Authors

Discussion

Recent advances in automated patch-clamp instrumentation has opened up a range of potential high-throughput screening (HTS) strategies for ion channel targets. Automated patch-clamp, using instruments such as the Sophion Qube 384, gives a functional readout but is associated with longer timelines than more traditional fluorescence-based assays. Using a fluorescence-based assay for a large screening campaign and then automated patch-clamp in follow-up activities is an attractive approach to rapidly progress through an HTS campaign. However, it is vital that the HTS assay translates to the functional readout which is used to triage hit material.

In this example, assays have been established for a potassium ion channel using fluorescence-based (using a Molecular Devices FLIPR Penta) and automated patch-clamp platforms (on a Sophion Qube 384). During the development of the fluorescence-based assay, reagent kits manufactured by both ThermoFisher and Molecular Devices were tested. The Molecular Devices potassium assay kit was found to be more predictive of activity in the automated patch-clamp assay than the Thermo FluxORII assay reagents.

Notably, compounds with activity when tested in the automated patch-clamp assay, or with the Molecular Devices assay reagents, were inactive when tested in the fluorescence-based assay using the Thermo FluxORII reagents. The effect does not appear to be uniform, based on differing pIC₅₀ shifts for commercially available controls, nor limited to an individual subset of compounds, based on the hit rates emerging from pilot screens conducted with both sets of assay reagents.

The data highlight the importance of thorough assay validation and translation to a functional readout. Furthermore, some commonly used detection kits, although giving similar assay performance, may result in an increased false negative rate and mislead downstream hit triage/early SAR determination.