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BioAssay: AID 588724

SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9 III

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(43)
 
 
Active(35)
 
 
Inactive(8)
 
 
 Tested Substances
 Tested Substances
All(45)
 
 
Active(36)
 
 
Inactive(9)
 
 
AID: 588724
Data Source: Johns Hopkins Ion Channel Center (KCNK9_Inh_SAR_CRC_3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-30
Hold-until Date: 2012-10-24
Modify Date: 2012-10-24

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 35
Related Experiments
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AIDNameTypeComment
488922Primary cell-based screen for identification of compounds that inhibit the two-pore domain potassium channel KCNK9Screeningdepositor-specified cross reference: Primary cell-based screen for identification of compounds that inhibit the two-pore domain potassium
488964Summary of probe development for inhibitors of the two-pore domain potassium channel KCNK9Summarydepositor-specified cross reference: Summary of probe development for inhibitors of the two-pore domain potassium channel KCNK9
492992Confirmatory screen for identification of compounds that inhibit the two-pore domain potassium channel (KCNK9)Screeningdepositor-specified cross reference: Confirmatory screen for identification of compounds that inhibit the two-pore domain potassium chann
492997Second counter screen for compounds that modulate the two-pore domain potassium channel (KCNK9)Screeningdepositor-specified cross reference: Second counter screen for compounds that modulate the two-pore domain potassium channel (KCNK9)
504846SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9Confirmatorydepositor-specified cross reference
492993Specificity screen against Kir2.1 for compounds that modulate the two-pore domain potassium channel (KCNK9)Screeningsame project related to Summary assay
504902SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRCConfirmatorysame project related to Summary assay
504920SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC for hERG SpecificityConfirmatorysame project related to Summary assay
504922SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRCConfirmatorysame project related to Summary assay
540321SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
540322SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9 IIConfirmatorysame project related to Summary assay
540323SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
540324SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
588741SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in hERG expressing cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588759SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588760SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588761SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNQ2 expressing cells: FluxOR Assay CRCConfirmatorysame project related to Summary assay
588776SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNK3 expressing cells: FluxOR Assay CRC 1Confirmatorysame project related to Summary assay
588798SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNQ2 expressing cells: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
588800SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in KCNQ2 expressing cells: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
623881SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623883SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against hERG: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623884SAR Analysis for the identification of selective inhibitors of KCNK9 against parental cells: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623886SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNQ2: FluxOR Assay CRCConfirmatorysame project related to Summary assay
623887SAR analysis for compounds that inhibit the two-pore domain potassium channel KCNK9 4Confirmatorysame project related to Summary assay
623894SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNK3: FluxOR Assay CRC 3Confirmatorysame project related to Summary assay
623898SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9): Automated ElectrophysiologyConfirmatorysame project related to Summary assay
623912SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNK3: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
623913SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNQ2: FluxOR Assay CRC 4Confirmatorysame project related to Summary assay
623914SAR Analysis for the identification of inhibitors of the two-pore domain potassium channel KCNK9 - Selectivity assay against KCNQ2: FluxOR Assay CRC 2Confirmatorysame project related to Summary assay
623915SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9) -selectivity assay against KCNK3: Automated ElectrophysiologyConfirmatorysame project related to Summary assay
624121SAR analysis for the identification of selective inhibitors of the two-pore domain potassium channel (KCNK9): Manual ElectrophysiologyOthersame project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC)
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 compounds that inhibit the two-pore domain potassium channel KCNK9 III


Description:

The purpose of this assay is to test dose dependent responses for compounds that inhibit KCNK9. The same protocol is employed for this assay as the primary screen (AID: 488922), except that each compound is tested at multiple concentrations. A HEK293 cell line which stably expresses KCNK9 was employed, and channel activity monitored by use of a thallium sensitive dye (FluxORtrade mark). Compounds that inhibit KCNK9 result in less accumulation of intracellular thallium. This decrease in thallium will result in a change in the fluorescence of the Flux OR 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 300,000 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; controls are assay buffer (IC0), and ICmax of SID17386958
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' prime factors
16. IC50 and Hill Constant calculation from replicates was generated using Microcol Origin 6.0
17. Outcome assignment: If the test compound causes inhibition of KCNK9 in any concentration tested, and a dose response is generated, the compound is considered to be active (outcome=2).
If the test compound does not cause inhibition of KCNK9 at any concentration tested or a dose response is not generated, the compound is designated as inactive (outcome=1).
18. 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. KCNK9-expressing HEK293 Cells (provided by Sojin Shikano, PhD, DVM, University of Illinois at Chicago)
2. Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Mediatech, Cat#10-013-CV)
3. Fetal Bovine Serum (Gibco, Cat#26140)
4. L-Glutamine (Invitrogen, Cat#25030081)
5. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
6. CellStripper (Mediatech, Cat#25-056-Cl)
7. Blasticidin S (Research Products Internationl Corp., Cat#B12150)
8. Hygromycin (Mediatech, Cat#30-240-CR)
9. HEPES (Sigma, Cat#H4034)
10. 10XHBSS (Invitrogen, Cat#14065056)
11. Tetracycline (SIGMA, Cat#T7660)
12. SID 17386958 (Chembridge, Cat#8926102)
13. FluxOR detection kit (Invitrogen, Cat#F10017)
14. Triple-layer flask (VWR, Cat#62407-082)
15. 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*IC50 (uM)FloatμM
2NNumber of replicates for KCNK9 ratio measurementsInteger
3Hill ConstantHill constant of fittingFloat
4Ratio at 30uM (30μM**)Ratio for KCNK9 at the specified compound concentrationFloat
5SD at 30uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
6Ratio at 10uM (10μM**)Ratio for KCNK9 at the specified compound concentrationFloat
7SD at 10uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
8Ratio at 3.33uM (3.33μM**)Ratio for KCNK9 at the specified compound concentrationFloat
9SD at 3.33uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
10Ratio at 1.11uM (1.11μM**)Ratio for KCNK9 at the specified compound concentrationFloat
11SD at 1.11uMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
12Ratio at 370nM (0.37μM**)Ratio for KCNK9 at the specified compound concentrationFloat
13SD at 370nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
14Ratio at 123nM (0.123μM**)Ratio for KCNK9 at the specified compound concentrationFloat
15SD at 123nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
16Ratio at 41.1nM (0.041μM**)Ratio for KCNK9 at the specified compound concentrationFloat
17SD at 41.1nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
18Ratio at 13.7nM (0.013μM**)Ratio for KCNK9 at the specified compound concentrationFloat
19SD at 13.7nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
20Ratio at 4.57nM (0.00457μM**)Ratio for KCNK9 at the specified compound concentrationFloat
21SD at 4.57nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat
22Ratio at 1.52nM (0.00152μM**)Ratio for KCNK9 at the specified compound concentrationFloat
23SD at 1.52nMStandard Deviation of measurement of KCNK9 ratio at the specified concentrationFloat

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

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