Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Confirmatory, Confirmatory Screening, Single Concentration Activity Observed

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.

Name: Confirmatory screen for compounds that potentiate KCNQ2 potassium channels

Description:
See the related primary HTS assay (PubChem AID:2239). This assay serves as a confirmatory screen to verify compound activity from the primary screen.

Assay overview:

The purpose of this assay is to validate compounds identified as active in primary screen assay (PubChem AID 2239). This assay employs the same experimental conditions as presented in the primary screen assay. Compounds were tested in duplicates and their effects were evaluated by the calculated fluorescence ratio percentage, normalized with negative control. If the compound causes 3SD (of negative controls) or more activity increase (from mean of negative controls) in both duplicates, and is active in the primary screen, the compound is considered to be active.

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)-3*STDEVRatio(NC))/(AvgRatio(NC)-AvgRatio(Blank))
Percentage(%): percentage change of compound readout over those of negative controls (vehicle control) and 3 times of its standard deviation.
Ratio(cmpd): Ratio of the test compound.
AvgRatio(NC): Ratio average of the negative controls with stimulus buffer.
STDEVRatio(NC): Standard deviation of ratio of negative controls
Ratio(Blank): Ratio of the blank control without stimulus buffer.

17. Outcome assignment:
If the compound causes 3SD (of negative controls) or more activity increase (from mean of negative controls) in both duplicates, and is active in the primary screen, the compound is considered to be active. (Value=2) Otherwise, it is designated as inactive (Value=1).

18. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned a score between 0 and 100 by calculation of INT(Percentage(%)).
Percentage(%) is the percentage average of the duplicates of the test compound over the 3SD of negative controls. They are normalized to the smallest (0%) and the largest (100%).

List of reagents

1. KCNQ2-CHO cell line (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