High throughput screening technologies for ion channels

Medicilon provides a wide range of ion channel assays to quickly and accurately provide results for drug discovery and development research worldwide. We offer ion channel screening services on a variety of different high quality electrophysiology platforms. Email:marketing@medicilon.com.cn web:www.medicilon.com

Introduction Ion channels are a very important membrane protein family involved in a variety of fundamental physiological processes. Their malfunction causes a variety of human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. Traditionally, patch clamp electrophysiology is the gold standard for ion channel studies. However, the method is labor-intensive with a low throughput and requires highly trained staff to perform the experiments. Ion channels are difficult targets to be investigated using high throughput approaches, which hinders the use of ion channels compared with other targets. Recently, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Consequently, HTS was designed to identify active compounds for ion channel targets, which are of great interest to academic and industrial researchers. The methodologies for studying ion channels can be divided into non-electrophysiological and electrophysiological methods. This review will summarize the current technologies and commonly used screening methods for different ion channel classes. High throughput screening technologies In the past, HTS methods for ion channels have been extensively developed and applied to most ion channels. In chronological order, the approaches include: the ligand binding assay, flux-based assay, fluorescence-based assay and automated electrophysiological assay. Ligand binding assays Ligand binding assays have been widely used to screen for ion channel modulators. However, these assays are not considered as functional assays because they detect the binding affinity of a compound to an ion channel rather than the ability of altering channel function. Ligand binding assays require a previous knowledge of the target binding sites and of the formation of a radio-labeled ligand which is specific to those binding sites. Activity of the test compound is indicated by the displacement of the labeled ligand. Consequently, conventional instrumentation may be used, in which throughput represents its major strength. Because the method only discovers compounds that influence radioligand binding, it misses allosteric modulators of ion channels. Binding assays identify affinity data but do not identify the functional change of ion channels. For example, an agonist cannot be distinguished from an antagonist in a binding assay. Secondary assays are necessary to determine if the compound is an agonist, antagonist or neither. Furthermore, the scope of binding assay is limited by the availability and affinity of radio-labeled ligands. The sensitivity of a binding assay is often determined by the affinity of a known labeled ligand. A high-affinity ligand may not allow the detection of weak binders. However, the use of a low-affinity ligand can lead to an increased detection of nonspecific binding. When the ligand affinity is within a certain range, the IC50 values obtained from binding assays have a reasonable correlation with those obtained from patch clamping. Thus far, the assay format has been rarely used for general screening but is still good for identifying modulators specific to some given ligands. Then the cells and supernatant are collected for radioactive counting. However, radioactive-efflux assays suffer from the inconvenience and cost associated with the handling of radioactive materials. Additionally, it is necessary to use different radioisotopes for channels that are selective for different ions. Therefore, a nonradioactive Rb+ efflux assay was developed that uses atomic absorption spectroscopy to detect rubidium. The flux assay is a format preferred by many screening laboratories because it measures ionic flux that better correlates with the activity. This assay technology is widely applied in the pharmaceutical industry for both drug discovery and hERG-related drugsafety screening to identify potential QT liabilities that might cause lethal arrhythmias. However, these assays have the disadvantages of low temporal resolution (typically from seconds to minutes), uncontrolled membrane potential, less information content compared with voltage-clamping and lower throughput compared with fluorescence-based assays. Furthermore, this assay generates a very weak signal for some ion channels, which requires a high level of channel expression to achieve an acceptable signal-to-noise ratio. Fluorescence-based assays Fluorescence-based methods do not directly measure ionic current. Rather, they measure either the membrane-potentialdependent or ion-concentration-dependent changes of fluorescence signals as a result of ionic flux. Because fluorescencebased methods produce a robust and homogeneous cell population measurement, these assays are similar to those for other protein classes. Therefore, more instrument choices and expertise are available. Consequently, these assays are relatively easy to implement and to optimize to achieve a higher throughput. Voltage-sensitive dye assays Fluorescent voltage-sensitive dyes measure voltage changes across the cellular membrane using either the potential-dependent accumulation and redistribution or the fluorescence resonance energy transfer (FRET) mechanism. Oxonol derivatives, such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol, are lipophilic and negatively-charged dyes that display an increased quantum yield in an aqueous environment upon binding to hydrophobic intracellular molecules instead of weak fluorescence. Because the change in fluorescence occurs minutes after the change in membrane potential, these dyes are best suited to detect a steady state instead of kinetic changes of membrane potential. These dyes cannot detect second and sub-second resolution changes of membrane potential.