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

描述(由申请人提供)：临床上至关重要但严重肾毒性的抗真菌两性霉素B具有独特的作用机制；它不是结合到大分子靶标上，而是在酵母膜中自组装成膜离子通道。AMB的有效性来自于它对酵母膜中的麦角固醇的亲和力。然而，AMB具有严重的肾毒性，因为它与人细胞膜上的胆固醇有竞争性的亲和力。由于缺乏合适的模型膜来研究AMB，直接导致了对其甾醇专一性缺乏详细的分子理解，从而严重限制了设计更有效和/或肾毒性较小的衍生物。典型的模型膜是脂质体，然而，Amb在脂质体中形成了非常大的“超聚集体”。有趣的是，膜蛋白结构的分析也受到聚集倾向和糟糕的膜模型的限制。近年来，纳米级的盘状脂质双层(纳米盘)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/麦角甾醇通道在结构和化学计量方面的差异。定量分析以及UV和CD光谱分析将确定在每种甾醇存在下AMB/纳米盘的比率。AMB的UV和CD光谱将作为所结合AMB的物理状态的探针。固体核磁共振实验将被用来确定胆固醇和麦角甾醇的通道长度偏好。ISCSIP旋转回波双共振SS核磁共振将鉴定那些与脂质中的P原子相互作用的AMB碳原子。最后，对纳米盘中AMB的SS核磁共振分析将允许确定AMB/麦角甾醇和AMB/胆固醇结合所涉及的特定原子，这是通过AMB碳与甾醇结合时13C化学位移的变化来测量的。总而言之，这些研究将阐明胆固醇和麦角甾醇Amb通道复合体的差异，这将作为合理设计更有效、肾毒性较低的Amb衍生物的起点。公共卫生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。实验将探索两性霉素B与细胞膜和其中存在的甾醇的相互作用，以促进对AMB有效性和毒性的基本机制的理解。这些实验的结果将使制备更有效和/或毒性更低的两性霉素B衍生物成为可能。

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.