Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel

Murphy, Steven M.; Palmer, Marc; Poole, Michelle Fontilla; Padegimas, Linas; Hunady, Karen; Danzig, Joel; Gill, Sikander; Gill, Rajwant; Ting, Anthony E.; Sherf, Bruce A.; Brunden, Kurt R.; Stricker–Krongrad, Alain

doi:10.1016/j.vascn.2005.10.003

Cited by 37 publications

(26 citation statements)

References 59 publications

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“…hERG, a representative of promiscuous targets, attracts a wide range of structurally diverse compounds to block the channel due to the relative large inner cavity compared to the other members of Kv channel family [36,40,41] . In the current screening, hERG channels were inhibited by substantially diverse compounds in the MLSMR collection with a hit rate of 27% at 10 µmol/L when 3-SD criterion was used for hERG inhibitor hit selection.…”

Section: Wwwchinapharcom Yu Hb Et Almentioning

confidence: 99%

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

Zou

Wang

et al. 2016

Acta Pharmacol Sin

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Aim: hERG potassium channels display miscellaneous interactions with diverse chemical scaffolds. In this study we assessed the hERG inhibition in a large compound library of diverse chemical entities and provided data for better understanding of the mechanisms underlying promiscuity of hERG inhibition.Methods: Approximately 300 000 compounds contained in Molecular Library Small Molecular Repository (MLSMR) library were tested. Compound profiling was conducted on hERG-CHO cells using the automated patch-clamp platform-IonWorks Quattro TM .Results: The compound library was tested at 1 and 10 μmol/L. IC 50 values were predicted using a modified 4-parameter logistic model. Inhibitor hits were binned into three groups based on their potency: high (IC 50 <1 μmol/L), intermediate (1 μmol/L< IC 50 <10 μmol/L), and low (IC 50 >10 μmol/L) with hit rates of 1.64%, 9.17% and 16.63%, respectively. Six physiochemical properties of each compound were acquired and calculated using ACD software to evaluate the correlation between hERG inhibition and the properties: hERG inhibition was positively correlative to the physiochemical properties ALogP, molecular weight and RTB, and negatively correlative to TPSA. Conclusion: Based on a large diverse compound collection, this study provides experimental evidence to understand the promiscuity of hERG inhibition. This study further demonstrates that hERG liability compounds tend to be more hydrophobic, high-molecular, flexible and polarizable.Keywords: hERG; high-throughput screening; MLSMR library; automated electrophysiology; cardiotoxicity Acta Pharmacologica Sinica (2016) 37: 111−123; doi: 10.1038/aps.2015 Original Article IntroductionThe acquired long QT syndrome is both a threat to public health and a major stumbling block for drug development [1] . It is most often caused through unintended blockade of the cardiac repolarizing potassium channel, I Kr , encoded by the Human Ether-a-go-go related gene (hERG) [2] . Blockade of hERG channel was found to be associated with an increased duration of ventricular repolarization and prolongation of QT interval (long QT syndrome, or LQTS). hERG potassium channel displays promiscuous interactions with diverse chemical scaffolds [3] . Structurally and functionally unrelated drugs have been shown to block hERG channel, and some of these agents, including terfenadine, astemizole, droperidol and cisapride, etc, have been withdrawn from the market due to their potential to predispose individuals to sudden cardiac death. Due to the important implications in both drug discovery [4] and clinical practice, evaluation of hERG liability has been required for any new chemical entities by regulatory agencies according to the ICH S7B guideline since 2005 [5] .Manual patch clamp method is considered as gold standard for ion channel functional evaluation [6] . However the method is very labor-intensive with low throughput, and thus its application in drug discovery is limited. Ion flux assays with surrogate ions of Rubidium (Rb + ) or Thallium (Tl + ) have ...

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Section: Wwwchinapharcom Yu Hb Et Almentioning

confidence: 99%

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

Zou

Wang

et al. 2016

Acta Pharmacol Sin

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“…In two reports, using either HEK-293 or CHO-K1 cells stably expressing recombinant hERG channels, IC 50 values determined in a Rb + flux assay were approximately 10-fold higher than those determined by electrophysiology, but the rank order of compounds according to potency was similar for the two assay types. 29,30 Fluorescence-based assays. The development of improved fluorescent dyes and plate readers has provided another approach to high throughput screening of ion channel activities.…”

Section: Medium and High Throughput Herg Assaysmentioning

confidence: 99%

“…It has been possible, however, to select HEK-293 and CHO-K1 cell lines stably expressing recombinant hERG channels that give rise to a depolarization signal in response to addition of extracellular potassium (50-60 mM). 29,30 At least in the HEK-293 background, this signal is the sum of hERG-dependent and hERG-independent components; a complication that probably contributes to the low signal-to-noise ratio observed in this assay. In both cell lines, IC 50 values were significantly right-shifted compared to those determined by electrophysiology, regardless of whether DiBAC 4 (3) (Invitrogen, Carlsbad, CA), FMP or Blue Several higher throughput automated electrophysiological instruments have been developed and typically use a planar substrate with holes, illustrated in Figure 5, that replaces the traditional patch pipette.…”

Section: Medium and High Throughput Herg Assaysmentioning

confidence: 99%

“…4) are very robust and reproducible and IC 50 values typically correlate well with those determined in electrophysiology. 30,36,38,39 These three non-peptide radioligands, in analogy with most small molecule hERG blockers, are thought to bind in the large inner cavity of the hERG channel and require channel opening to access their binding sites. In contrast, the scorpion venom peptide BeKm-1 preferentially blocks channels in the closed state and binds to the extracellular S5-pore linker of the channel.…”

Section: Medium and High Throughput Herg Assaysmentioning

confidence: 99%

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Role of hERG potassium channel assays in drug development

Priest¹,

Bell²,

García³

2008

Channels

115

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“…Radioligand binding assays measure displacement by compounds of interest of radiolabeled high affinity blockers such as dofetilide [30,31], astemizole [32], and MK-499 [33]. The binding assay can use native myocytes, mammalian cell lines (HEK-293 cells) expressing hERG channels, or cell membranes (for example, HEK-293 cell membranes [31,34] [31,34]. Therefore, radioligand binding assays provide a useful initial screen for potential interactions of new chemical entities.…”

Section: Non-electrophysiological Approaches For Determining Herg Chamentioning

confidence: 99%

Preclinical Drug Safety and Cardiac Ion Channel Screening

Gintant

2011

Heart Rate and Rhythm

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A new chemical entity must demonstrate efficacy and safety in order to be considered a successful therapeutic agent. Towards that goal, it is best to discover drugs demonstrating a wide concentration range between (lower) preclinical efficacious plasma concentrations and (higher) plasma concentrations eliciting preclinical off-target adverse effects. Drugs possessing such characteristics provide greater flexibility in clinical trials and explorations into alternative indications. Thus, preclinical studies related to both efficacy and safety must be considered equally important during drug discovery efforts. Cardiac safety plays a key role in defining the safety of novel therapeutics, and defining a drug's therapeutic window/safety margin.Much emphasis has been placed in the past decade on detecting (and avoiding) the ability of non-cardiovascular drugs to delay or alter ventricular repolarization (acquired long QT syndrome [1]). This focus arises in part from the association of delayed repolarization to the rare but potentially life-threatening arrhythmia torsades de pointes. As torsades is a rare event, surrogate markers are employed to detect and avoid this potential proarrhythmic risk. Preclinically, the most recognized in vitro marker for delayed repolarization is drug block of I Kr (an outward repolarizing current that initiates and defines terminal ventricular repolarization). In humans, the a-subunit of the I Kr channel is encoded by the human ether-a-go-go related gene (hERG) gene. Block of hERG/I Kr along with prolongation of the QT interval (an interval on the ECG that generally represents the onset of ventricular depolarization and the termination of ventricular repolarization) form the presentday cornerstone for cardiac electrophysiologic safety testing. Due to the importance of testing hERG current as a surrogate marker of proarrhythmia, great strides have been made in developing and streamlining testing protocols/procedures to evaluate

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Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel

Cited by 37 publications

References 59 publications

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

Role of hERG potassium channel assays in drug development

Preclinical Drug Safety and Cardiac Ion Channel Screening

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