Department of Chemistry, Graduate School of Science , Osaka University , 1-1 Machikaneyama , Toyonaka , Osaka 560-0043 , Japan.
JST-ERATO Lipid Active Structure Project, Graduate School of Science , Osaka University , 1-1 Machikaneyama , Toyonaka , Osaka 560-0043 , Japan.
Biochemistry. 2019 Apr 30;58(17):2282-2291. doi: 10.1021/acs.biochem.9b00180. Epub 2019 Apr 18.
The clinically important antibiotic amphotericin B (AmB) is a membrane-active natural product that targets membrane sterol. The antimicrobial activity of AmB is generally attributed to its membrane permeabilization, which occurs when a pore is formed across a lipid bilayer. In this study, the molecular orientation of AmB was investigated using solid-state nuclear magnetic resonance (NMR) to better understand the mechanism of antifungal activity. The methyl ester of AmB (AME) labeled with NMR isotopes, d-AME, and its fluorinated and/or C-labeled derivatives were prepared. All of the AmB derivatives showed similar membrane-disrupting activities and ultraviolet spectra in phospholipid liposomes, suggesting that their molecular assemblies in membranes closely mimic those of AmB. Solid-state H NMR measurements of d-AME in a hydrated membrane showed that the mobility of AME molecules depends on concentration and temperature. At a 1:5:45 AME:Erg:dimyristoylphosphatidylcholine ratio, AME became sufficiently mobilized to observe the motional averaging of quadrupole coupling. On the basis of the rotational averaging effect of F chemical shift anisotropy, H quadrupolar splitting, and C-F dipolar coupling of 14β-F-AMEs, we deduced that the molecular axis of AME is predominantly parallel to the normal of a lipid bilayer. This result supports the barrel-stave model as a molecular assembly of AmB in membranes.
临床上重要的抗生素两性霉素 B（AmB）是一种靶向膜甾醇的膜活性天然产物。AmB 的抗菌活性通常归因于其跨脂双层形成孔时的膜通透性，在本研究中，使用固态核磁共振（NMR）研究了 AmB 的分子取向，以更好地理解其抗真菌活性的机制。用 NMR 同位素标记了两性霉素 B 的甲酯（AME），即 d-AME，及其氟化和/或 C 标记衍生物。所有 AmB 衍生物在磷脂脂质体中均显示出相似的膜破坏活性和紫外光谱，表明它们在膜中的分子组装与 AmB 非常相似。在水合膜中对 d-AME 的固态 H NMR 测量表明，AME 分子的流动性取决于浓度和温度。在 AME:Erg:dimyristoylphosphatidylcholine 的比例为 1:5:45 时，AME 变得足够移动，以观察到四极耦合的运动平均。基于 14β-F-AMEs 的 F 化学位移各向异性、H 四极分裂和 C-F 偶极耦合的旋转平均效应，我们推断 AME 的分子轴主要平行于脂质双层的法线。这一结果支持桶形棒模型作为 AmB 在膜中的分子组装。
