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BioAssay: AID 493047

Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ3 expressing cells

Assay Implementation: Haibo Yu Ph.D., Kaiping Xu M.S., Shunyou Long M.S., Meng Wu Ph.D., David Meyers Ph.D., Owen Mcmanus Ph.D. ..more
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 Tested Compounds
 Tested Compounds
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Inactive(5)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Inactive(5)
 
 
AID: 493047
Data Source: Johns Hopkins Ion Channel Center (KCNQ3_Act_Tl_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-01-20
Hold-until Date: 2011-12-06
Modify Date: 2011-12-06

Data Table ( Complete ):           View All Data
Target
Sequence: potassium voltage-gated channel subfamily KQT member 3 [Rattus norvegicus]
Description ..   

Gene:KCNQ3          More BioActivity Data..
Tested Compounds:
Related Experiments
Show more
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
2258Summary of probe development for potentiators of KCNQ2 potassium channelsSummarydepositor-specified cross reference: Summary assay for the KCNQ2 potentiators.
2287Confirmatory screen for compounds that potentiate KCNQ2 potassium channelsScreeningdepositor-specified cross reference: Confirmatory screen in duplicates for 1189 compounds and 804 of them are found active.
2282Counter screen for compounds that potentiate KCNQ2 potassium channelsScreeningsame project related to Summary assay
2283Specificity screen against KCNQ1 for compounds that potentiate KCNQ2 potassium channelsScreeningsame project related to Summary assay
2345Specificity screen against Kir2.1 for compounds that potentiate KCNQ2Screeningsame project related to Summary assay
2408Mode of action - current amplitude concentration response for ztz240, a potentiator of KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
2415Mode of Action - subtype specificity assay for ztz240, a potentiator of KCNQ2 potassium channelsScreeningsame project related to Summary assay
2432Mode of action assay - molecular determinants for ztz240, a potentiator of KCNQ2 potassium channelsScreeningsame project related to Summary assay
2443Mode of action - deactivation constant concentration response for ztz240, a potentiator of KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
2548Mode of action assay - current amplitude concentration response for derivatives of ZTZ240, a potentiator of KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
2558Mode of action - Automated patch clamp assay for KCNQ2 potentiators on Retigabine insensitive KCNQ2 Mutant W236L cell lineScreeningsame project related to Summary assay
2603Mode of action assay-Automated electrophysiology assay of compounds that potentiate KCNQ2 potassium channelConfirmatorysame project related to Summary assay
2654Dose response of Retigabine-insensitive compounds that potentiate KCNQ2 potassium channelConfirmatorysame project related to Summary assay
493037Mode of action assay-Confirmatory dose response assay for compounds that activate KCNQ2 potassium channelsConfirmatorysame project related to Summary assay
493038Mode of action assay-Dose response assay for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorysame project related to Summary assay
493039Mode of action assay-Dose response assay for KCNQ2 activators in the KCNQ2/KCNQ3 co-expressing cells on automated patch clampConfirmatorysame project related to Summary assay
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 clampConfirmatorysame project related to Summary assay
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 clampConfirmatorysame project related to Summary assay
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 clampConfirmatorysame project related to Summary assay
493046Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ5 expressing cellsConfirmatorysame project related to Summary assay
493113Mode of action assay-SAR analysis for compounds that activate KCNQ2 potassium channels on automated patch clampConfirmatorysame project related to Summary assay
504416Mode of action assay- Specificity dose response assay for the KCNQ2 activators in the KCNQ4 expressing cells by the Tl fluxConfirmatorysame project related to Summary assay
504417Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1 expressing cells by the Tl flux assayConfirmatorysame project related to Summary assay
504418Mode of action assay-Specificity dose response assay for the KCNQ2 activators in the KCNQ1/E1 expressing cells by the Tl fluxConfirmatorysame project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (KCNQ3_Act_Tl_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Name: Mode of action assay-Specificity test for the KCNQ2 activators in the KCNQ3 expressing cells

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., Kaiping Xu M.S., Shunyou Long M.S., Meng Wu Ph.D., David Meyers Ph.D., Owen Mcmanus 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. Isolation and characterization of bioactive chemical probes could form important pharmacological foundation, providing a great deal of insights into the structure and function.

The M-type channels are unique voltage-gated and ligand-regulated K+ channels with their distinct physiological and pharmacological characteristics. They are activated at a voltage near the threshold for action potential initiation and regulate membrane excitability. The KCNQ (or also called Kv7), members of Kv channel superfamily, includes five members, KCNQ1 to KCNQ5. Among them homodimer and/or heterodimer of KCNQ2 and KCNQ3 are believed components of the M-channel. The modulators of KCNQ2 are playing an important role in the neuronal function regulation. As the therapeutic targets, the KCNQ2 openers have great potential used to treat epilepsy, pain and anxiety et al.


Principle of the assay
The purpose of the assay is to examine the non-specific effects of KCNQ2 activators on the KCNQ3 expressing cells. It employs the same experimental conditions as presented in the primary screen assay, except in multiple concentrations. Compounds were tested in triplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio.

Keywords:

KCNQ2, KCNQ3, agonist, activator, potentiator, Concentration Response Curve, Thallium, FluxOR, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Protocol for the KCNQ3 screen:
1. Cell culture: CHO-K1 cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 ug/ml streptomycin.
2. 24 hr before the test, CHO-K1 cells were transiently transfected with KCNQ3 plasmids and Lipofectamine LTX with Plus reagents.
3. Remove medium and add 25 ul /well of 1x FluxOR solution to cells.
4. Incubate 90 minutes at room temperature (RT) in darkness.
5. Prepare 7.5X compound plates and control plates on the Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0), ICmax of ZTZ240 (all with DMSO concentrations matched to that of test compounds).
6. Remove FluxOR dye solution and add 20 ul /well of assay buffer to cells.
7. Add 4 ul of 7.5x compound stock into the cell plates via Cybi-Well system.
8. Incubate all cell plates for 20 minutes at RT in darkness.
9. Prepare 5x stimulus buffer containing 12.5 mM K2SO4 and 12.5 mM Tl2SO4.
10. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader.
11. Measure fluorescence for 10 seconds at 1Hz to establish baseline.
12. Depolarize cells with 6 ul/well of stimulus buffer and continue measuring fluorescence for 110 seconds.
13. Calculate ratio readout as F(max-min)/F0.
14. Calculate the percentage of tested compounds with the following formula:
Percentage (%)=100* (Ratio(cmpd)- AvgRatio(NC))/(AvgRatio(NC)-AvgRatio(Blank))
Percentage(%): percentage change of compound readout over those of negative controls (vehicle control)
Ratio(cmpd): Ratio of the test compound.
AvgRatio(NC): Ratio average of the negative controls with stimulus buffer.
Ratio(Blank): Ratio of the blank control without stimulus buffer.

15. Outcome assignment:
If the test compound causes potentiation or inhibition effect on the KCNQ3 in any concentrations tested and the dose response is generated, the compound is considered to be active.
If the test compound does not cause potentiation or inhibition effect on the KCNQ3 in any concentrations tested or a dose response is not generated, the compound is designated as inactive.
16. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.

List of reagents

1. CHO-K1 cell lines (provided by JHICC)
2. PBS: pH7.4 (Gibco, Cat#10010)
3. Medium: DMEM/F12 50/50 (Mediatech, Cat#15-090-CV)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. 200 mM L-Glutamine (Gibco, Cat#25030)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. 0.05% Trypsin-EDTA (Gibco, Cat#25300)
8. Geneticin: (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. ZTZ-240 (Synthesized)
11. FluxOR detection kit (Invitrogen, Cat #F10017): FluxOR, assay buffer and stimulus buffer.
12. Triple-layer flask (VWR, Cat #62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
Comment
Comments
Possible artifacts of this assay may include, but are not limited to: unintended chemicals or dust in or under the wells of the microtiter plate, or compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The condition is optimal for screening for compounds that modulate KCNQ2 potassium channels, not for the assay of the KCNQ3 modulators. Normalization is to this set of data and cannot be used for comparison with other counter screens.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: CHO-K1
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1potentiation at 14nM (0.014μM**)% potentiation for KCNQ3 at the specified concentrationFloat
2SD at 14nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
3potentiation at 41nM (0.041μM**)% potentiation for KCNQ3 at the specified concentrationFloat
4SD at 41nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
5Potentiation at 120nM (0.12μM**)% potentiation for KCNQ3 at the specified concentrationFloat
6SD at 120nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
7Potentiation at 370nM (0.37μM**)% potentiation for KCNQ3 at the specified concentrationFloat
8SD at 370nMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
9Potentiation at 1.1uM (1.1μM**)% potentiation for KCNQ3 at the specified concentrationFloat
10SD at 1.1uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
11Potentiation at 3.3uM (3.3μM**)% potentiation for KCNQ3 at the specified concentrationFloat
12SD at 3.3uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
13Potentiation at 10uM (10μM**)% potentiation for KCNQ3 at the specified concentrationFloat
14SD at 10uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
15Potentiation at 30uM (30μM**)% potentiation for KCNQ3 at the specified concentrationFloat
16SD at 30uMStandard Deviation of measurement for % potentiation at the specified concentrationFloat
17EC50*mean EC50 of compound potentiation of on KCNQ3FloatμM
18EC50 SDStandard deviation of mean of EC50FloatμM
19Nnumber of repeats for EC50 of compound potentiationInteger
20HillHill constantFloat
21Hill SDStandard deviation of Hill constantFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA027716-01

Data Table (Concise)
Data Table ( Complete ):     View All Data
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