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

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 (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (kM ) and maximal velocity (Vmax ) 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 kM 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.

A high-throughput screening assay for mutant isocitrate dehydrogenase 1 using acoustic droplet ejection mass spectrometry.

Acoustic droplet ejection mass spectrometry (ADE-MS) has recently emerged as a promising label-free, MS-based readout method for high throughput screening (HTS) campaigns in early pharmaceutical drug discovery, since it enables high-speed analysis directly from 384- or 1536-well plates. In this manuscript we describe our characterization of an ADE-MS based high sample content enzymatic assay for mutant isocitrate dehydrogenase 1 (IDH1) R132H with a strong focus on assay development. IDH1 R132H has become a very attractive therapeutic target in the field of antitumor drug discovery, and several pharmaceutical companies have attempted to develop novel small molecule inhibitors against mutant IDH1. With the development of an mIDH1 ADE-MS based HTS assay and a detailed comparison of this new readout technique to the commonly used fluorescence intensity mIDH1 assay, we demonstrated good correlation of both methods and were able to identify new potent inhibitors of mIDH1.

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds.

Classical high-throughput screening (HTS) technologies for the analysis of ionic currents across biological membranes can be performed using fluorescence-based, radioactive, and mass spectrometry (MS)-based uptake assays. These assays provide rapid results for pharmacological HTS, but the underlying, indirect analytical character of these assays can be linked to high false-positive hit rates. Thus, orthogonal and secondary assays using more biological target-based technologies are indispensable for further compound validation and optimization. Direct assay technologies for transporter proteins are electrophysiology-based, but are also complex, time-consuming, and not well applicable for automated profiling purposes. In contrast to conventional patch clamp systems, solid supported membrane (SSM)-based electrophysiology is a sensitive, membrane-based method for transporter analysis, and current technical developments target the demand for automated, accelerated, and sensitive assays for transporter-directed compound screening. In this study, the suitability of the SSM-based technique for pharmacological compound identification and optimization was evaluated performing cell-free SSM-based measurements with the electrogenic amino acid transporter B0AT1 (SLC6A19). Electrophysiological characterization of leucine-induced currents demonstrated that the observed signals were specific to B0AT1. Moreover, B0AT1-dependent responses were successfully inhibited using an established in-house tool compound. Evaluation of current stability and data reproducibility verified the robustness and reliability of the applied assay. Active compounds from primary screens of large compound libraries were validated, and false-positive hits were identified. These results clearly demonstrate the suitability of the SSM-based technique as a direct electrophysiological method for rapid and automated identification of small molecules that can inhibit B0AT1 activity.

Triantennary GalNAc Molecular Imaging Probes for Monitoring Hepatocyte Function in a Rat Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can lead to irreversible liver cirrhosis and cancer. Early diagnosis of NASH is vital to detect disease before it becomes life-threatening, yet noninvasively differentiating NASH from simple steatosis is challenging. Herein, bifunctional probes have been developed that target the hepatocyte-specific asialoglycoprotein receptor (ASGPR), the expression of which decreases during NASH progression. The results show that the probes allow longitudinal, noninvasive monitoring of ASGPR levels by positron emission tomography in the newly developed rat model of NASH. The probes open new possibilities for research into early diagnosis of NASH and development of drugs to slow or reverse its progression.

Functional Studies of Sodium Channels: From Target to Compound Identification.

Over the last six decades, voltage-gated sodium (Nav ) channels have attracted a great deal of scientific and pharmaceutical interest, driving fundamental advances in both biology and technology. The structure and physiological function of these channels have been extensively studied; clinical and genetic data have uncovered their implication in diseases such as epilepsy, arrhythmias, and pain, bringing them into focus as current and future drug targets. While different techniques have been established to record the activity of Nav channels, proper determination of their properties still presents serious challenges, depending upon the experimental conditions and the desired subtype of channel to be characterized. The aim of this unit is to review the characteristics of Nav channels, their properties, the cells in which they can be studied, and the currently available techniques. Topics covered include the determination of Nav -channel biophysical properties as well as the use of toxins to discriminate between subtypes using electrophysiological or optical methods. Perspectives on the development of high-throughput screening assays with their advantages and limitations are also discussed to allow a better understanding of the challenges encountered in voltage-gated sodium channel preclinical drug discovery. © 2016 by John Wiley & Sons, Inc.

Measuring interference of drug-like molecules with the respiratory chain: toward the early identification of mitochondrial uncouplers in lead finding.

The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I-V proteins in the ETC. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts.

Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids.

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.

Establishment of cell-free electrophysiology for ion transporters: application for pharmacological profiling.

Ion transporters are emerging targets of increasing importance to the pharmaceutical industry because of their relevance to a wide range of numerous indications of cardiovascular, metabolic, and inflammatory diseases. However, traditional ion transporter assay technologies using radioactive or fluorescent ligands and substrates or manual patch clamping suffer from several problems: limited sensitivity and robustness, significant numbers of false positives and false negatives, and cost. The authors describe a novel method for the measurement of ion transporters using cell-free electrophysiology based on the SURFE (2) R (surface electrogenic event reader) technology platform. The main advantages of the method described here are high sensitivity and simple handling. Material for assays is mainly a simple membrane preparation, which can be stored over weeks and months. Thus, the application of the method does not depend on a permanently running cell-culture lab. The application of the technology itself uses a bench-top system and chips loaded with membrane fragments. The SURFE (2) R technology was used to establish an Na+/Ca2+-exchanger assay. The assay performance, as judged by the Z' value of 0.73 and the signal-to-background ratio of 7.6, suggests that this is a reliable and robust assay. The authors compared the technology with patch-clamp experiments: The measurement of activity of 17 different inhibitors and the determination of an IC (50)value indicated a good correlation between SURFE (2) R technology and patch clamp results. Using the SURFE (2) R technology, results were obtained with 20 times higher throughput and required less-qualified personnel compared with manual patch clamping.

Inhibition of Na+-H+ exchange by cariporide reduces inflammation and heart failure in rabbits with myocardial infarction.

The aim of this study was to assess the effects of the Na+-H+ exchange inhibitor cariporide on left ventricular (LV) morphology and function as well as inflammation in rabbits with heart failure. Rabbits with myocardial infarction (MI) and sham controls were randomized to receive either standard chow or chow supplemented with cariporide for 9 weeks. LV morphology was determined by echocardiography. LV systolic and diastolic function was assessed under load-dependent and -independent conditions by analysis of LV pressure-volume loops using piezo-electric crystals. Plasma concentrations of C-reactive protein and aldosterone were measured. Rabbits with MI developed LV dilatation that was reduced by cariporide. Systolic and diastolic LV function was impaired in rabbits with MI when compared to sham, as indicated by a decreased dP/dtmax (MI: 3537 +/- 718 mmHg s(-1), sham: 5839 +/- 247 mmHg s(-1), P < 0.05), the load-independent preload recruitable stroke work (PRSW)(MI: 22 +/-7 mmHg, sham: 81 +/- 23 mmHg, P < 0.05) and a reduction in the time constant of relaxation tau (tau) (MI: 27+/-1 ms, sham: 17+/-1 ms, P < 0.05), and significantly improved by cariporide (dP/dtmax: 4586 +/- 374 mmHg s(-1), PRSW: 67 +/- 18 mmHg, tau: 20 +/- 2 ms; P < 0.05 vs MI/control). Induction of MI was associated with an increase in aldosterone and CRP, indicating activation of the neurohormonal and the inflammatory system that were largely reduced by cariporide. Cariporide improves LV morphology and function post MI and suppresses inflammation and neurohormonal activation in congestive heart failure (CHF). Na+-H+ exchange inhibition may represent a new pharmaceutical approach for the treatment of CHF.

Inhibition of human TREK-1 channels by bupivacaine.

UNLABELLED: Human TWIK-related K(+) channels (TREK-1) stabilize the membrane potential (mp) of neurons and have a major role in the regulation of membrane excitability. In view of their physiological significance, interaction of bupivacaine with TREK-1 channels may be clinically important. Our aim was to characterize with the patch-clamp technique the properties of human TREK-1 channels and the effects of bupivacaine on these channels expressed in Chinese hamster ovary (CHO) cells. Transfection of CHO cells with TREK-1 channels (CHO(TREK-1) cells) hyperpolarized the mp from -33 +/- 13 to -78 +/- 4 mV. The channels were stimulated by intracellular acidosis. Inhibition of TREK-1 channels by bupivacaine was reversible, concentration-dependent, voltage-independent, and increased with intracellular acidosis. Bupivacaine depolarized the mp of CHO(TREK-1) cells in a reversible and concentration-dependent manner. Concentrations for channel inhibition and membrane depolarization were not linearly related (50% inhibitory concentration value for channel inhibition 370 +/- 20 micro M, Hill coefficient 1.8 +/- 0.1, n = 51; 50% inhibitory concentration value for membrane depolarization 856 +/- 14 micro M, Hill coefficient 2.4 +/- 0.1, mean +/- SEM, n = 27). The results suggest that protonated bupivacaine elicits the observed effects via a site of interaction accessible from the intracellular space. Inhibition of TREK-1 channels and consecutive depolarization of the cell membrane by bupivacaine may contribute to blockade of neuronal signal conduction during regional anesthesia. IMPLICATIONS: The interaction of bupivacaine with human TREK-1 channels was studied with the patch-clamp technique. Bupivacaine inhibited TREK-1 channels and depolarized the membrane potential of cells expressing TREK-1 channels in a concentration-dependent and reversible manner. Both effects may contribute to conductance block caused by bupivacaine.