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BioAssay: AID 504920

SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC for hERG Specificity

Assay Implementation: Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D. Owen Mcmanus, Ph. D. ..more
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Inactive(5)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Inactive(5)
 
 
AID: 504920
Data Source: Johns Hopkins Ion Channel Center (K9_Inh_SAR_hERG_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-06
Hold-until Date: 2012-06-30
Modify Date: 2012-07-04

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
488964Summary of probe development for inhibitors of the two-pore domain potassium channel KCNK9summarySummary of probe development for inhibitors of the two-pore domain potassium channel KCNK9
488922Primary cell-based screen for identification of compounds that inhibit the two-pore domain potassium channel KCNK9screeningPrimary cell-based screen for identification of compounds that inhibit the two-pore domain potassium channel KCNK9
92992Inhibition of ADP induced aggregation of human platelets after 15 min of incubation of the drug with PRP prior to the addition of ADPconfirmatoryConfirmatory screen for identification of compounds that inhibit the two-pore domain potassium channel (KCNK9)
492997Second counter screen for compounds that modulate the two-pore domain potassium channel (KCNK9)screeningSecond counter screen for compounds that modulate the two-pore domain potassium channel (KCNK9)
540323SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC 2confirmatory
588741SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC 3confirmatory
623883SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against hERG: FluxOR Assay CRC 4confirmatory
Description:
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Confirmatory, Concentration-Response Relationship 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: Meng Wu, Ph.D., Johns Hopkins University, School of Medicine
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03 MH090849-01
Grant Proposal PI: Meng Wu, Ph.D., Johns Hopkins University, School of Medicine
Assay Implementation: Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D. Owen Mcmanus, Ph. D.

Name: SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC for hERG Specificity


Description:

The purpose of this assay is to assess the specificity of compounds identified as inhibitors of KCNK9. This specificity screen is to eliminate any compounds that interact with the voltage-gated potassium channel, hERG. Compound inhibition of hERG was tested in at least duplicate using a thallium sensitive dye.
Protocol
Protocol:
1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50ug/ml streptomycin,and 500ug/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 the dark
6. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer containing IC70 of dofetilide; controls are assay buffer, IC70, and ICmax of dofetilide (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 the dark
10. Prepare 5x stimulus buffer containing 25 mM K2SO4 and 7 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. Add 6 ul/well of stimulus buffer onto cells 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' factors
16. IC50 and Hill Constant calculation from replicates was generated using Microcol Origin 6.0
17. Outcome assignment: If the test compound causes a maximum inhibition of hERG greater than 30% in any concentration tested and a dose response curve is generated, the compound is considered to be active (outcome=2). If the test compound does not cause inhibition of hERG at any concentration tested or a dose response is not generated, the compound is designated as inactive (outcome=1).
16. Score assignment: Compounds with an IC50 less than 1uM are given a score of 100, 1uM-5uM a score of 75, 5uM-10uM a score of 50, 10uM-20uM a score of 25 and any compound with an IC50 greater than 20uM or those that are designated inactive in the outcome are given a score of 0.

List of reagents
1. hERG 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. 0.25% Trypsin-EDTA (Gibco, Cat#25300)
8. Geneticin: (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. Dofetilide (Fisher, NC9753685)
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
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals, or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, 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. The conditions of this assay are optimal for screening for compounds that modulate KCNK9 channels, NOT for the assay of hERG modulators. Normalization is to this set of data and cannot be used for comparison with other counter screens.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Ratio at 30uM (30μM**)Ratio for KCNK9 at the specified compound concentrationFloat
2SD at 30uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
3Ratio at 10uM (10μM**)Ratio for KCNK9 at the specified compound concentrationFloat
4SD at 10uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
5Ratio at 3.33uM (3.33μM**)Ratio for KCNK9 at the specified compound concentrationFloat
6SD at 3.33uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
7Ratio at 1.11uM (1.11μM**)Ratio for KCNK9 at the specified compound concentrationFloat
8SD at 1.11uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
9Ratio at 370nM (0.37μM**)Ratio for KCNK9 at the specified compound concentrationFloat
10SD at 370nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
11Ratio at 123nM (0.123μM**)Ratio for KCNK9 at the specified compound concentrationFloat
12SD at 123nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
13Ratio at 41.1nM (0.041μM**)Ratio for KCNK9 at the specified compound concentrationFloat
14SD at 41.1nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
15Ratio at 13.7nM (0.013μM**)Ratio for KCNK9 at the specified compound concentrationFloat
16SD at 13.7nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
17Ratio at 4.57nM (0.00457μM**)Ratio for KCNK9 at the specified compound concentrationFloat
18SD at 4.57nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
19Ratio at 1.52nM (0.00152μM**)Ratio for KCNK9 at the specified compound concentrationFloat
20SD at 1.52nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
21NNumber of replicates for KCNK9 ratio measurementsInteger
22Hill ConstantHill constant of fittingFloat
23IC50*IC50 (uM)FloatμM

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH090849-01

Data Table (Concise)
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