The obtained ic 50 s values were compared with their manual patch clamp counterparts. Patch clamp technique is the gold standard for carrying. Pdf identifying modulators of herg channel activity using. Currently, the conventional wholecell patch clamp assay is the. In this assay, cho herg cells or hek293 herg cells are used to specifically assess the effect of test compounds on the herg channel. Inhibition of activity in the herg channel has been linked to cardiac safety issues for many approved drugs. Availability of cuttingedge gold standard assays was ranked as what most motivates the selection of an ion channel safety liability. Fhnd004 is a newly synthesized epidermal growth factor receptor egfr inhibitor for the treatment of nonsmall cell lung cancer nsclc. The voltage clamp was difficult both teoretically and practically, not a lot of people could have experience doing that. Protocols are available for single dose testing and doseresponse curves. 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. Hts, single concentration dose response studies, selectivity profiling. Traditionally, electrophysiologists have relied on the goldstandard assay of patch clamp electrophysiology in mammalian cells or two electrode voltage clamp. Conventional patch clamping, now also referred to as manual patchclamp to.
Patchclamp studies in the evaluation of suppressive. Dec 07, 2014 whole cell patch clamp recordings from morphologically digitimer ds2a duration. Placement of the patch clamp electrodes using micromanipulators is a time. The comprehensive in vitro proarrhythmia assay cipa. 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. View verification of cipa recommended voltage protocols in patchclamp assay for herg. Pdf automated tight seal electrophysiology for assessing. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patch clamp electrophysiology studies. Patch clamp electrophysiology, voltage clamp, action. Jul 12, 2011 the development of the planar patch clamp technology was a major breakthrough in dealing with alqts during drug development. Why are there current fluctuations in herg patch clamp.
Electrophysiology herg currents were measured by means of the patch clamp technique in the wholecell configuration. Manual patch clamping is the goldstandard for the investigation of ion channel activity. I am doing herg voltage clamp protocol using port a patch but i am unable to get. Role of herg potassium channel assays in drug development. Ionworks barracuda automated patch clamp system molecular. Evaluation of the rubidium efflux assay for preclinical. Automated patch clamp devices have enhanced our understanding of ion channels activities in the brain, heart and other excitable systems. Manual patch clamp evaluation of herg channel pharmacology. Why are there current fluctuations in herg patch clamp assay using port a patch nanion. Recently, a voltage clamp device that uses planar electrode. Comparison to a herg rubidium efflux screen steve sorota, xuesong zhang, michael margulis, kristal tucker, and tony priestley abstract. 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 impact of automated patch clamping apc on ion channel screening is now. 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. Qpatch ii a barcode scanner on the gripper arm and stacking of plates on the work plane provides faster and more robust automation. The present study examined the effects of gentamicin 10, 25, and 50. The qpatch performs up to 48 independent patch clamp experiments in parallel in a 48channel chip array with microfluidic chambers qplate. Sep 02, 2014 automated patch clamp electrophysiology is an emerging technique for identifying cardiac ion channel liabilities. The gold standard to carry out the glp herg assay is the technique so called patch clamp voltage clamp method wholecell configuration. Cardiac electrophysiology has been developed from ecg to membrane ionic currents via the action potential, or technically surface electrical integrated measurement to patch clamp techniques, vial micro electrode technique. Another concern with regard to the automated platforms is the potential for results that are inconsistent with those from conventional voltage clamp assays. Why are there current fluctuations in herg patch clamp assay using. Completed herg channel pharmacology using 12 cipatraining drugs and manual patch clamp rigs. Introduction the wholecell patch clamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a. Qpatch ii is designed to overcome the constant pressure to increase lab efficiency while maintaining medium and high throughput and high data quality.
Since patch clamp testing is costly and because numerous, chemically diverse scaffolds block herg, strategies to mitigate potential cardiac liability during earlystage drug development typically employ a binding assay to predict the ability of a compound to block herg current in the functional patch clamp assay. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels. 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. The intercellline reproducibility of herg assay using the. This assay has identified the ic 50 values of known herg channel blockers tightly correlated to and within a narrow range of the published values determined by radioligand binding assays and patch clamp recordings. A comparison of the performance and application differences. Therefore, unless very low concentrations of test compounds are included in the compound titrations, the full assay window will not be.
The electrode is tightly sealed to the patch of the membrane and the cell remains intact. The patch clamp rig includes a glass micropipette, which contains both an ionic solution and a chlorinated silver electrode for measuring voltages and currents. Its the pore forming subunit of a potassium ion channel that conducts the rapid component of the delayed rectifier potassium current known as ikr. S2218 did not significantly inhibit the herg ion channel current herg electrophysiology patch clamp assay 30. Patch clamp electrophysiology instruments used to evaluate ion channel behavior. View verification of cipa recommended voltage protocols in patch clamp assay for herg, cav1.
Outsourced ion channel testing trends technology networks. Jul 27, 2017 however, the multiwell plate format of automatized patch clamp does not allow visual detection of potential microprecipitation of poorly soluble compounds. Manual patch clamp of the herg potassium channel to evaluate potential cardiac liability. A single cell is then positioned on the hole by suction and a tight connection gigaseal is formed. 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. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Automated patch clamp manual patch clamp twoelectrode voltage clamp cardiac safety panel. Early identification of herg liability in drug discovery.
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. Applied strict data quality criteria and compared results generated by 3 electrophysiologists using different styles of manual patch clamp rigs. For the biotechs who are not familiar with herg assay. Industry leading flexibility and performance in high throughput patch clamp assays. Channels 89 a major limitation of all automated electrophysiology platforms is the high cost of the instruments and consumables. Manual patch clamp assay manual patch clamping is the goldstandard for the investigation of ion channel activity. This method is considered comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. 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. Performance of the predictor fluorescence polarization. The qpatch htx is a highthroughput and automated electrophysiology platform. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies.
Normalised concentrationresponse curve of antagonist cisapride against the herg current activated by. Panel c shows a family of herg currents from an experiment described above. Sucrose gap method with 2 electrodes was the standard voltage clamp technique when used the cardiac tissue. 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. Creative bioarray uses the stateoftheart automated qpatchht system to provide a higherthroughput herg safety assay with better consistency at a. Precompound current and postcompound current are measured by patch clamp, and applied to the calculation of herg inhibition. Instead of positioning a pipette on an adherent cell, cell suspension is pipetted on a chip containing a microstructured aperture. The intercellline reproducibility of herg assay using the wholecell patch clamping 1.
Automated patch clamp an overview sciencedirect topics. The patch clamp technique is commonly used in electrophysiological. Trpa1 in a cellbased system in conjunction with automated intracellular fluorescence multiwell cellular screening assays as well as automated patch clamp screening assays have enabled the. Placement of the patch clamp electrodes using micromanipulators is a time consuming and complicated. Inhibition of herg current at 1 um by patch clamp electrophysiology assay.
Planar patch clamp is a novel method developed for high throughput electrophysiology. Whole cell patch clamp was used to record currents at 37c. Both filtration and homogeneous flashblue herg binding assay have been optimized with 125 ibekm1 panels 711. Many factors can impact data quality hence reproducibility. The qplate is in a standard microtitre plate mtp format. That technique required a huge effort, training, luckiness and patience, but good results acceptable for publication were not easily obtained. The control tail current amplitude from a patch clamp assay is analogous to the negative control value in the predictor herg fp assay. The intercellline reproducibility of herg assay using. Biochemical herg assays thermo fisher scientific us. 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. During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell. Ion channel targets remain a top priority for many pharma and biotechs with most looking to increase their screening efforts in 2006. The introduction of parallel patch clamp instruments offers the promise of moderatethroughput, highfidelity voltage clamp for drug screening assays. Wholecell patch clamp recording was performed on wild type.
We provide manual patch clamp electrophysiology and multi electrode array assays, in conjunction with. Patch clamp assay from single neuron down to single channel, thanks to the different recording configurations wholecell, perforated patch, cellattached, outsideout, patch clamp techniques provide the highest resolution for electrophysiological recordings in ion channel screening process. Using automated patch clamp electrophysiology platforms in paina. The patch clamp technique allows the investigation of a small set or even single ion channels. While the patch clamp method works very well for identifying compounds that block potassium ion flow through herg channels, it requires a high resistance seal be made.
Figure 2 main assay technologies used to study ion channels targets in drug. Acroscell experienced technicians allow us to provide customized assays. This ion channel contributes to the repolarization phase of. Bsys periodically tests cells for presence of mycoplasma by means of highly sensitive pcr based assays. The qplate which contains the 16 patch clamp sites, each constituting a complete patch clamp unit with silicon chip, flow channels, electrodes, and waste reservoir kutchinsky et al. 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. Automated tight seal electrophysiology for assessing the. Using the patch clamp technique, scientists ask questions about the nature of the potential and current. The effects of gentamicin and penicillinstreptomycin on the. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long.
In this case the cell bath has a precisely drilled approximately 1 micron diameter hole in its bottom, and the fluid compartment under the hole functions as the amplifiers intracellular electrode. Characterization of herg channel blockers using the flipr. The assay should be conducted by manual patch clamp rather than automated patch clamp and variants, and be conducted at physiological temperature to reproduce the invivo kinetics of activation. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Identifying modulators of herg channel activity using the patchxpress planar patch clamp article pdf available in journal of biomolecular screening 102. Characterization of a herg screen using the ionworks ht. This type of electrode is distinct from the sharp microelectrode used to puncture cells in traditional intracellular recordings, in that it is sealed onto the surface of. Throughput according to our customers needs from one compound to hts screening. Predictor herg fluorescence polarization assay frequently.
Jan 26, 2012 in this work, a systematic comparison of diverse panel of reference drugs spanning a broad herg potency range was performed to validate the patchliner for temperaturecontrolled herg assay. Utility of herg assays as surrogate markers of delayed. The qpatch htx and the syncropatch 384pe systems deliver high quality. However limitations in fluid control can sometimes hinder high throughput drug discovery and bring complicated assays to a halt.
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. Automated patchclamp methods for the herg cardiac potassium. Special note for comparison to electrophysiology data. 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 during the drug discovery. From ecg to ionic currents hergworkshops jimdopage. Recording of voltagedependent herg current activated upon depolarization, and deactivated upon repolarization. A single ion channel conducts around 10 million ions per second. Traditionally, electrophysiologists have relied on the goldstandard assay of patchclamp electrophysiology in mammalian cells or two electrode voltage clamp in xenopus oocytes. Examples of the use of qpatch in basic characterization. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and. Electrophysiology is the most reliable in vitro screening method for identifying potential cardiac herg liabilities, but only the recent advent of planar electrode based voltage clamp electrophysiology promises sufficient throughput to support the drug testing needs of most drug discovery programs.
View automated planar electrode electrophysiology in drug discovery. An invitro patch clamp assay using hek293 cells transfected with the human erg gene, which encodes the channel allowing the ikr potassium current. Patchclamp studies in the evaluation of suppressive effects. Cardiac panel automated patch clamp assays, nonglp herg ic50 assays and herg ion channel assays were the aspects of ion channel safety liability testing survey respondents most want to outsource today.
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