SAR Analysis for the identification of selective inhibitors of the two-pore domain potassium channel KCNK9 in Kir2.1 expressing cells: FluxOR Assay CRC 3
Assay Implementation: Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu, Ph.D., Owen Mcmanus, Ph.D. ..more
BioActive Compound: 1
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 Kir2.1 expressing cells: FluxOR Assay CRC 3
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 inwardly-rectifying channel, Kir2.1. The protocol as in AID 1672 is used for this assay except each compound is tested at 10 concentrations. Compound inhibition of KIR2.1 was tested in at least duplicate using a thallium sensitive dye.
1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, 15 ug/mL Blasticidin S and 400 ug/mL hygromycin
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose 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 IC100 of Chlorpromaizne (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 Kir2.1 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 Kir2.1 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. Kir2.1 HEK293 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. G418 (Geneticin): (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. Chlorpromazine hydrochloride (Sigma, C8138)
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)6663 and Lot #8163495)
14. 10x HBSS (Gibco, Cat#14065)14. Triple-layer flask (VWR, Cat #62407-082)
15. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
Data interpretation: If the compound is active in this assay, it is NOT likely that it is to be specific hits for KCNK9 target. Only the compounds, which are NOT active in this assay, will be further persued for KCNK9 specific inhibitors.
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 Kir2.1 modulators. Normalization is to this set of data and cannot be used for comparison with other counter screens.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)