Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel.
BACKGROUND
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 (3)H-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was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points.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 (3)H-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 muM) 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).


CONCLUSIONS
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.
Murphy
Steven M
SM
Athersys, Inc., 3201 Carnegie Avenue, Cleveland, OH 44115, USA.
Palmer
Marc
M
Poole
Michelle Fontilla
MF
Padegimas
Linas
L
Hunady
Karen
K
Danzig
Joel
J
Gill
Sikander
S
Gill
Rajwant
R
Ting
Anthony
A
Sherf
Bruce
B
Brunden
Kurt
K
Stricker-Krongrad
Alain
A
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D011869:Radioligand Assay / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D011994:Recombinant Proteins