Solid‐Supported Membrane (SSM)‐Based Electrophysiology Assays Using Surface Electrogenic Event Reader Technology (SURFE²R) in Early Drug Discovery

Abstract

This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE 2 R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE 2 R 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 cellline-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 SURFE 2 R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC 50 or IC 50 values, respectively) and kinetic functional assays to calculate apparent affinity (k M ) and maximal velocity (V max ) of substrate uptake.

Advancing drug discovery through assay development: a survey of tool compounds within the human solute carrier superfamily

“…Generally, we encountered several obstacles for cell-based SLC assay development, such as the difficulty to develop an assay in the absence of known substrates for the target transporter, electroneutrality of the target, or poor plasma membrane localization, among others ( Dvorak et al, 2021 ). On the other hand, in vitro assays for SLCs require purified protein embedded in liposomes ( Johnson and Lee, 2015 ; Drew et al, 2021 ) or membrane preparations ( Bazzone and Barthmes, 2020 ; Bazzone et al, 2022 ; Pommereau et al, 2023 ). Although RESOLUTE has developed robust methods to express and purify SLCs for these purposes ( Raturi et al, 2023 ), these approaches have a higher cost and labor requirement than cell-based approaches and require specialized know-how.…”

“…Three years after the publication of the review, the consortium partners have deployed and further developed several of the presented assay technologies. We have set-up 119 assays for 74 SLCs, and some of these assays have been published in original research articles ( Sijben et al, 2021a ; Sijben et al, 2021b ; Sijben et al, 2022a ; Mocking et al, 2022 ; Bongers et al, 2023 ; Pommereau et al, 2023 ) or in the open-access repositories Zenodo, PubChem, and ChEMBL.…”

A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development

“…However, to date, around 150 peer-reviewed publications incorporating SSME recordings have been published, with several of them investigating drug transport [ 50 , 63 ] or how compounds modulate the activities of channels, transporters, and ion pumps [ 36 , 64 , 65 ]. Sanofi recently published detailed protocols on the utilization of SSME in early drug discovery [ 35 ]. To conclude, APC, SSME, and LPC serve as complementary technologies due to their distinct capabilities and advantages, with SSME filling a gap, enabling electrophysiological investigation with decent throughput on non-treated lysosomes without the requirement of a running cell culture.…”

“…(4) In contrast to LPC, SSME recordings are HTS-compatible. The SSME platforms used in this study, SURFE 2 R N1 and SURFE 2 R 96SE (Nanion Technologies), offer a data throughput ranging from 150 data points per day to a mid-range of secondary HTS (5-10,000 data points per day), respectively, and are being used for transporter drug screening purposes [35,36].…”