Validation of a [3H]astemizole binding assay in HEK293 cells expressing HERG K+ channels.
A radioligand binding assay for the HERG (human ether-a-go-go-related gene) K(+) channel was developed to identify compounds which may have inhibitory activity and potential cardiotoxicity. Pharmacological characterization of the [(3)H]astemizole binding assay for HERG K(+) channels was performed using HERG-expressing HEK293 cells. The assay conditions employed yielded 90% specific binding using 10 microg/well of membrane protein with 1.5 nM of [(3)H]astemizole at 25 degrees C. The K(d) and B(max) values were 5.91 +/- 0.81 nM and 6.36 +/- 0.26 pmol/mg, respectively. The intraassay and interassay variations were 11.4% and 14.9%, respectively. Binding affinities for 32 reference compounds (including dofetilide, cisapride, and terfenadine) with diverse structures demonstrated a similar potency rank order for HERG inhibition to that reported in the literature. Moreover, the [(3)H]astemizole binding data demonstrated a rank order of affinity that was highly correlated to that of inhibitory potency in the electrophysiological studies for HERG in HEK293 (r(SP) = 0.91, P<0.05). In conclusion, the [(3)H]astemizole binding assay is rapid and capable of detecting HERG inhibitors.
Chiu
Peter J S
PJ
MDS Pharma Services, 22011 30th Drive SE, Bothell, WA 98021-4444, USA. peter.chiu@mdsps.com
Marcoe
Karen F
KF
Bounds
Sidney E
SE
Lin
Chun-Hsiung
CH
Feng
Jin-Jye
JJ
Lin
Atsui
A
Cheng
Fong-Chi
FC
Crumb
William J
WJ
Mitchell
Richard
R
D016589:Astemizole / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D008024:Ligands; D018408:Patch-Clamp Techniques; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism*; D011869:Radioligand Assay; D013997:Time Factors; D014162:Transfection; D014316:Tritium
D002121:Calcium Channel Blockers; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D008024:Ligands; D024642:Potassium Channels, Voltage-Gated; D014316:Tritium; D016589:Astemizole