Kinetic profiling of drug–target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein–coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor (β₂AR). The assay involves temporally-resolved imaging of the binding of individual β₂AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of β₂AR and suspended alprenolol (antagonist) and fenoterol (agonist). The set-up combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

中文翻译：

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单分子动态溶液中抑制测定：一种对天然膜中与 GPCR 结合的候选药物进行完整动力学分析的方法

使用基于表面的无标记技术对药物-靶标相互作用的动力学分析对于水溶性药物靶标已经很好地建立，但对于一般的膜蛋白，尤其是 G 蛋白偶联受体 (GPCR)，很难执行。这是因为 GPCR 的表面固定往往会改变它们的配置和功能，导致目标覆盖率低和非特异性结合。我们在这里描述了一种新的分析方法，用于对药物与 GPCR 人 β 2 肾上腺素能受体 (β ₂ AR) 的结合进行动力学分析。该测定涉及个体 β ₂结合的时间分辨成像在筛选药物存在的情况下，将含有 AR 的细胞膜衍生脂质体转化为表面固定的配体。这种方法允许确定 β ₂ AR 和悬浮的阿普洛尔（拮抗剂）和非诺特罗（激动剂）的结合和解离常数。该设置将 384 孔板传感器芯片与自动液体处理相结合，检测只需几分钟即可完成，非常适合药物筛选活动。