当前位置: X-MOL 学术bioRxiv. Biophys. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes
bioRxiv - Biophysics Pub Date : 2021-09-16 , DOI: 10.1101/2021.09.16.460640
Tim Kaminski , Vladimir P. Zhdanov , Fredrik Höök

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:

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。
更新日期:2021-09-19
down
wechat
bug