Abstract
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.
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Acknowledgements
We gratefully acknowledge the financial support from the National Natural Science Foundation of China Grant for Excellent Key Laboratory (81123004), the National Natural Science Foundation of China (61327014, 61175103 and 91413122), Shanghai Municipal Science and Technology Commission (13JC1406700) and the External Cooperation Program of BIC, Chinese Academy of Sciences (1536631KYSB20130003).
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Supplementary Table S1 is available at Acta Pharmacologica Sinica's website.
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Supplementary Information, Table S1
Potentiation activity of validated hits on KCNQ2 currents. (DOCX 252 kb)
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Yue, Jf., Qiao, Gh., Liu, N. et al. Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques. Acta Pharmacol Sin 37, 105–110 (2016). https://doi.org/10.1038/aps.2015.142
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DOI: https://doi.org/10.1038/aps.2015.142
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