Results of herg assay on qpatch with ic 50 only protocol it plot the. Patch clamp technique is the gold standard for carrying. Therefore, the in vitro herg channel patchclamp assay is commonly used as an. 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. In this chapter we describe bench procedures for the culture and preparation of hergexpressing cho cells for recording on an automated patchclamp workstation. Assay performance was validated with a panel of established herg channel blockers.
Covid19 is an emerging, rapidly evolving situation. Data analysis is performed using softmax pro software and graphpad prism. Radioisotope based cyp and herg assays available for profiling and screening. Why are there current fluctuations in herg patch clamp assay using port a patch nanion. The protocol described in this unit is designed to assess the effects of new. A stimulation protocol for herg channel measurements. Introduction the wholecell patchclamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a. Early identification of herg liability in drug discovery programs by.
The current fluctuations are so high that i cant even test a. Frontiers early identification of herg liability in drug. Implementation of a kinetic herg assay protocol using. First reported in 2010 using the conventional whole cell patch clamp technique milnes et al. I am doing herg voltage clamp protocol using port a patch but i am unable to get a steady current. Electrophysiology can provide detailed and quantitative information on the potency and mechanism of herg block by a test compound. Automated patch clamp apc allows assessing the effects of compounds on the herg channel in the preclinical development process, enabling an early evaluation in drug development. Fluxor assay kit life technologies following the manufacturers assay protocol. Inhibition of herg current at 1 um by patch clamp electrophysiology assay. Data was gathered at ambient temperature since this is most relevant to most high throughput platforms on.
Wholecell configuration of the patchclamp technique in. We demonstrate its use on the nanion syncropatch 384pe, a 384well automated patchclamp platform, by applying it to chinese. Pdf early identification of herg liability in drug. That technique required a huge effort, training, luckiness and patience, but good results acceptable for. Measuring kinetics and potency of herg block for cipa. The performance of the dynaflow system in this study demonstrates its suitability for conducting safety studies. Early identification of herg liability in drug discovery. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.
The qpatch performs up to 48 independent patch clamp experiments in parallel in a 48channel chip array with microfluidic chambers qplate. Verification of cipa recommended voltage protocols in. The patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. Rbcs cyp and herg assays offer radiolabeled data at an affordable price. 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. A homogeneous herg binding assay was developed using flashblue gpcr beads and 125 ibekm1 0.
We present a new 15 second protocol to characterize herg kv11. Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the herg channel. 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. Why are there current fluctuations in herg patch clamp assay using. Deactivation of herg channels was analyzed with a protocol shown in the inset of figure 4d. The qpatch htx and the syncropatch 384pe systems deliver high quality. The assay protocol contains cell type specific settings for establishing the patch clamp recording configuration, as well as voltage protocols to. The herg safety service is performed by 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. To investigate the effects of fhnd004 on i herg, wholecell patch clamp recordings were performed on hek293 cells heterologously expressing herg channels. Pdf early identification of herg liability in drug discovery. With its vast experimental freedom and gigaseal data quality, the patchliner is one of the most versatile patch clamp instruments. The comprehensive in vitro proarrhythmia assay cipa initiative was established to develop a new paradigm for assessing proarrhythmic risk, building on the emergence of new technologies and an expanded understanding of torsadogenic mechanisms beyond herg block.
The results were obtained by eliciting herg current responses with a voltage pulse protocol. The intercellline reproducibility of herg assay using. Example of currents recorded from dividing hergtrex 293 cells in patchclamp assay. The predictor herg fluoresence polarization assay uses a proprietary fluorescent tracer and membrane preparation to create a highly reproducible assay. That technique required a huge effort, training, luckiness and patience, but good results acceptable for publication were not easily obtained.
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. Special note for comparison to electrophysiology data. Role of herg potassium channel assays in drug development. Wholecell configuration of the patchclamp technique in the herg. This ion channel sometimes simply denoted as herg is best known for its contribution to the electrical activity of the heart. Unattended runs like we show here on the qube are a crucial advantage by saving time and resources.
The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies. In vitro electrophysiological safety studies have become an integral part of the drug development process since, in many instances, compound. Data were acquired using a step depolarisation protocol using manual patchclamp at ambient temperature. Although patchclamp experiments represent the state of the art for measuring herg blockade, such experiments are relatively unwieldy and expensive. Gigaseal hts patch clamp cipaspecified cardiac ion channels recorded at high throughput gigaseal hts patch clamp high throughput recordings of cardiac ion channels at physiological temperature. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or longterm exposure on the amplitude of ikr in human embryonic kidney 293 hek293 cells stably transfected with the herg channel wholecell configuration of the patchclamp technique. Manual patch clamp evaluation of herg channel pharmacology. The closest to this ideal would be voltage clamp recordings in cardiac myocytes using a. Medium and high throughput herg assays the ideal herg assay provides a linear measure of channel activity under physiologically relevant conditions. Patch clamp technique is the gold standard for carrying out the herg assay. The metrion biosciences team recently produced an application note reporting the successful validation of the milnes voltage protocol for herg screening using a qpatch 48 automated patch clamp apc assay platform. Characterization of herg channel blockers using the flipr. The qpatch htx is a highthroughput and automated electrophysiology platform. However, the multiwell plate format of automatized patchclamp does not allow visual detection of potential microprecipitation of poorly soluble compounds.
Investigation of miscellaneous herg inhibition in large diverse compound collection using automated patchclamp assay. Early identification of herg liability in drug discovery programs by automated patch clamp. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Interference with herg channel function may occur by binding of drugs to either the open, closed or inactivated states of the channel 5. The comprehensive in vitro proarrhythmia assay cipa. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. Therefore, unless very low concentrations of test compounds are included in the compound titrations, the full assay window will not be. The control tail current amplitude from a patch clamp assay is analogous to the negative control value in the predictor herg fp assay. Request the cipa assay protocol for the syncropatch 384pe here.
Verification of cipa recommended voltage protocols in patchclamp assay for herg, cav1. Cipa herg protocol this protocol was used for herg studies on the patchliner and syncropatch 384pe. Investigation of miscellaneous herg inhibition in large. Automated electrophysiological patch clamp allows assessment of herg channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. The impact of automated patch clamping apc on ion channel screening is now evident, particularly in early noncompliant herg liability testing, where apc is rapidly becoming the new gold standard technology. The intercellline reproducibility of herg assay using the wholecell patchclamping 1. 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.
Ion channel targets remain a top priority for many pharma and biotechs with most looking to increase their screening efforts in 2006. Manual wholecell patchclamping of the herg cardiac k channel. The flipr potassium assay kit figure 1 contains a thalliumsensitive indicator dye. Automated patchclamp methods for the herg cardiac potassium. 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. The predictor herg fluorescence polarization assay kit provides a set of validated components to perform herg channel biochemical binding studies in the absence of radioligand. These machines can execute traditional voltageclamp protocols aimed at specific. One of the unique advantages of such voltage clamp recordings is the ability to control membrane potential. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel.
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