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. Manual patch clamp evaluation of herg channel pharmacology. Protocols are available for single dose testing and doseresponse curves. 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. Ion channel targets remain a top priority for many pharma and biotechs with most looking to increase their screening efforts in 2006. Evaluation of a highthroughput fluorescence assay method for. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patch clamp electrophysiology studies. Traditionally herg screening is performed with a whole cell voltage patch clamp apparatus.
Pdf improved throughput of patchxpress herg assay using. Please email us to discuss which platform is most appropriate according to your needs, or get in touch with the contact person on the relevant subpage. Automated patchclamp methods for the herg cardiac potassium channel. Many factors can impact data quality hence reproducibility. This method is considered comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel. The flipr potassium assay kit figure 1 contains a thalliumsensitive indicator dye. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies. While the technology is an essential method in the field of. This method is considered the gold standard in regards to testing ion channels. Investigation of miscellaneous herg inhibition in large diverse compound collection using automated patchclamp assay. Screening system for druginduced arrhythmogenic risk. An in vitro electrophysiological assay system, which can assess compound effects and thus show cardiotoxicity including arrhythmia risks of test drugs, is an essential method in the field of drug development and toxicology.
The ionworkstm ht system delivers high quality, accurate and sensitive data which is comparable with the traditional single cell patch clamp method. In addition to confirming the activity of potential hits from high or medium throughput screens, manual patchclamping can be used to assess mechanism of action of compounds and to determine the effects of compounds on the. The voltage clamp was difficult both teoretically and practically, not a lot of people could have experience doing that. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels. Manual patch clamp assay manual patch clamping is the goldstandard for the investigation of ion channel activity. Currently, the conventional wholecell patch clamp assay is the most reliable method available to accurately determine the activity of compounds at the human. The patch clamp technique allows the investigation of a small set or even single ion channels.
Solubility assessment and online exposure confirmation in. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Wholecell configuration of the patchclamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval. To develop an hts assay for herg using an automated patch clamp. To identify potential herg liabilities early in drug discovery programs and thus avoid problems with herg channel interactions for latestage compounds, it has become common practice in drug discovery programs to start testing compounds relatively early. The predictor herg fluorescence polarization assay kit provides. However, it is often seen that the responsible scientist from the biotech company is not familiar with patch clamp, or voltage clamp with single electrode. Covid19 is an emerging, rapidly evolving situation. Throughput according to our customers needs from one compound to hts screening. Consequently, for in vitro assay formats based on a constant flow of test compoundcontaining buffer, as used in a classical patchclamp assay, a method is described which provides online, almost realtime monitoring of compound exposure during the whole experiment. The human etheragogo related gene herg encodes the inward rectifying. For 12 drugs reported to have varying cardiotoxicity risks, doseinhibition curves were determined for six ion channels using automated patch clamp systems. Hts, single concentration dose response studies, selectivity profiling.
Implementation of a kinetic herg assay protocol using. Cell and tissue culture, patch clamp technique, drug delivery, electrophysiology, live cell imaging, transfection, cell proliferation assay, electrocardiogram. Investigation of miscellaneous herg inhibition in large diverse. Comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. In the automated patchclamp measurements performed here, kv currents were. Invitrogens predictor herg fluorescence polarization. However, the multiwell plate format of automatized patchclamp does not allow visual detection of potential microprecipitation of poorly soluble compounds. 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. This lecture explains about the patch clamp technique process and the set up and recording with patch clamp technique. Investigation of miscellaneous herg inhibition in large diverse compound collection using automated patch clamp assay.
Patch clamp technique is the gold standard for carrying. The intercellline reproducibility of herg assay using the wholecell patchclamping 1. In this study, highthroughput electrophysiological recordings of human embryonic kidney hek 293 cells and chinese hamster ovary cho cells stably expressing. Validation of a multiple compound addition assay for herg ion. Although patch clamp experiments represent the state of the art for measuring herg blockade, such experiments are relatively unwieldy and expensive. The multiplecompound addition assay has the added benefit of generating cumulative concentrationresponse data similar to those generated in conventional patch clamp assays. 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 when displaced by compounds that bind to the channel. The data obtained with the fluorescence assay were in qualitative agreement with those from patchclamp electrophysiological analysis. The intercellline reproducibility of herg assay using.
Characterization of herg channel blockers using the flipr. 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 qpatch htx and the syncropatch 384pe systems deliver high quality. Electrophysiological analysis of mammalian cells expressing. 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. Evaluation of the rubidium efflux assay for preclinical. The qpatch performs up to 48 independent patch clamp experiments in parallel in a 48channel chip array with microfluidic chambers qplate. In this study we show that 1 the biophysical and pharmacological properties of herg current recorded with kf are similar to those with. The intercellline reproducibility of herg assay using the wholecell patch clamping 1. Evaluation of a highthroughput fluorescence assay method.
Validation of a multiple compound addition assay for herg. The closest to this ideal would be voltage clamp recordings in cardiac myocytes using a. Automated patch clamp an overview sciencedirect topics. Medium and high throughput herg assays the ideal herg assay provides a linear measure of channel activity under physiologically relevant conditions. Role of herg potassium channel assays in drug development. Inhibition of herg current at 1 um by patch clamp electrophysiology assay. Cyprotexs herg safety assay employs the ionworkstm ht system molecular devices as an automated patch clamp electrophysiology measurement. Hts screening of herg blockers using 2ndgeneration. The patch clamp method allows for the simultaneous assessment of ion channel inhibition activity of e. We also show that the sensitivity of the assay can be improved by adding a. Implementation of a kinetic herg assay protocol using qpatch. Patch clamp technique is the gold standard for carrying out the herg assay.
We introduce a novel screening system for druginduced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. The correlation between data obtained with automated patchclamp qpatch 16 and manual patchclamp was high. The patchclamp method allows for the simultaneous assessment of ion channel inhibition activity of e. Manual patch clamp method is considered as gold standard for ion. Solubility assessment and online exposure confirmation in a. First reported in 2010 using the conventional whole cell patch clamp technique milnes et al. 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. Applied strict data quality criteria and compared results generated by 3 electrophysiologists using different styles of manual patch clamp rigs. Invitrogens predictor herg fluorescence polarization assay. 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. Manual patch clamping is the goldstandard for the investigation of ion channel activity.
Biochemical herg assays thermo fisher scientific us. Consequently, for in vitro assay formats based on a constant flow of test compoundcontaining buffer, as used in a classical patch clamp assay, a method is described which provides online, almost realtime monitoring of compound exposure during the whole experiment utilizing enhanced sensitivity uv spectroscopy. Pdf automated patchclamp methods for the herg cardiac. Both filtration and homogeneous flashblue herg binding assay have been optimized with 125 ibekm1 panels 711. Normalised concentrationresponse curve of antagonist cisapride against the herg current activated by. Sucrose gap method with 2 electrodes was the standard voltage clamp technique when used the cardiac tissue. In this chapter we describe bench procedures for the culture and preparation of hergexpressing cho cells for recording on an automated patchclamp workstation. Early identification of herg liability in drug discovery. Detection limit of herg activity by ht herg assay using the fmp dye and patch clamp pc. Hts screening of herg blockers using 2ndgenerationautomated. Dec 16, 2015 this study is proposing a highthroughput screening system for investigating herg channel inhibition using an automated multiwell patch clamp technology. The cell harvesting for the automated patch clamp experiment should be carried.
This method is broadly applicable to many ion channel assays, particularly those which study compound block of voltagegated ion channels. Since manual patch clamping has long been regarded as the gold standard for evaluating herg safety by fda and other regulatory institutions, creative biolabs provides firstclass manual patch clamp electrophysiology measurement of herg inhibition with high accuracy. Introduction the wholecell patch clamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a potential risk of qtinterval prolongation that. Patch clamp technique is the gold standard for carrying out.
That technique required a huge effort, training, luckiness and patience, but good results acceptable for publication were not easily obtained. 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. Patch clamp technique method electrophysiology technique. A single ion channel conducts around 10 million ions per second. Introduction the wholecell patchclamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a. This method is broadly applicable to many ion channel assays. Creative bioarray uses the stateoftheart automated qpatchht system to provide a higherthroughput herg safety assay with better consistency at a. Completed herg channel pharmacology using 12 cipatraining drugs and manual patch clamp rigs. The qpatch htx is a highthroughput and automated electrophysiology platform. Performance of the predictor fluorescence polarization. Investigation of miscellaneous herg inhibition in large. Detection limit of herg activity by htherg assay using the fmp dye and patch clamp pc.
A homogeneous herg binding assay was developed using flashblue gpcr beads and 125 ibekm1 0. This study is proposing a highthroughput screening system for investigating herg channel inhibition using an automated multiwellpatchclamp technology. An in vitro electrophysiological assay system, which can assess compound effects. The scorpion toxin bekm1 5, 6 blocks herg preferentially through closed channels 2, 7. Therefore, a method that allows for more economical and higher throughput screening of panels of compounds for herg blockade is both necessary and desirable. Electrophysiological analysis of mammalian cells expressing herg. 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. The intercellline reproducibility of herg assay using the. This ion channel sometimes simply denoted as herg is best known for its contribution to the electrical activity of the heart. Wholecell configuration of the patchclamp technique in the herg. Automated patchclamp methods for the herg cardiac potassium. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. Although patchclamp experiments represent the state of the art for measuring herg blockade, such experiments are relatively unwieldy and expensive. The gold standard to carry out the glp herg assay is the technique so called patch clamp voltage clamp method wholecell configuration.
Optimization of the assay conditions buffers, incubation time, and assay plate has provided a highthroughput assay for the rapid determination of herg channel affinity. 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. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for herg. Therefore, a method that allows for more economical and higher throughput screening of panels of compounds for. Methods cell culture and cell preparation for patchclamp analysis in. Performance of the predictor fluorescence polarization assay. Improved throughput of patchxpress herg assay using. Seventythree percent of the compounds were identified as herg active in both assays correlation of ic 50. For the biotechs who are not familiar with herg assay.
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