Evaluation of a high-throughput fluorescence assay method for HERG potassium channel inhibition.
The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z' factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependent activity of the antagonists.
Dorn
Arnulf
A
Hoffmann-La Roche Ltd., Pharma Research, Basel, Switzerland. arnulf.dorn@roche.com
Hermann
Francis
F
Ebneth
Andreas
A
Bothmann
Hendrick
H
Trube
Gerhard
G
Christensen
Klaus
K
Apfel
Christian
C
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D005456:Fluorescent Dyes / Q000737:chemistry; D018408:Patch-Clamp Techniques; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D005456:Fluorescent Dyes; D024642:Potassium Channels, Voltage-Gated