Bookmark and Share
BioAssay: AID 2594

Confirmation dose response assay for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

Assay Implementation: Meng Wu Ph.D., Shunyou Long M.S., Haibo Yu Ph.D., Hao-ran Wang Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D. ..more
_
   
 Tested Compounds
 Tested Compounds
All(25)
 
 
Active(22)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(25)
 
 
Active(22)
 
 
Inactive(3)
 
 
AID: 2594
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_inhibitors_Tl_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-17

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: inward rectifier potassium channel 2 [Mus musculus]
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 22
Related Experiments
Show more
AIDNameTypeProbeComment
1672Primary cell-based high-throughput screening assay for identification of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening depositor-specified cross reference: Primary HTS of 305616 compounds where 2592 were identified as active.
1843Summary of probe development for inhibitors/blockers of inward-rectifying potassium ion channel Kir2.1Summary3 depositor-specified cross reference: Summary assay for Kir2.1 inhibitor assays.
2032Confirmatory screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2105Counter screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2236hERG counter screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2329KCNK9 specificity screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Screening same project related to Summary assay
2345Specificity screen against Kir2.1 for compounds that potentiate KCNQ2Screening same project related to Summary assay
2404Manual electrophysiological patch clamp assay and ROMK specificity of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Confirmatory same project related to Summary assay
2581Automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Confirmatory same project related to Summary assay
2591Manual electrophysiological patch clamp assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Confirmatory same project related to Summary assay
463252Secondary automated electrophysiology assay of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1Other same project related to Summary assay
504555Dose response assay on hERG potassium channel for Kir2.1 inhibitors on automated electrophysiologyConfirmatory same project related to Summary assay
504557Confirmation dose response assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1.Confirmatory same project related to Summary assay
504559Dose response assay for compounds that inhibit inward-rectifying potassium channel Kir2.1 on automated electrophysiologyConfirmatory same project related to Summary assay
504693Dose response assay on hERG potassium channel for Kir2.1 inhibitors on automated electrophysiology (II)Confirmatory same project related to Summary assay
504694Dose response assay for compounds that inhibit inward-rectifying potassium channel Kir2.1 on automated electrophysiology (II)Confirmatory same project related to Summary assay
504695Confirmation dose response assay for SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1 (II)Confirmatory same project related to Summary assay
504828Manual electrophysiological patch clamp assay for extended SAR compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1_1Confirmatory same project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Confirmatory, Confirmatory Screening, Multiple 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: Elena Makhina Ph.D., University of Pittsburgh
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA026212-01
Grant Proposal PI: Elena Makhina Ph.D., University of Pittsburgh
Assay Implementation: Meng Wu Ph.D., Shunyou Long M.S., Haibo Yu Ph.D., Hao-ran Wang Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D.

Name: Confirmation dose response assay for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1.

Description:

See the related essay (PubChem AID: 1672).
Protocol
Assay overview:
The purpose of this assay is for the confirmation dose responses for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1, that identified in the primary screen assay (PubChem AID 1672) and consequent specificity screens (PubChem AID 1843). It employs the same experimental conditions as presented in the primary screen assay, except in multiple concentrations. Compounds were tested in triplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio.
Protocol for the Kir2.1 project:
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: Cells were re-suspended in DMEM/F12 medium with 10% FBS at 300,000 cells/ml and added to the 384 well microtiter plates at 50 ul/well
3. Incubate overnight at 37#C and 5% CO2
4. Remove medium and add 25 ul/well of 1x FluxOR solution
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
8. Add 4 ul of 7.5x compound stock into the cell plates using the 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. Outcome assignment:
If the test compound causes inhibition of the Kir2.1 in any concentrations tested AND a dose response is generated, the compound is considered to be active.
If the test compound does not cause inhibition of the Kir2.1 current in any concentrations tested OR a dose response is not generated, the compound is designated as inactive.
17. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
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)
Comment
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals, dust in or on wells of the microtiter plate, or 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.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ratio at 57uM (57μM**)Ratio for Kir2.1 at the specified compound concentration Float
2SD at 57uMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
3ratio at 19uM (19μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
4SD at 19uMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
5ratio at 6.3uM (6.3μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
6SD at 6.3uMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
7ratio at 2.1uM (2.1μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
8SD at 2.1uMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
9ratio at 0.71uM (0.71μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
10SD at 0.71uMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
11ratio at 235nM (0.235μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
12SD at 235nMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
13ratio at 78nM (0.078μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
14SD at 78nMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
15ratio at 26nM (0.026μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
16SD at 26nMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
17ratio at 8.7nM (0.0087μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
18SD at 8.7nMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
19ratio at 2.9nM (0.0029μM**)Ratio for Kir2.1 at the specified compound concentrationFloat
20SD at 2.9nMStandard Deviation of measurement of Kir2.1 ratio at the specified concentrationFloat
21NNumber of replicates for Kir2.1 ratio measurementsInteger
22IC50 *IC50 (uM)FloatμM
23IC50 SDStandard deviation of IC50FloatμM
24HillHill constant of fittingFloat
25Hill SDStandard deviation of Hill constantFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA026212-01

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