Ion channels are involved in various basic physiological processes, and their failure can lead to a variety of human diseases. Therefore, ion channels represent an attractive class of drug targets and an important class of off-target proteins, which can be used for in vitro pharmacological analysis. Over the past few decades, rapid advances in functional assays and instrument development have enabled high-throughput screening (HTS) activities in an ever-expanding list of channel types. In chronological order, HTS methods for ion channels include ligand binding assays, flux-based assays, fluorescence-based assays, and automated electrophysiological assays. Ligand binding assays Ligand binding assays have been widely used to screen ion channel modulators. However, these assays are not considered functional assays because they measure the binding affinity of the compound to the ion channel, rather than the ability to change the channel's function. Ligand binding assays require prior knowledge of target binding sites and the formation of radiolabeled ligands specific to these binding sites. The activity of the test compound is indicated by the displacement of the labeled ligand. Therefore, conventional instruments can be used, where throughput represents its main advantage. Because this method only finds that the compound affects radioligand binding, it ignores allosteric modulators of ion channels. The binding assay can identify affinity data, but cannot identify changes in ion channel function. Ion flux Ion flux measurement has been successfully applied to directly access functional changes in ion channel activity. Radioisotopes have been used to track the cell inflow or outflow of specific ions (such as 22 Na +, 45 Ca 2+ and 86 Rb +), and are used to study Na +, Ca 2+ and K + channels, respectively. The commonly used measurement format is 86 Rb + efflux of K + channel or non-selective cation channel. This detection technology is widely used in the pharmaceutical industry for two drug discovery and hERG-related drug safety screening to determine potential QT liabilities that may cause fatal arrhythmia. Fluorescenc Fluorescence-based methods cannot directly measure ion currents. They measure membrane potential-dependent or ion-concentration-dependent changes in fluorescence signals due to ion flux. Because fluorescence-based methods can produce reliable and uniform cell population measurements, these assays are similar to those of other protein classes. Therefore, more instrument options and expertise can be applied. These assays are relatively easy to implement and optimize to achieve higher throughput. Patch clamp Patch clamp has been widely regarded as the gold standard for direct recording of ion channel activity. This technology can provide high-quality and physiologically relevant data of ion channel functions at the single cell or single channel (in a small piece of membrane) level. For pharmacological testing of compounds, it provides a standard for measuring the potency of the interaction between the compound and the channel. Although the conventional patch clamp provides a direct, information-rich real-time method to study channel functions, it has very low throughput and labor-intensive characteristics. Combination At present, the combination of fluorescence-based screening technology and automatic patch clamp has become the most commonly used method for ion channel targeted drug discovery. As costs decrease and technology advances, automated electrophysiology will become the main form of measurement for most ion channel subtypes. For different ion channel subclasses, high-throughput screening methods differ due to ion selectivity, channel activation kinetics, and consideration of whether ligands are required. Advances and improvements in ion channel HTS technology have accelerated the discovery of ion channel drugs. These measurements have relatively low time resolution and information content, but can be robust and low-cost. The electrophysiological method has the most direct method to measure ion channel activity and also provides flexibility for the analysis and optimization of each channel type. The combination of non-electrophysiological and electrophysiological HTS methods provides an integrated and cost-effective method for ion channel drug discovery and ensures the generation of high-quality data.

screeningDrug development