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BioAssay: AID 2258

Summary of probe development for potentiators of KCNQ2 potassium channels

Assay Implementation: Haibo Yu Ph.D., Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott, Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
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AID: 2258
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ2_pot_Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-01-15
Modify Date: 2013-03-27
Target
Sequence: potassium voltage-gated channel KQT-like protein 2 [Rattus norvegicus]
Description ..   
Protein Family: KCNQ voltage-gated potassium channel

Gene:KCNQ2     Related Protein 3D Structures     More BioActivity Data..
Related Experiments
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AIDNameTypeComment
2239Primary cell-based high-throughput screening assay for identification of compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Primary HTS assay for KCNQ2 potentiators with 305606 compounds tested, and 1644 are found to be acti
2282Counter screen for compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Counter screen assay against the parental CHO-K1 cell for 1189 compounds, of which 513 are active.
2283Specificity screen against KCNQ1 for compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Counter screen assay against KCNQ1 channel for 1189 compounds, and among them 331 are active.
2287Confirmatory screen for compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Confirmatory screen assay in duplicate for 1189 compounds identified through HTS, and 804 are found
2345Specificity screen against Kir2.1 for compounds that potentiate KCNQ2Screeningdepositor-specified cross reference: Counter screen assay against Kir2.1 potassium channel for 1189 compounds and 18 are active.
2408Mode of action - current amplitude concentration response for ztz240, a potentiator of KCNQ2 potassium channelsConfirmatorydepositor-specified cross reference: Measurement of current amplitude concentration response of compound ztz240 using manual patch clamp.
2415Mode of Action - subtype specificity assay for ztz240, a potentiator of KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Manual patch clamp measurement of compound ztz240 specificity in KCNQ2 and subtype channels.
2432Mode of action assay - molecular determinants for ztz240, a potentiator of KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Manual patch clamp measurement of current response for compound ztz240 in KCNQ2 and its mutant chann
2443Mode of action - deactivation constant concentration response for ztz240, a potentiator of KCNQ2 potassium channelsConfirmatorydepositor-specified cross reference: Manual patch clamp measurement of deactivation constant concentration response for ztz240 compound i
2548Mode of action assay - current amplitude concentration response for derivatives of ZTZ240, a potentiator of KCNQ2 potassium channelsConfirmatorydepositor-specified cross reference: current amplitude concentration response measurement of 4 ztz240 derivative compounds using automate
2558Mode of action - Automated patch clamp assay for KCNQ2 potentiators on Retigabine insensitive KCNQ2 Mutant W236L cell lineScreeningdepositor-specified cross reference: Screen for KCNQ2 potentiators in mutant W236L cell line using automated patch clamp for 936 compound
2603Mode of action assay-Automated electrophysiology assay of compounds that potentiate KCNQ2 potassium channelConfirmatorydepositor-specified cross reference: Current amplitude concentration response for 12 active KCNQ2 potentiators using automated patch clam
2654Dose response of Retigabine-insensitive compounds that potentiate KCNQ2 potassium channelConfirmatorydepositor-specified cross reference: Current amplitude concentration response for 2 active KCNQ2 potentiators with dissimilar structure t
493037Mode of action assay-Confirmatory dose response assay for compounds that activate KCNQ2 potassium channelsConfirmatorydepositor-specified cross reference
493038Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorydepositor-specified cross reference: Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on aut
493039Mode of action assay-Dose response assay for KCNQ2 activators in the KCNQ2/KCNQ3 co-expressing cells on automated patch clampConfirmatorydepositor-specified cross reference: Mode of action assay-Dose response assay for KCNQ2 activators in the KCNQ2/KCNQ3 co-expressing cells
493042Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1/KCNE1 expressing cells on automated patch clampConfirmatorydepositor-specified cross reference: Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 pot
493043Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ4 expressing cells on automated patch clampConfirmatorydepositor-specified cross reference: Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 pot
493044Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 potassium channels in the KCNQ1 expressing cells on automated patch clampConfirmatorydepositor-specified cross reference: Mode of action assay-Dose response assay for the identification of selective activators of KCNQ2 pot
493046Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ5 expressing cellsConfirmatorydepositor-specified cross reference: Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ5 expressing cells.
493047Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ3 expressing cellsConfirmatorydepositor-specified cross reference: Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ3 expressing cells.
493113Mode of action assay-SAR analysis for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorydepositor-specified cross reference: Mode of action assay-SAR analysis for compounds that activate KCNQ2 potassium channels on automated
504416Mode of action assay- Specificity dose response assay for the KCNQ2 activators in the KCNQ4 expressing cells by the Tl fluxConfirmatorydepositor-specified cross reference: Mode of action assay- Specificity dose response assay for the KCNQ2 activators in the KCNQ4 expressi
504417Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1 expressing cells by the Tl flux assayConfirmatorydepositor-specified cross reference: Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1 expressin
504418Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1/E1 expressing cells by the Tl fluxConfirmatorydepositor-specified cross reference: Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1/E1 expres
588635SAR-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiologyOthersame project related to Summary assay
Description:
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Min Li, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA027716-01
Grant Proposal PI: Min Li, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Haibo Yu Ph.D., Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott, Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.

Description:

Voltage-gated potassium (K) channels are critical for neuronal function in excitable tissues such as brain and heart. They are also found in non-excitable tissues important for other functions such as hormone secretion, oxygen-sensing and immune responses. There are more than 100 genes in human genome encoding different but homologous potassium channels. Voltage-gated K+ channels, exemplified by the Shaker K+ channel, share considerable sequence similarity. Isolation and characterization of bioactive chemical probes could form important pharmacological foundation, providing a great deal of insights into the structure and function.

The KCNQ (or also called Kv7) channel family includes five members, KCNQ1 to KCNQ5; KCNQ are members of Kv channel superfamily[1-6]. Different from other Kv channel classes, KCNQ channels commonly display a sub-threshold opening (i.e., at a voltage near resting potential) and are regulated by GPCR signaling, e.g., muscarinic receptors [7, 8]. KCNQ2-5 channels are primarily expressed in the brain, while KCNQ1 is abundantly expressed in cardiac tissue. Among congenital long QT patients, a disease which affects 1 in 2,500 live births, the most prevalent mutations are within the KCNQ1 allele. Mutations in KCNQ2 and KCNQ3 are known to cause benign familial neonatal convulsion (BFNC), a rare form of epilepsy [9].

In the mid-90s, a compound known as D-23129, or retigabine, was initially developed to target GABAnergic signaling [10, 11]. Despite its poor effect on the GABA pathway, retigabine was shown to have potent anti-convulsant activity. In 1997, retigabine was also found to induce the opening of voltage-gated potassium channels at resting membrane potentials, and since then, through a variety of other studies, this has been established to be its main mechanism of action. Retigabine, acting on KCNQ2/3 potassium channels which code M-currents, is now being used for treatment of epilepsy. None of the anti-convulsants that are in clinical use today have a comparable mechanism of action, i.e., through ligand activation of voltage-gated potassium channels. Several potentiation compounds are synthesized according to the scaffold of retigabine. However, considering the poor potency of retigabine and several other undesirable characteristics including its broad action on KCNQ2, 3, 4, and 5 [12], it is not clear whether these related structures will fundamentally improve therapeutic efficacy. Hence, it argues for the performance of a non-biased screen for new structures with potentiation activity.

Systemic compound screens for M-current have not been reported. Because of the rapid progress of molecular cloning and functional characterization, KCNQ2 has been validated as the key molecular target of M-current. It is therefore feasible to design non-biased high-throughput screens specifically targeting to KCNQ2 channels. This justifies use of the Tl+-based fluorescence assay in 384-well format by FDSS for the identification of modulatory compounds acting on KCNQ2 from a large compound library.

References:
1. Charlier, C. et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat. Genet. 1998.?18, 53-55, PMID: 9425900
2. Gutman, G.A. et al. International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol. Rev. 2003. 55, 583-586, PMID: 14657415
3. Kubisch, C. et al. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell. 1999. 96, 437-446, PMID: 10025409
4. Schroeder, B.C., Hechenberger, M., Weinreich, F., Kubisch, C. & Jentsch, T.J. KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents. J. Biol. Chem. 2000, 275, 24089-24095,PMID: 10816588.
5. Singh, N.A. et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat. Genet. 1998.18, 25-29, PMID: 9425895.
6. Wang, Q. et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat. Genet. 1996, 12, 17-23, PMID: 8528244.
7. Brown, D.A. et al. Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature. 1980. 283, 673-676. PMID: 6965523.
8. Marrion, N.V. Control of M-current. Annu. Rev. Physiol.1997.59, 483-504, PMID: 9074774.
9. Maljevic, S. et al. Nervous system KV7 disorders: breakdown of a subthreshold brake. J Physiol. 2008. 586, 1791-1801. PMID: 18238816.
10. Rostock, A. et al. D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures. Epilepsy Res. 1996. 23, 211-223. PMID: 8739124
11. Tober, C., Rostock, A., Rundfeldt, C. & Bartsch, R. D-23129: a potent anticonvulsant in the amygdala kindling model of complex partial seizures. Eur. J. Pharmacol. 1996. 303, 163-169. PMID: 8813562
12. Gribkoff, V.K. (2003) The therapeutic potential of neuronal KCNQ channel modulators. Expert Opin. Ther. Targets. 2003. 7, 737-748. PMID: 14640909.
Protocol
Please see the related bioassays (e.g., AID 2239) for details of assay protocols.
Comment
Result of this screening campaign has yielded a probe compound, ML213.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML213
PubChem Substance ID (SID) for probe 1: 104223736
PubChem Compound ID (CID) for probe 1: 3111211
Probe type for probe 1: Activator
IC50/EC50 (nM) for probe 1: <366
Target for probe 1: KCNQ2 (multiple variants) (gi: 18959272)
Anti-target for probe 1: KCNQ1_Act_Tl, KCNQ3_Tl
Fold selectivity for probe 1: 2.2-80
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK133435/ (ID: 3025446)
Grant number for probe 1: DA027716-01
NCBI Book chapter title for probe 1: A small molecule activator of KCNQ2 and KCNQ4 channels
Additional Information
Grant Number: 1 R03 DA027716-01

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