UV, CD, and NMR studies of Amphotericin B in nanoscale discoidal lipid bilayers

纳米级盘状脂质双层中两性霉素 B 的 UV、CD 和 NMR 研究

• 批准号: 8436306
• 负责人: Thomas Michael Anderson
• 金额: $ 4.69万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-16 至 2014-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436306
• 关键词: Affinity; Amphotericin B; Antifungal Agents; Binding; Carbon; Cell membrane; Chemicals; Cholesterol; Complex; Dependence; Development; Dimerization; Effectiveness; Environment; Ergosterol; Health; High Pressure Liquid Chromatography; Human; Ion Channel; Kidney; Length; Lighting; Lipid Bilayers; Lipids; Liposomes; Measures; Membrane; Membrane Proteins; Molecular; Monitor; Nephrotoxic; Pharmacologic Substance; Preparation; Research; Resolution; Specificity; Sterols; Structural Protein; Structure; System; Tail; Therapeutic Index; Toxic effect; Yeasts; base; design; hemiketal; improved; intermolecular interaction; membrane model; mycosamine; nanodisk; nanoscale; physical state; preference; protein structure; research study; solid state nuclear magnetic resonance; stoichiometry; success

DESCRIPTION (provided by applicant): The clinically vital, but severely nephrotoxic, antifungal Amphotericin B has a unique mechanism of action; rather than bind to a macromolecular target, it self-assembles into membrane ion channels in yeast membranes. The effectiveness of AmB arises from its affinity for ergosterol in yeast membranes. However, AmB is severely nephrotoxic due to a competing affinity for cholesterol in human cell membranes. Lack of a suitable model membrane for studying AmB has resulted directly in a lack of detailed molecular understanding of its sterol specificity, thus severely limiting the design of more effective and /or less nephrotoxic derivatives. The typical model membrane employed is the liposome, however, AmB forms extremely large "hyper-aggregates" in liposomes. Interestingly, analysis of membrane protein structure has also been limited by tendency for aggregation and poor model membranes. Recently, nanoscale discoidal lipid bilayers (nanodiscs) have proven to be effective model membranes for studying structure of monomeric membrane proteins. We propose to harness the advantages of the nanodisc to analyze differences in structure and stoichiometry of the AmB/cholesterol and AmB/ergosterol channels. Quantitative analysis and UV and CD spectroscopic analysis will determine the AmB/nanodisc ratio in the presence of each sterol. UV and CD spectra of AmB will serve as a probe of the physical state of the incorporated AmB. Solid state NMR experiments (SSNMR) will be used to determine the channel length preference for cholesterol and ergosterol. ISCSIP rotational echo double resonance SSNMR will identify those AmB carbon atoms interacting with P atoms of the lipid. Finally, SSNMR analysis of AmB in the nanodisc will allow determination of specific atoms involved in AmB/ergosterol and AmB/cholesterol binding, as measured by changes in 13C chemical shift of the AmB carbons upon binding the sterol. Collectively, these studies will illuminate differences in the cholesterol and ergosterol AmB channel complexes which will serve as a starting point for the rational design of more effective, less nephrotoxic AmB derivatives.

描述（由申请人提供）：临床上至关重要但严重的肾毒性，抗真菌性两性霉素B具有独特的作用机理；它没有与大分子靶标结合，而是将自组合成酵母膜中的膜离子通道。 AMB的有效性源于其对酵母膜中麦角固醇的亲和力。但是，由于人类细胞膜中胆固醇的竞争亲和力，AMB具有严重的肾毒性。缺乏用于研究AMB的合适模型膜直接导致缺乏对其固醇特异性的详细分子理解，从而严重限制了更有效和 /或更少的肾毒性衍生物的设计。使用的典型模型膜是脂质体，但是，AMB在脂质体中形成了极大的“超聚集”。有趣的是，膜蛋白结构的分析也受到聚集和模型膜差的趋势的限制。最近，被证明是研究单体膜蛋白结构的有效模型膜。我们建议利用纳米盘的优势分析AMB/胆固醇和AMB/Ergosterol通道的结构和化学计量的差异。定量分析以及紫外线和CD光谱分析将在每个固醇存在下确定AMB/Nanodisc的比率。 AMB的紫外线和CD光谱将作为对综合AMB的物理状态的探测。固态NMR实验（SSNMR）将用于确定胆固醇和麦角固醇的通道长度偏好。 ISCSIP旋转回声双共振SSNMR将识别与脂质P原子相互作用的AMB碳原子。最后，纳米盘中AM的SSNMR分析将允许确定AMB/麦角固醇和AMB/胆固醇结合所涉及的特定原子，如通过结合固醇时AMB碳的13C化学移位所测量的。总的来说，这些研究将阐明胆固醇和麦角固醇AMB通道复合物的差异，这将成为合理设计更有效，较少肾毒性AMB衍生剂的起点。