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
BioActive Compounds: 35
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
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.
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)
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)