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

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Abstract

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 V½ 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.

Introduction

Drug-induced prolongation of the cardiac action potential, which is manifested as a prolongation of the QT interval on an electrocardiogram, has led to the withdrawal of a number of medicines from the market owing to evidence that this is associated with a potentially fatal cardiac arrhythmia called Torsades de Pointes (Shah, 2005). As a result, regulatory agencies have focused on the QT interval prolongation issue, culminating in both clinical and pre-clinical guidance documents (Anon, 2005a, Anon, 2005b). The consequences for drug development of a compound prolonging the QT interval in man in the so-called “thorough QT/QTc study” make it imperative that during the drug discovery phase the risk of QT interval prolongation is minimised.

It is widely accepted that for most compounds the primary molecular mechanism for QT interval prolongation is direct inhibition of the rapid component of the delayed rectifier potassium current (IKr), which plays a key role in action potential repolarisation. It is a logical undertaking, therefore, to test compounds for IKr inhibition early in the drug discovery process and, where possible, design-out any activity. Unfortunately, the need to routinely isolate myocytes, the low amplitude of the native IKr current, the presence of contaminating currents and the low-throughput nature of microelectrode-based electrophysiology make this approach impractical. Instead, heterologous systems have been developed expressing the human ether-a-go-go-related gene (hERG) that encodes the pore-forming α sub-units of the channel carrying IKr. These hERG-expressing cell lines or their membrane fragments can be used in assays that rapidly but indirectly assess compound effects on hERG via measurement of radioligand binding, rubidium efflux, fluorescence or luminescence (Angelo et al., 2003, Chiu et al., 2004, Diaz et al., 2004, Finlayson et al., 2001, Murphy et al., 2005, Netzer et al., 2003, Sorota et al., 2005, Tang et al., 2001, Wang et al., 2003, Wible et al., 2005). However, an electrophysiology-based test is ultimately required to allow a direct assessment of effects on channel function. The rate-limiting step in generating such data in a timeframe that can influence chemical design during drug discovery is the low-throughput of conventional patch clamp electrophysiology. This has, to some extent, been removed by the invention of a 384-well plate-based planar patch clamp system (IonWorks™ HT) (Schroeder, Neagle, Trezise, & Worley, 2003). Here we describe the optimisation and validation of an IonWorks™ HT assay for the hERG-encoded potassium channel by comparing the data with those generated using conventional electrophysiology. We have also compared IonWorks™ HT data with those from an Rb+ efflux assay. The data demonstrate the utility of the IonWorks™ HT assay as a means of providing quantitative potency information based on a direct measurement of channel function. These data are comparable to those from conventional electrophysiology and can be obtained in a timeframe short enough to influence compound design.

Section snippets

For IonWorks™ HT

The hERG-expressing Chinese hamster ovary K1 (CHO) cells described by Persson, Carlsson, Duker, and Jacobson (2005) were grown to semi-confluence at 37 °C in a humidified environment (5% CO2) in F-12 Ham medium containing l-glutamine, 10% foetal calf serum (FCS) and 0.6 mg/ml hygromycin (all Sigma-Aldrich). Prior to use, the monolayer was washed using a pre-warmed (37 °C) 3 ml aliquot of Versene 1:5000 (Invitrogen). After aspiration of this solution the flask was incubated at 37 °C in an

Choice of cell line for IonWorks™ HT experiments

In order to evaluate the utility of IonWorks™ HT for estimating compound potency at hERG, we compared measurements from CHO-hERG cells in IonWorks™ HT with those from HEK-hERG cells made using conventional electrophysiology. The conventional whole-cell data pre-dated the development of the IonWorks™ HT assay but our attempts to produce a robust assay with the same HEK-hERG cells in IonWorks™ HT were unsuccessful: an insufficient proportion of recordings yielded hERG tail currents of an

Principal finding

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 influence of cell concentration on potency may be a consequence of compounds partitioning into cell membranes, with high cell concentrations having the greatest effect on free compound concentration. Similar reasoning has been used to explain the under-estimation of hERG

Acknowledgements

The authors would like to thank Dr. A Harmer for technical assistance, and Dr. M Main and Dr. MJ Waring for their critical evaluation of the manuscript.

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