Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques.

AIM

To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators.

METHODS

KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique.

RESULTS

From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively.

CONCLUSION

The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators.