Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and NaV1.5 blockade

Introduction

The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa_V1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture.

Methods

Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds.

Results

Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC₅₀s (paired values denote constantly cultured and frozen, respectively) of 4.8 ± 0.4 and 5.1 ± 0.4, 1.4 ± 0.1 and 1.1 ± 0.1, 24.4 ± 2.4 and 21.9 ± 1.8, 2.1 ± 0.4 and 2.1 ± 0.1, 5.2 ± 0.4 and 4.0 ± 0.2 μM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa_V1.5 with IC₅₀s of 46.6 ± 4.3 and 28.0 ± 2.3, 7.6 ± 0.7 and 6.2 ± 0.5, 153.5 ± 13.0 and 106.0 ± 4.7, 5.5 ± 0.5 and 4.8 ± 0.2 μM. Voltage dependences of activation (V_1/2) for hERG were statistically identical, 0.4 ± 0.8 mV and 2.5 ± 0.5 mV. In hNa_V1.5, the V_1/2 of inactivation and activation were statistically identical, −82.7 ± 0.1 mV versus − 84.9 ± 0.3 mV, −47.5 ± 0.3 mV versus − 45.0 ± 0.6 mV. Current density in both conditions in hERG experiments was similar, 47.0 ± 4.1 pA versus 42.3 ± 6.0 pA/pF.

Discussion

hERG and hNa_V1.5 screens run using frozen cells have statistically identical IC₅₀s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.