Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks HT.
BACKGROUND
Regulatory and competitive pressure to reduce the QT interval prolongation risk of potential new drugs has led to focus on methods to test for inhibition of the human ether-a-go-go-related gene (hERG)-encoded K+ channel, the primary molecular target underlying this safety issue. Here we describe the validation of a method that combines medium-throughput with direct assessment of channel function.


METHODS
The electrophysiological and pharmacological properties of hERG were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and an indirect assay (Rb+ efflux).


RESULTS
Basic electrophysiological properties of hERG were similar whether recorded conventionally (HEK cells) or using IonWorks HT (CHO cells): for example, tail current V1/2 -12.1+/-5.0 mV (32) for conventional and -9.5+/-6.0 mV (46) for IonWorks HT (mean+/-S.D. (n)). A key finding was that as the number of cells per well was increased in IonWorks HT, the potency reported for a given compound decreased. Using the lowest possible cell concentration (250,000 cells/ml) and 89 compounds spanning a broad potency range, the pIC50 values from IonWorks HT (CHO-hERG) were found to correlate well with those obtained using conventional methodology (HEK-hERG)(r=0.90; p<0.001). Further validation using CHO-hERG cells with both methods confirmed the correlation (r=0.94; p<0.001). In contrast, a comparison of IonWorks HT and Rb+ efflux data with 649 compounds using CHO-hERG cells showed that the indirect assay consistently reported compounds as being, on average, 6-fold less potent, though the differences varied depending on chemical series.


CONCLUSIONS
The main finding of this work is that providing a relatively low cell concentration is used in IonWorks HT, the potency information generated correlates well with that determined using conventional electrophysiology. The effect on potency of increasing cell concentration may relate to a reduced free concentration of test compound owing to partitioning into cell membranes. In summary, the IonWorks HT hERG assay can generate pIC50 values based on a direct assessment of channel function in a timeframe short enough to influence chemical design.
Bridgland-Taylor
M H
MH
Safety Assessment UK, AstraZeneca R&D Alderley Park, Macclesfield, SK10 4TG, UK.
Hargreaves
A C
AC
Easter
A
A
Orme
A
A
Henthorn
D C
DC
Ding
M
M
Davis
A M
AM
Small
B G
BG
Heapy
C G
CG
Abi-Gerges
N
N
Persson
F
F
Jacobson
I
I
Sullivan
M
M
Albertson
N
N
Hammond
T G
TG
Sullivan
E
E
Valentin
J-P
JP
Pollard
C E
CE
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology / Q000295:instrumentation*; D051638:Ether-A-Go-Go Potassium Channels / Q000187:drug effects*; D006801:Humans; D018408:Patch-Clamp Techniques / Q000295:instrumentation*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D015203:Reproducibility of Results; D012413:Rubidium / Q000378:metabolism
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D026902:Potassium Channel Blockers; D012413:Rubidium