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

Yue, Jinfeng; Qiao, Guan-Hua; Liu, Geoffrey; Gao, Zhaobing

doi:10.1038/aps.2015.142

Cited by 14 publications

(6 citation statements)

References 27 publications

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“…Before the HTS, HEK293 cell lines stably expressing human TASK-3 or its closest homolog TASK-1 were established, because selective active compounds for the TASK subfamily would have a greater tendency to exhibit selectivity among the K2P family. Then, the channel activity was measured by a thallium flux assay as in previous work (Yu et al, 2015;Yue et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

A Small-Molecule Compound Selectively Activates K2P Channel TASK-3 by Acting at Two Distant Clusters of Residues

Tian

Qiu

Lan

et al. 2019

Molecular Pharmacology

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The TASK-3 channel is a member of the K2P family that is important for the maintenance of the resting membrane potential. Previous studies have demonstrated that the TASK-3 channel is involved in several physiologic and pathologic processes, including sleep/wake control, cognition, and epilepsy. However, there is still a lack of selective pharmacological tools for TASK-3, which limits further research on channel function. In this work, using a high-throughput screen, we discovered that N-(2-((4-nitro-2-(trifluoromethyl)phenyl)amino)ethyl)benzamide (NPBA) showed excellent potency and selectivity as a novel TASK-3 activator. The molecular determinants of NPBA activation were then investigated by combining chimera and mutagenesis analysis. Two distant clusters of residues located at the extracellular end of the second transmembrane domain (A105 and A108) and the intracellular end of the third transmembrane domain (E157) were found to be critical for NPBA activation. We then compared the essentials of the actions of NPBA with inhalation anesthetics that nonselectively activate TASK-3 and found that they may activate TASK-3 channels through different mechanisms. Finally, we transplanted the three residues A105, A108, and E157 into the TASK-1 channel, which resists NPBA activation, and the constructed mutant TASK-1(G105A, V108A, A157E) showed dramatically increased activation by NPBA, confirming the importance of these two distant clusters of residues. SIGNIFICANCE STATEMENTTASK-3 channels conduct potassium and are involved in various physiological and pathological processes. However, the lack of selective modulators has hindered efforts to increase our understanding of the physiological roles of TASK-3 channels. By using a high-throughput screen, we identified NPBA as a potent and selective TASK-3 activator, and we show that NPBA is a more potent activator than terbinafine, the only reported TASK-3 selective activator to date. We also show here that NPBA has outstanding selectivity for TAS-3 channels. These characteristics make NPBA a promising pharmacological probe for research focused on defining TASK-3 channel function(s). In addition, we identified two distant clusters of residues as determinants of NPBA activation providing new molecular clues for the understanding of the gating mechanism of K2P channels.

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Section: Resultsmentioning

confidence: 99%

A Small-Molecule Compound Selectively Activates K2P Channel TASK-3 by Acting at Two Distant Clusters of Residues

Tian

Qiu

Lan

et al. 2019

Molecular Pharmacology

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“…For example, the reagents or equipment used in these methods are more expensive and technically demanding for the experimenter. More importantly, the extremely low intracellular content of Ca 2+ determines the di culty of its direct detection [21,22]. Therefore, it is di cult to meet the needs of largescale high-throughput drug screening by the above methods.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a CaCC-based Cell Model and Method for High-throughput Screening of M3 Receptor Drugs

Liu

Xiao-hong

Hong

et al. 2022

Cell Biochem Biophys

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Muscarinic acetylcholine receptor subtype 3 (M3 receptor) is a G Protein-Coupled Receptor (GPCR) that mediates many important physiological functions. Currently, no M3 receptor drugs with high speci city, high activity, and few side effects have been developed, and there is a lack of methods suitable for highthroughput screening of drugs with GPCRs. In this study, we established an e cient and sensitive drug cell screening model and method for targeting M3 receptors based on calcium-activated chloride channels (CaCCs). This screening model consists of Fischer rat thyroid follicular epithelial (FRT) cells that endogenously express M3 receptors, CaCCs, and the indicator YFP-H148Q/I152L. We veri ed that the model can sensitively detect changes in intracellular Ca 2+ concentration using uorescence quenching kinetics experiments, con rmed the screening function of the model by applying available M3 receptor drugs, and also evaluated the good performance of the model in high-throughput screening.

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“…In 2016, GlaxoSmithKline declared that the production will be discontinued after June 2017 and the product will no longer be commercially available (GlaxoSmithKline 2016). Several research groups are searching for retigabine analogues and other small-molecule compounds with a high selectivity to the Kv7.2/3 channel to reduce the risk of adverse effects (Wickenden et al 2008, Peretz et al 2010, Hu et al 2013, Yue et al 2016, Wang et al 2017). …”

Section: Kv Channels As New Targets For Antiepileptic Drugsmentioning

confidence: 99%

Molecular Mechanisms of Resin Acids and Their Derivatives on the Opening of a Potassium Channel

Ottosson¹

2017

Linköping University Medical Dissertations

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

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

Cited by 14 publications

References 27 publications

A Small-Molecule Compound Selectively Activates K2P Channel TASK-3 by Acting at Two Distant Clusters of Residues

A Small-Molecule Compound Selectively Activates K2P Channel TASK-3 by Acting at Two Distant Clusters of Residues

Establishment of a CaCC-based Cell Model and Method for High-throughput Screening of M3 Receptor Drugs

Molecular Mechanisms of Resin Acids and Their Derivatives on the Opening of a Potassium Channel

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