AID	Name	Type	Comment
2239	Primary cell-based high-throughput screening assay for identification of compounds that potentiate KCNQ2 potassium channels	screening	Primary HTS assay for KCNQ2 potentiators with 305606 compounds tested, and 1644 are found to be active.
2287	Confirmatory screen for compounds that potentiate KCNQ2 potassium channels	screening	Confirmatory screen in duplicates for 1189 compounds and 804 of them are found active.
2258	Summary of probe development for potentiators of KCNQ2 potassium channels	summary	Summary assay for the KCNQ2 potentiators.

Data Source: Johns Hopkins Ion Channel Center (KCNQ2_Act_Tl_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Name: Mode of action assay-Confirmatory dose response assay for compounds that activate KCNQ2 potassium channels

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.

Name: Mode of action assay-Confirmatory dose response assay for compounds that activate KCNQ2 potassium channels


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 the confirmatory dose response for the compounds identified as active in the primary screen (PubChem AID: 2239) on the KCNQ2 potassium channel. 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,agonist, activator, potentiator, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.

Protocol for the KCNQ2 screen:
1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 ug/ml streptomycin, and 500 ug/ml G418.
2. Cell plating: Add 50 ul/well of 120,000 cells/ml re-suspended in DMEM/F12 medium with 10% FBS.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 25 ul /well of 1x FluxOR solution to cells.
5. Incubate 90 minutes at room temperature (RT) in darkness.
6. 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).
7. Remove FluxOR dye solution and add 20 ul /well of assay buffer to cells.
8. Add 4 ul of 7.5x compound stock into the cell plates via Cybi-Well system.
9. Incubate all cell plates for 20 minutes at RT in darkness.
10. Prepare 5x stimulus buffer containing 12.5 mM K2SO4 and 12.5 mM Tl2SO4.
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader.
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline.
13. Depolarize cells with 6 ul/well of stimulus buffer and continue measuring fluorescence for 110 seconds.
14. Calculate ratio readout as F(max-min)/F0.
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z'.
16. 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.

17. Outcome assignment:
If the test compound causes potentiation effect on KCNQ2 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 effect on KCNQ2 in any concentrations tested or a dose response is not generated, the compound is designated as inactive.

18. 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. KCNQ2-CHO 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)

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, compounds that non-specifically modulate the cell host or the targeted activity, compounds that induce K/Tl flux independent of KCNQ2, compounds that directly interact with the Tl sensitive dye molecule, and 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.

Grant Number: 1 R03 DA027716-01