子殘基和細菌膜的脂質之間形成了顯著的鹽橋，從而降低了它們的抗菌胜肽內部的陽離子-π相互作用。因此，芳香族殘基就更加容易與脂質之間形成陽離子-π作用，利用這個作用力讓AMP在穿膜的過程可以彎曲脂質的頭部基團以形成環形孔，從而提升抗菌活性。另外我們也觀察到AMP會與脂質頭部基團結合形成穩定的π - 陽離子（膽鹼）- π基序，其在結構和能量上都非常穩定。另一方面，在帶中性電荷的真核細胞質膜中，AMP則傾向 “關閉”它們的活性模式：AMP 的陽離子殘基和真核細胞膜的中性電荷脂質之間形成的鹽橋要少得多，因此顯著的抗菌胜肽內形成陽離子-π對。在這種模式下，AMP 與帶中性電荷的脂質膜的相互作用要少得許多，這在能量上不利於它的穿膜反應，從而降低它們的溶血性。我們認為我們所提出的這種新的AMP 作用機制是有助於未來設計和優化具有低毒性且強效殺菌力的 AMP 的發展。

With the failure of antibiotics, the discovery of next-generation antibacterial compounds based on chemically and structurally abundant antimicrobial peptides (AMPs) is promising. One of the crucial challenges for systemic rational design and optimization of de novo AMPs is that molecular mechanisms of antimicrobial activity and cytotoxicity are poorly understood. Here, we present designable action modes of AMPs for their antimicrobial activity and cytotoxicity derived from the molecular dynamics (MD) simulations in conjunction with umbrella sampling (US) for the membrane translocation of two AMPs with broad-spectrum in vitro activity, arenicin-3 and AA139. We show inside the anionic bacterial membrane, cationic and aromatic AMPs “turn on” their antimicrobial active mode: Significant salt bridges between cationic residues of AMPs and lipids of the bacterial membrane are formed and thus unpair their intra-peptide cation- interactions. Consequently, aromatic residues form lipid-specific cation- pairs, which can bend the head groups of lipids for forming toroidal pores and thus promote antimicrobial activity. A stablecation(choline) motif binding the head group of lipid, which is structurally and energetically suitable for being lipid head groups is observed. On the other hand, within the neutrally charged eukaryotic plasma membranes, cationic and aromatic AMPs “turn off” their active mode: much fewer salt bridges between cationic residues of AMPs and neutrally charged lipids of eukaryotic membranes are formed, and consequently significant intra-peptide cation- pairs are formed. In this mode, AMPs have much fewer interactions with a neutrally-charged lipid membrane, which is energetically unfavorable for their membrane translocation and thus reduces their cytotoxicity. Our proposed action mechanisms of AMPs give us comprehensible guides for rational design and optimization of potent AMPs with low toxicity.

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