The report also examines assay technologies preferences used today in the study of ion channels targets today figure 2. To develop an hts assay for herg using an automated patch clamp platform, ionworks quattro, two different herg cell lines chok1 and hek293 as parental cells were compared. Glp patch clamp studies including the herg assay use the standard reference manual patch clamp method. This ion channel sometimes simply denoted as herg is best known for its contribution to the electrical activity of the heart. Oct 16, 2016 the patch clamp technique is a refinement of the voltage clamp. Automated patchclamp methods for the herg cardiac potassium. However, the throughput 400 data points per hour is too low for primary screening of large compound libraries.
Frontiers early identification of herg liability in drug. The scorpion toxin bekm1 5, 6 blocks herg preferentially through closed channels 2, 7. Planar patch clamp has been a tremendous advance for identifying compounds with herg liability dubin et al. While this article focuses on herg channel recordings using the qpatch sophion as, copenhagen, denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms. Predictor herg fluorescence polarization assay frequently. A number of herg assays to assess cardiac safety have been developed e. Fluorescencebased assays predominate in primary screening of full diversity libraries, with greatest use made of fluorescentbased membrane potential assays. Blockade of the human ether agogorelated gene herg.
Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels herg currents are recorded from stably transfected cho cells. Compound effects were tested using dual compound additions at 1. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels. Fluorescence alternatives to popular biological assays. Traditionally herg screening is performed with a whole cell voltage patch clamp apparatus. Role of herg potassium channel assays in drug development. Growing interest in outsourcing ion channel services. Early identification of herg liability in drug discovery programs by. The results were obtained by eliciting herg current responses with a voltage pulse protocol. Using the assay, ic50 values for over 10 known herg channel blockers compared well to published patch clamp and radioligand binding data figure 1 and table 1.
Examples of compounds that show undesired herg channel activity. Whilst this was able to identify most of the actives in the test set it had a relatively. Predictor herg fluorescence polarization assay kit performed. May 31, 2018 wholecell patch clamp recording was performed on wild type wt, mutant herg channels heterologously expressed in hek 293 cells or ikr endogenously expressed in hl1 cells, respectively. Jul 27, 2017 in this chapter we describe bench procedures for the culture and preparation of herg expressing cho cells for recording on an automated patch clamp workstation. Among all existing automated electrophysiology systems for herg screening, the ionworks quattro automated patch clamp system had the highest throughput and lowest data point cost. The human etheragogorelated gene herg encodes the poreforming subunit of the rapidly activating delayed rectifier potassium channel ikr. In addition, we evaluated 19 blinded compounds at 10 m in the rb efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. Throughput according to our customers needs from one compound to hts screening.
Both filtration and homogeneous flashblue herg binding assay have been optimized with 125 ibekm1 panels 711. The results show that the majority of respondents are seeking a whole cell. Electrophysiological analysis of mammalian cells expressing herg. View verification of cipa recommended voltage protocols in patch clamp assay for herg, cav1. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in. The chantest platform covers the entire preclinical ion channel spectrum from hits to leads, lead selection and optimisation and glp ind applications. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel. Addressing herg activity while maintaining favorable.
Early identification of herg liability in drug discovery. Manual patch clamp evaluation of herg channel pharmacology. Evaluation of the rubidium efflux assay for preclinical. Competitive binding assay of 10 known herg blockers using the predictor herg fluorescence polarization assay. When lead compounds in the benzamide series, 1a and 1b, were screened for herg activity in an automated patch clamp assay, little or no activity herg safety service is performed at our parent company, evotec, and is a cellbased assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. The expertise of our technicians allows us to provide customized assays. Investigation of miscellaneous herg inhibition in large. The assay is based on the principle of fluorescence polarization, where a redshifted fluorescent tracer displays a high polarization when bound to the herg channel and a low polarization. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.
Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels herg currents are recorded from stably transfected cho cells protocols are available for single dose testing and doseresponse curves throughput according to our customers needs herg patch clamp. Seven reference herg blockers are examined in a cellbased assay, and the results are compared to values obtained using the ionworks barracuda plus automated patch clamp system. In this assay, cho herg cells or hek293 herg cells are used to specifically assess the effect of test compounds on the herg channel. Automated patchclamp methods for the herg cardiac potassium channel. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. Automated patch clamp an overview sciencedirect topics. We also show that the sensitivity of the assay can be. Sep 02, 2014 while this article focuses on herg channel recordings using the qpatch sophion as, copenhagen, denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms. We also show that the sensitivity of the assay can be improved by adding a. Protocols are available for single dose testing and doseresponse curves. While the patch clamp method works very well for identifying compounds that block potassium ion flow through.
Pdf automated patchclamp methods for the herg cardiac. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patch clamp electrophysiology studies. Biochemical herg assays thermo fisher scientific us. Precompound current and postcompound current are measured by patch clamp, and applied to the calculation of herg inhibition. Channels 89 a major limitation of all automated electrophysiology platforms is the high cost of the instruments and consumables. Examples of compounds that show undesired herg chan.
Since activation and inactivation of herg is dependent on membrane potential, voltage clamp recordings can differentiate between compounds that preferentially interact with different states of the channel. Pdf early identification of herg liability in drug. Manual patch clamp of the herg potassium channel to evaluate potential cardiac liability. Another concern with regard to the automated platforms is the potential for results that are inconsistent with those from conventional voltage clamp assays. A more costeffective, faster alternative to the herg patch clamp assay is the predictor herg cellular fp assay from life technologies this application saves money and time by allowing potential drugs to be prescreened before undergoing more tedious patch clamp screening. The herg safety service is performed at our parent company, evotec, and is a cellbased assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. Apr 16, 2020 one standard assay for early testing of the torsadogenic potential of compounds is glp standard patch clamp electrophysiology on the heterogeneously expressed herg channel. Automated patch clamp assays have been successfully used in a. When lead compounds in the benzamide series, 1a and 1b, were screened for herg activity in an automated patch clamp assay, little or no activity herg current at 1 um by patch clamp electrophysiology assay. Covid19 is an emerging, rapidly evolving situation. In this assay, choherg cells or hek293herg cells are used to specifically assess the effect of test compounds on the herg channel. The performance of the dynaflow system in this study demonstrates its suitability for conducting safety studies.
Pdf early identification of herg liability in drug discovery. In the automated patchclamp measurements performed here. Drugmediated or medical conditionmediated disruption of herg function is the primary cause of acquired longqt syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. This method is considered comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. Validation of the ionworks barracuda system for herg ion. The impact of automated patch clamping apc on ion channel screening is. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies. The polarization data are shown in figure 2, with the data summarized and compared with patch clamp or radioligand displacement data that have been previously reported in table 1. Figure 2 main assay technologies used to study ion channels targets in drug discovery. Addressing herg activity while maintaining favorable potency.
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