Screening for Small Molecules' Bilayer-Modifying Potential Using a Gramicidin-Based Fluorescence Assay
Screening for Small Molecules' Bilayer-Modifying Potential Using a Gramicidin-Based Fluorescence Assay
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DOI：
10.1089/adt.2009.0250
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发表时间：
2010-08-01
影响因子：
1.8
通讯作者：
Andersen, Olaf S.
中科院分区：
文献类型：
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作者：
Ingolfsson, Helgi I.;Andersen, Olaf S.
许多用于调节生物功能的药物和其他小分子都是两亲物，它们吸附在双层/溶液界面上，从而改变脂质双层特性。这很重要，因为膜蛋白通过疏水相互作用与其宿主双层能量耦合。因此，双层特性的变化改变了膜蛋白的功能，这为“脱靶”药物作用提供了可能的机制。我们之前已经证明，由线性短杆菌肽形成的通道是脂质双层特性变化的合适探针，正如双层跨越蛋白所经历的那样。我们现在报告了一种基于短杆菌肽的荧光测定法，用于检测双层特性的变化。该测定基于测量由于短杆菌肽通道可渗透猝灭剂的进入而导致负载荧光团的大单层囊泡中荧光猝灭的时间过程。该方法具有可扩展性，适用于机制研究和双层扰动、潜在脱靶效应的高通量筛选，我们使用辣椒素 (Cap) 和其他化合物对此进行了说明。
Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides a possible mechanism for "off-target" drug effects. We have previously shown that channels formed by the linear gramicidins are suitable probes for changes in lipid bilayer properties, as experienced by bilayer-spanning proteins. We now report a gramicidin-based fluorescence assay for changes in bilayer properties. The assay is based on measuring the time course of fluorescence quenching in fluorophore-loaded large unilamellar vesicles, due to entry of a gramicidin channel-permeable quencher. The method is scalable and suitable for both mechanistic studies and high-throughput screening for bilayer-perturbing, potential off-target effects, which we illustrate using capsaicin (Cap) and other compounds.