Sanofi, Integrated Drug Discovery, Industriepark Hoechst, Frankfurt am Main, Germany.
Curr Protoc. 2023 Mar;3(3):e650. doi: 10.1002/cpz1.650.
This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE²R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE²R allows the use of the standard 96-well format, making it an ideal platform for tertiary assays in a drug-discovery campaign. The assays are performed with cell-line-derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE²R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC or IC values, respectively) and kinetic functional assays to calculate apparent affinity (k ) and maximal velocity (V ) of substrate uptake. © 2023 Sanofi. Current Protocols published by Wiley Periodicals LLC. PROTOCOL GROUP 1: Sample preparation for SSM-based electrophysiology assays Support Protocol 1: Production of cell batches Support Protocol 2: Simple isolation of cell membranes Alternate Protocol 1: Isolation of cell membranes with sucrose gradient pre-purification Support Protocol 3: Production and isolation of liposomes Support Protocol 4: Preparation of sensor with isolated cell membranes Alternate Protocol 2: Preparation of sensor with isolated proteoliposomes PROTOCOL GROUP 2: Determination of assay parameters for SSM-based electrophysiology assay Support Protocol 5: Assay with stable buffer Alternate Protocol 3: Assay with ion gradient Support Protocol 6: Determination of membrane/liposome concentration Support Protocol 7: Determination of substrate dependency k PROTOCOL GROUP 3: Determination of advanced assay parameters for SSM-based electrophysiology assays Support Protocol 8: Assessment of ion concentration dependency Support Protocol 9: Assessment of pH dependency Support Protocol 10: Assessment of DMSO dependency Support Protocol 11: Assessment of signal stability with multiple activations PROTOCOL GROUP 4: Compound testing through SSM-based electrophysiology assays using SURFE²R apparatus Support Protocol 12: Assessment of signal specificity of a published inhibitor or unknown compound(s) Support Protocol 13: Compound wash-out Support Protocol 14: Statistical analysis.
本文详细介绍了基于固体支撑膜 (SSM) 的电生理学测定法 (SURFE²R) 的操作程序和故障排除技巧，用于测量产生电的溶质载体转运蛋白 (SLC)，并评估调节其活性的化合物的作用。SURFE²R 允许使用标准的 96 孔格式，使其成为药物发现过程中三级测定的理想平台。该测定法使用来自细胞培养物的膜级分或含有感兴趣转运蛋白的蛋白脂囊泡进行。描述了三种主要方案来从细胞培养物中分离膜级分并生成含有感兴趣转运蛋白的蛋白脂囊泡。此外，还包括 SURFE²R 单浓度和剂量反应实验的详细方案，以测量测试化合物刺激或抑制转运蛋白功能的效力（分别为 EC 或 IC 值）和动力学功能测定法，以计算底物摄取的表观亲和力 (k) 和最大速度 (V)。 © 2023 赛诺菲。由 Wiley Periodicals LLC 出版的《当代协议》。第 1 组方案：基于 SSM 的电生理学测定法的样品制备 支持方案 1：细胞批次的生产 支持方案 2：简单的细胞膜分离 备选方案 1：用蔗糖梯度预纯化分离细胞膜 支持方案 3：脂质体的生产和分离 支持方案 4：用分离的细胞膜制备传感器 备选方案 2：用分离的蛋白脂囊泡制备传感器 第 2 组方案：基于 SSM 的电生理学测定法的测定参数确定 支持方案 5：稳定缓冲液的测定 备选方案 3：离子梯度的测定 支持方案 6：膜/脂囊泡浓度的测定 支持方案 7：底物依赖性 k 的测定 第 3 组方案：基于 SSM 的电生理学测定法的高级测定参数确定 支持方案 8：离子浓度依赖性的评估 支持方案 9：pH 值依赖性的评估 支持方案 10：DMSO 依赖性的评估 支持方案 11：多次激活时信号稳定性的评估 第 4 组方案：使用 SURFE²R 仪器通过基于 SSM 的电生理学测定法进行化合物测试 支持方案 12：评估已发表抑制剂或未知化合物 (s) 的信号特异性 支持方案 13：化合物洗脱 支持方案 14：统计分析。
