Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks

The safety implications of blocking the human cardiac Na + channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function. The electrophysiological and pharmacological properties of hNav1.5 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 canine cardiac Purkinje Fibre action potential upstroke data. Activation curve parameters for hNav1.5 in IonWorks™ HT were not statistically different ( p > 0.05) from those generated using conventional electrophysiology. IonWorks™ HT V 1/2 = − 22 ± 0.8 mV, slope = 6.9 ± 0.2 ( n = 11); conventional electrophysiology V 1/2 = − 20 ±1.6 mV, slope = 6.4 ± 0.3 ( n = 11). Potency values for a range of hNav1.5 blockers determined using IonWorks™ HT correlated closely with those obtained using conventional electrophysiology ( R = 0.967, p < 0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity ( V max) showed that the IonWorks™ HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks™ HT or IonWorks™ Quattro, although the latter yielded more consistent data. The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na + channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.