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: 8042702
• 负责人: Thomas Michael Anderson
• 金额: $ 4.64万
• 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/8042702
• 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. PUBLIC HEALTH RELEVANCE The proposed research will study the mechanism of action of the clinically vital, yet toxic, antifungal drug amphotericin B (AmB). Experiments will probe the interaction of amphotericin B with cell membranes and sterols present therein to advance understanding of the fundamental mechanism of AmB effectiveness and toxicity. Results of these experiments will enable preparation of amphotericin B derivatives that are more effective and/or less toxic.

描述（由申请人提供）：具有临床意义但具有严重肾毒性的抗真菌两性霉素 B 具有独特的作用机制；它不是与大分子靶标结合，而是自组装到酵母膜中的膜离子通道中。 AmB 的有效性源自其对酵母膜中麦角甾醇的亲和力。然而，由于与人体细胞膜中胆固醇的竞争亲和力，AmB 具有严重的肾毒性。缺乏适合研究 AmB 的模型膜直接导致对其甾醇特异性缺乏详细的分子了解，从而严重限制了更有效和/或肾毒性较小的衍生物的设计。所采用的典型模型膜是脂质体，然而，AmB 在脂质体中形成极大的“超聚集体”。有趣的是，膜蛋白结构的分析也受到聚集趋势和不良模型膜的限制。最近，纳米级盘状脂质双层（nanodiscs）已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来分析 AmB/胆固醇和 AmB/麦角甾醇通道的结构和化学计量差异。定量分析以及 UV 和 CD 光谱分析将确定每种甾醇存在下的 AmB/纳米圆盘比率。 AmB 的 UV 和 CD 光谱将作为掺入的 AmB 物理状态的探针。固态核磁共振实验 (SSNMR) 将用于确定胆固醇和麦角甾醇的通道长度偏好。 ICSIP 旋转回波双共振 SSNMR 将识别那些与脂质 P 原子相互作用的 AmB 碳原子。最后，纳米圆盘中 AmB 的 SSNMR 分析将能够确定参与 AmB/麦角甾醇和 AmB/胆固醇结合的特定原子，通过结合甾醇时 AmB 碳的 13C 化学位移的变化来测量。总的来说，这些研究将阐明胆固醇和麦角甾醇 AmB 通道复合物的差异，这将作为合理设计更有效、肾毒性更小的 AmB 衍生物的起点。公共健康相关性 拟议的研究将研究临床上重要但有毒的抗真菌药物两性霉素 B (AmB) 的作用机制。实验将探讨两性霉素 B 与细胞膜和其中存在的甾醇的相互作用，以加深对两性霉素 B 有效性和毒性的基本机制的理解。这些实验的结果将能够制备更有效和/或毒性更低的两性霉素B衍生物。