Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel
Introduction: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report e...
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creator | Murphy, Steven M. Palmer, Marc Poole, Michelle Fontilla Padegimas, Linas Hunady, Karen Danzig, Joel Gill, Sikander Gill, Rajwant Ting, Anthony Sherf, Bruce Brunden, Kurt Stricker-Krongrad, Alain |
description | Introduction: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery.
Methods: Competitive binding assays were developed using
3H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points.
Results: Binding assays using HEK293-hERG membranes and
3H-dofetilide were robust (
Z′
=
0.69
±
0.015, mean
±
S.E.M.), highly reproducible (test–retest slope
=
1.04,
r
2
=
0.98), and correlated well with IC
50 values obtained by patch clamp (slope
=
0.98,
r
2
=
0.89). Binding assays using IMR-32 membranes were less sensitive (
Z′
=
0.4
±
0.03, mean
±
S.E.M., false negative rate
=
0.4) but still correlated well with patch clamp data (slope
=
1.06,
r
2
=
0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC
50
<
0.1 μM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate
=
0.5). Finally, the rubidium efflux assay gave highly reproducible results (
Z′
=
0.80
±
0.02, mean
±
S.E.M.) that correlated with patch clamp IC
50 values (slope
=
0.87,
r
2
=
0.73).
Discussion: The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery. |
doi_str_mv | 10.1016/j.vascn.2005.10.003 |
format | Article |
fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_68621853</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S1056871905001383</els_id><sourcerecordid>21046061</sourcerecordid><originalsourceid>FETCH-LOGICAL-c303t-fc7c314985583b6127d2c4d9b622619c8b8aaa0c851a6281a6c32081fa82d70f3</originalsourceid><addsrcrecordid>eNqFkU9v1DAQxS1ERUvhEyAhn7hl8Z_EcQ4cULUtSJWQKpC4WRN70vUqsRc7WdFvj9NdiRtc7NH4N-On9wh5x9mGM64-7jdHyDZsBGNN6WwYky_IFdetrGqtf74sNWtUpVveXZLXOe9ZITpevyKXXEmhONdXZNoeYVxg9jHQONBhCXatYaQQHO19cD48UsgZnjL1gVocx0zx9yFhzusT-nmHiSa0cSo4hJnGRDG4-IghLpnutg931O4gBBzfkIsBxoxvz_c1-XG7_X7zpbr_dvf15vN9ZSWTczXY1kped7pptOwVF60TtnZdr0SR3VndawBgVjcclNDlsFIwzQfQwrVskNfkw2nvIcVfC-bZTD6v0iFg0WSUVoLrRv4XFJzViileQHkCbYo5JxzMIfkJ0pPhzKxxmL15jsOscazNYnaZen9ev_QTur8zZ_8L8OkEYHHj6DGZbD0Gi84XR2fjov_nB38AWoSddA</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>21046061</pqid></control><display><type>article</type><title>Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel</title><source>MEDLINE</source><source>ScienceDirect Journals (5 years ago - present)</source><creator>Murphy, Steven M. ; Palmer, Marc ; Poole, Michelle Fontilla ; Padegimas, Linas ; Hunady, Karen ; Danzig, Joel ; Gill, Sikander ; Gill, Rajwant ; Ting, Anthony ; Sherf, Bruce ; Brunden, Kurt ; Stricker-Krongrad, Alain</creator><creatorcontrib>Murphy, Steven M. ; Palmer, Marc ; Poole, Michelle Fontilla ; Padegimas, Linas ; Hunady, Karen ; Danzig, Joel ; Gill, Sikander ; Gill, Rajwant ; Ting, Anthony ; Sherf, Bruce ; Brunden, Kurt ; Stricker-Krongrad, Alain</creatorcontrib><description>Introduction: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery.
Methods: Competitive binding assays were developed using
3H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points.
Results: Binding assays using HEK293-hERG membranes and
3H-dofetilide were robust (
Z′
=
0.69
±
0.015, mean
±
S.E.M.), highly reproducible (test–retest slope
=
1.04,
r
2
=
0.98), and correlated well with IC
50 values obtained by patch clamp (slope
=
0.98,
r
2
=
0.89). Binding assays using IMR-32 membranes were less sensitive (
Z′
=
0.4
±
0.03, mean
±
S.E.M., false negative rate
=
0.4) but still correlated well with patch clamp data (slope
=
1.06,
r
2
=
0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC
50
<
0.1 μM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate
=
0.5). Finally, the rubidium efflux assay gave highly reproducible results (
Z′
=
0.80
±
0.02, mean
±
S.E.M.) that correlated with patch clamp IC
50 values (slope
=
0.87,
r
2
=
0.73).
Discussion: The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.</description><identifier>ISSN: 1056-8719</identifier><identifier>EISSN: 1873-488X</identifier><identifier>DOI: 10.1016/j.vascn.2005.10.003</identifier><identifier>PMID: 16326118</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>3H-dofetilide ; Animals ; Arrhythmias ; Binding assay ; Cell Line ; CHO Cells ; Cricetinae ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels - biosynthesis ; Ether-A-Go-Go Potassium Channels - metabolism ; hERG ; Humans ; Membrane potential dye ; Protein Binding - physiology ; QT prolongation ; Radioligand Assay - methods ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - metabolism ; Rubidium efflux ; Torsades de Pointes</subject><ispartof>Journal of pharmacological and toxicological methods, 2006-07, Vol.54 (1), p.42-55</ispartof><rights>2005 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c303t-fc7c314985583b6127d2c4d9b622619c8b8aaa0c851a6281a6c32081fa82d70f3</citedby><cites>FETCH-LOGICAL-c303t-fc7c314985583b6127d2c4d9b622619c8b8aaa0c851a6281a6c32081fa82d70f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.vascn.2005.10.003$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27922,27923,45993</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16326118$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Murphy, Steven M.</creatorcontrib><creatorcontrib>Palmer, Marc</creatorcontrib><creatorcontrib>Poole, Michelle Fontilla</creatorcontrib><creatorcontrib>Padegimas, Linas</creatorcontrib><creatorcontrib>Hunady, Karen</creatorcontrib><creatorcontrib>Danzig, Joel</creatorcontrib><creatorcontrib>Gill, Sikander</creatorcontrib><creatorcontrib>Gill, Rajwant</creatorcontrib><creatorcontrib>Ting, Anthony</creatorcontrib><creatorcontrib>Sherf, Bruce</creatorcontrib><creatorcontrib>Brunden, Kurt</creatorcontrib><creatorcontrib>Stricker-Krongrad, Alain</creatorcontrib><title>Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel</title><title>Journal of pharmacological and toxicological methods</title><addtitle>J Pharmacol Toxicol Methods</addtitle><description>Introduction: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery.
Methods: Competitive binding assays were developed using
3H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points.
Results: Binding assays using HEK293-hERG membranes and
3H-dofetilide were robust (
Z′
=
0.69
±
0.015, mean
±
S.E.M.), highly reproducible (test–retest slope
=
1.04,
r
2
=
0.98), and correlated well with IC
50 values obtained by patch clamp (slope
=
0.98,
r
2
=
0.89). Binding assays using IMR-32 membranes were less sensitive (
Z′
=
0.4
±
0.03, mean
±
S.E.M., false negative rate
=
0.4) but still correlated well with patch clamp data (slope
=
1.06,
r
2
=
0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC
50
<
0.1 μM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate
=
0.5). Finally, the rubidium efflux assay gave highly reproducible results (
Z′
=
0.80
±
0.02, mean
±
S.E.M.) that correlated with patch clamp IC
50 values (slope
=
0.87,
r
2
=
0.73).
Discussion: The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.</description><subject>3H-dofetilide</subject><subject>Animals</subject><subject>Arrhythmias</subject><subject>Binding assay</subject><subject>Cell Line</subject><subject>CHO Cells</subject><subject>Cricetinae</subject><subject>ERG1 Potassium Channel</subject><subject>Ether-A-Go-Go Potassium Channels - biosynthesis</subject><subject>Ether-A-Go-Go Potassium Channels - metabolism</subject><subject>hERG</subject><subject>Humans</subject><subject>Membrane potential dye</subject><subject>Protein Binding - physiology</subject><subject>QT prolongation</subject><subject>Radioligand Assay - methods</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - metabolism</subject><subject>Rubidium efflux</subject><subject>Torsades de Pointes</subject><issn>1056-8719</issn><issn>1873-488X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9v1DAQxS1ERUvhEyAhn7hl8Z_EcQ4cULUtSJWQKpC4WRN70vUqsRc7WdFvj9NdiRtc7NH4N-On9wh5x9mGM64-7jdHyDZsBGNN6WwYky_IFdetrGqtf74sNWtUpVveXZLXOe9ZITpevyKXXEmhONdXZNoeYVxg9jHQONBhCXatYaQQHO19cD48UsgZnjL1gVocx0zx9yFhzusT-nmHiSa0cSo4hJnGRDG4-IghLpnutg931O4gBBzfkIsBxoxvz_c1-XG7_X7zpbr_dvf15vN9ZSWTczXY1kped7pptOwVF60TtnZdr0SR3VndawBgVjcclNDlsFIwzQfQwrVskNfkw2nvIcVfC-bZTD6v0iFg0WSUVoLrRv4XFJzViileQHkCbYo5JxzMIfkJ0pPhzKxxmL15jsOscazNYnaZen9ev_QTur8zZ_8L8OkEYHHj6DGZbD0Gi84XR2fjov_nB38AWoSddA</recordid><startdate>200607</startdate><enddate>200607</enddate><creator>Murphy, Steven M.</creator><creator>Palmer, Marc</creator><creator>Poole, Michelle Fontilla</creator><creator>Padegimas, Linas</creator><creator>Hunady, Karen</creator><creator>Danzig, Joel</creator><creator>Gill, Sikander</creator><creator>Gill, Rajwant</creator><creator>Ting, Anthony</creator><creator>Sherf, Bruce</creator><creator>Brunden, Kurt</creator><creator>Stricker-Krongrad, Alain</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U7</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>200607</creationdate><title>Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel</title><author>Murphy, Steven M. ; Palmer, Marc ; Poole, Michelle Fontilla ; Padegimas, Linas ; Hunady, Karen ; Danzig, Joel ; Gill, Sikander ; Gill, Rajwant ; Ting, Anthony ; Sherf, Bruce ; Brunden, Kurt ; Stricker-Krongrad, Alain</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c303t-fc7c314985583b6127d2c4d9b622619c8b8aaa0c851a6281a6c32081fa82d70f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>3H-dofetilide</topic><topic>Animals</topic><topic>Arrhythmias</topic><topic>Binding assay</topic><topic>Cell Line</topic><topic>CHO Cells</topic><topic>Cricetinae</topic><topic>ERG1 Potassium Channel</topic><topic>Ether-A-Go-Go Potassium Channels - biosynthesis</topic><topic>Ether-A-Go-Go Potassium Channels - metabolism</topic><topic>hERG</topic><topic>Humans</topic><topic>Membrane potential dye</topic><topic>Protein Binding - physiology</topic><topic>QT prolongation</topic><topic>Radioligand Assay - methods</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - metabolism</topic><topic>Rubidium efflux</topic><topic>Torsades de Pointes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Murphy, Steven M.</creatorcontrib><creatorcontrib>Palmer, Marc</creatorcontrib><creatorcontrib>Poole, Michelle Fontilla</creatorcontrib><creatorcontrib>Padegimas, Linas</creatorcontrib><creatorcontrib>Hunady, Karen</creatorcontrib><creatorcontrib>Danzig, Joel</creatorcontrib><creatorcontrib>Gill, Sikander</creatorcontrib><creatorcontrib>Gill, Rajwant</creatorcontrib><creatorcontrib>Ting, Anthony</creatorcontrib><creatorcontrib>Sherf, Bruce</creatorcontrib><creatorcontrib>Brunden, Kurt</creatorcontrib><creatorcontrib>Stricker-Krongrad, Alain</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Toxicology Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of pharmacological and toxicological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Murphy, Steven M.</au><au>Palmer, Marc</au><au>Poole, Michelle Fontilla</au><au>Padegimas, Linas</au><au>Hunady, Karen</au><au>Danzig, Joel</au><au>Gill, Sikander</au><au>Gill, Rajwant</au><au>Ting, Anthony</au><au>Sherf, Bruce</au><au>Brunden, Kurt</au><au>Stricker-Krongrad, Alain</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel</atitle><jtitle>Journal of pharmacological and toxicological methods</jtitle><addtitle>J Pharmacol Toxicol Methods</addtitle><date>2006-07</date><risdate>2006</risdate><volume>54</volume><issue>1</issue><spage>42</spage><epage>55</epage><pages>42-55</pages><issn>1056-8719</issn><eissn>1873-488X</eissn><abstract>Introduction: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery.
Methods: Competitive binding assays were developed using
3H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points.
Results: Binding assays using HEK293-hERG membranes and
3H-dofetilide were robust (
Z′
=
0.69
±
0.015, mean
±
S.E.M.), highly reproducible (test–retest slope
=
1.04,
r
2
=
0.98), and correlated well with IC
50 values obtained by patch clamp (slope
=
0.98,
r
2
=
0.89). Binding assays using IMR-32 membranes were less sensitive (
Z′
=
0.4
±
0.03, mean
±
S.E.M., false negative rate
=
0.4) but still correlated well with patch clamp data (slope
=
1.06,
r
2
=
0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC
50
<
0.1 μM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate
=
0.5). Finally, the rubidium efflux assay gave highly reproducible results (
Z′
=
0.80
±
0.02, mean
±
S.E.M.) that correlated with patch clamp IC
50 values (slope
=
0.87,
r
2
=
0.73).
Discussion: The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>16326118</pmid><doi>10.1016/j.vascn.2005.10.003</doi><tpages>14</tpages></addata></record> |
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language | eng |
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source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
subjects | 3H-dofetilide Animals Arrhythmias Binding assay Cell Line CHO Cells Cricetinae ERG1 Potassium Channel Ether-A-Go-Go Potassium Channels - biosynthesis Ether-A-Go-Go Potassium Channels - metabolism hERG Humans Membrane potential dye Protein Binding - physiology QT prolongation Radioligand Assay - methods Recombinant Proteins - biosynthesis Recombinant Proteins - metabolism Rubidium efflux Torsades de Pointes |
title | Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel |
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