Jin-feng Yue1, Guan-hua Qiao1, Ni Liu2,3, Fa-jun Nan2, Zhao-bing Gao1. 1. CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, State Key Laboratory of Drug Research, Chinese Academy of Sciences, Shanghai 201203, China. 2. The National Center for Drug Screening, Shanghai Institute of Materia Medica, State Key Laboratory of Drug Research, Chinese Academy of Sciences, Shanghai 201203, China. 3. Department of Chemistry and Institute of Medicinal Chemistry, East China Normal University, Shanghai 200062, China.
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.
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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