Early identification of herg liability in drug discovery programs by. Both filtration and homogeneous flashblue herg binding assay have been optimized with 125 ibekm1 panels 711. 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. Throughput according to our customers needs from one compound to hts screening. Automated patchclamp methods for the herg cardiac potassium channel. Electrophysiological analysis of mammalian cells expressing herg. The scorpion toxin bekm1 5, 6 blocks herg preferentially through closed channels 2, 7. The expertise of our technicians allows us to provide customized assays. In a more recent study doi using 18 chemically diverse herg blockers from the literature, all evaluated with the same patch clamp assay identified similar features, a basic nitrogen red, an aromatic ring blue and two further lipophilic regions cyan. 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. Single concentration profiling and full concentration response curves 4 pt. In this assay, choherg cells or hek293herg cells are used to specifically assess the effect of test compounds on the herg channel. Characterization of herg channel blockers using the flipr. Compound effects were tested using dual compound additions at 1.
Addressing herg activity while maintaining favorable potency. Examples of compounds that show undesired herg channel activity. Automated patch clamp assays have been successfully used in a. Oct 16, 2016 the patch clamp technique is a refinement of the voltage clamp. View verification of cipa recommended voltage protocols in patch clamp assay for herg, cav1. Predictor herg fluorescence polarization assay kit performed. Whilst this was able to identify most of the actives in the test set it had a relatively. 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.
Precompound current and postcompound current are measured by patch clamp, and applied to the calculation of herg inhibition. Early identification of herg liability in drug discovery. Left and right panels show the representative recordings of herg current. Traditionally herg screening is performed with a whole cell voltage patch clamp apparatus. Covid19 is an emerging, rapidly evolving situation. In this assay, cho herg cells or hek293 herg cells are used to specifically assess the effect of test compounds on the herg channel. The results show that the majority of respondents are seeking a whole cell. Role of herg potassium channel assays in drug development. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies.
The predictor herg fluoresence polarization assay uses a proprietary fluorescent tracer and membrane preparation to create a highly reproducible assay. 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. 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. Pdf early identification of herg liability in drug. Fluorescence alternatives to popular biological assays.
Another concern with regard to the automated platforms is the potential for results that are inconsistent with those from conventional voltage clamp assays. Evaluation of the rubidium efflux assay for preclinical. The performance of the dynaflow system in this study demonstrates its suitability for conducting safety studies. Predictor herg fluorescence polarization assay frequently. 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.
Validation of the ionworks barracuda system for herg ion. Frontiers early identification of herg liability in drug. An in vitro electrophysiological assay system, which can assess compound effects. 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. Figure 2 main assay technologies used to study ion channels targets in drug discovery. Pdf early identification of herg liability in drug discovery. 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. Automated patch clamp an overview sciencedirect topics. Competitive binding assay of 10 known herg blockers using the predictor herg fluorescence polarization assay. This method is considered comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. Apr 27, 2017 creative bioarray uses the stateoftheart automated qpatchht system to provide a higherthroughput herg safety assay with better consistency at a lower cost. Investigation of miscellaneous herg inhibition in large. The impact of automated patch clamping apc on ion channel screening is.
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. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels herg currents are recorded from stably transfected cho cells. 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. Addressing herg activity while maintaining favorable. We also show that the sensitivity of the assay can be. Biochemical herg assays thermo fisher scientific us. Manual patch clamp evaluation of herg channel pharmacology. A number of herg assays to assess cardiac safety have been developed e. This ion channel sometimes simply denoted as herg is best known for its contribution to the electrical activity of the heart.
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 report also examines assay technologies preferences used today in the study of ion channels targets today figure 2. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. Channels 89 a major limitation of all automated electrophysiology platforms is the high cost of the instruments and consumables. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels. 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.
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. Blockade of the human ether agogorelated gene herg. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium. The chantest platform covers the entire preclinical ion channel spectrum from hits to leads, lead selection and optimisation and glp ind applications. 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. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patch clamp electrophysiology studies. Automated patchclamp methods for the herg cardiac potassium.
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. Planar patch clamp has been a tremendous advance for identifying compounds with herg liability dubin et al. 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. However, the throughput 400 data points per hour is too low for primary screening of large compound libraries. The human etheragogorelated gene herg encodes the poreforming subunit of the rapidly activating delayed rectifier potassium channel ikr. 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.
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. 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. Protocols are available for single dose testing and doseresponse curves. 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. 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 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 results were obtained by eliciting herg current responses with a voltage pulse protocol.
In the automated patchclamp measurements performed here. Glp patch clamp studies including the herg assay use the standard reference manual patch clamp method. Manual patch clamp of the herg potassium channel to evaluate potential cardiac liability. Fluorescencebased assays predominate in primary screening of full diversity libraries, with greatest use made of fluorescentbased membrane potential assays. Growing interest in outsourcing ion channel services.
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