Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks™ HT

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• 作者：M.H. Bridgland-Taylor ; A.C. Hargreaves ; A. Easter ; A. Orme ; D.C. Henthorn ; M. Ding ; A.M. Davis ; B.G. Small ; C.G. Heapy ; N. Abi-Gerges et al.
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2006
• 期刊代码：28_10568719
• 类别：pha
• 出版时间：September-October 2006
• 卷：54
• 期：2
• 页码：189-199
• 文件大小：418 K

摘要

Introduction

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 V_½ − 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.

Discussion

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.