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

性状（由申请方提供）：临床上至关重要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;它不与大分子靶点结合，而是自组装到酵母细胞膜的膜离子通道中。AmB的有效性源于其对酵母细胞膜中麦角固醇的亲和力。然而，AmB是严重的肾毒性，由于在人类细胞膜中的胆固醇的竞争性亲和力。缺乏一个合适的模型膜研究AmB直接导致缺乏详细的分子理解其甾醇特异性，从而严重限制了更有效和/或更少的肾毒性衍生物的设计。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模型膜差的限制。最近，纳米盘状脂质双层（纳米盘）已被证明是有效的模型膜用于研究单体膜蛋白的结构。我们建议利用nanodisc的优势来分析AmB/胆固醇和AmB/麦角固醇通道的结构和化学计量的差异。定量分析以及UV和CD光谱分析将确定每种固醇存在下的AmB/纳米盘比率。AmB的UV和CD光谱将用作掺入的AmB的物理状态的探针。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旋转回波双共振SSNMR将识别与脂质的P原子相互作用的那些AmB碳原子。最后，纳米盘中AmB的SSNMR分析将允许确定涉及AmB/麦角固醇和AmB/胆固醇结合的特定原子，如通过结合固醇时AmB碳的13 C化学位移的变化所测量的。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，这将作为合理设计更有效、肾毒性更低的AmB衍生物的起点。

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.