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BioAssay: AID 2236

hERG counter screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

Assay Implementation: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Haibo Yu Ph.D., Beiyan Zou Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
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 Tested Compounds
 Tested Compounds
All(320)
 
 
Active(74)
 
 
Inactive(246)
 
 
 Tested Substances
 Tested Substances
All(320)
 
 
Active(74)
 
 
Inactive(246)
 
 
AID: 2236
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_Counter_hERG CHO)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-01-11

Data Table ( Complete ):           Active    All
Target
Sequence: putative potassium channel subunit [Homo sapiens]
Description ..   
Protein Family: CAP_ED

Gene:KCNH2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 74
Depositor Specified Assays
AIDNameTypeComment
1672Primary cell-based high-throughput screening assay for identification of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1screeningPrimary HTS assay for potassium channel Kir2.1 where 305616 compounds were screened.
1843Summary of probe development for inhibitors/blockers of inward-rectifying potassium ion channel Kir2.1summary
Description:
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 MH084087-01
Grant Proposal PI: Elena Makhina Ph.D., University of Pittsburgh
Assay Implementation: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Haibo Yu Ph.D., Beiyan Zou Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.

Name: hERG counter screen 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 to counter screen the compounds that identified in the primary screen assay (PubChem AID 1672) against their non-specific effects on hERG (KCNH2 potassium voltage-gated channel, subfamily H (eag-related), member 2 [Homo sapiens]) CHO cells. It employs the same experimental conditions as presented in the primary screen assay, except the usage of hERG stably expressed CHO cells, instead of Kir2.1-HEK293 cells. Compounds were tested in duplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio percentage normalized with negative controls without compounds. If the compound causes 3SD (of negative controls) or more activity decrease in both duplicates, the compound is considered to be active that might have non-specific effects on the hERG stably expressed CHO cells. It is NOT specific to inward rectifying potassium ion channel Kir2.1.

Protocol for hERG counter screen of 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: 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; 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. Calculate the percentages of tested compounds with the following formula:
Percentage (%) = (Ratio(cpd)- Ratio(Blank))/(Ratio(NC) - Ratio(Blank))*100
Where:
Percentage (%):Percentage of the test compound readout over those of negative controls at a concentration of 10 uM
Ratio(cpd): Ratio of the test compound
Ratio(Blank): Ratio of the blank control without stimulus buffer
Ratio(NC): Ratio of the negative controls with stimulus buffer
17. Outcome assignment:
If the compound causes 3SD (of negative controls) or more Percentage (%) decrease AND the fluorescence readout of the compound is within 3SD of the fluorescence of the negative controls, the compound is considered to be active.
If the compound is active in both duplicates, the compound is confirmed as active as a non-specific modulator for the hERG stably expressed CHO cells in the Outcome. Otherwise, it is designated as inactive.
18. Score assignment:
An inactive test compound is assigned the score as 0.
An active test compound is assigned a score between 4 and 100 by calculation of Integer(Abs((1.75-(Log10([AveragePercentage])))*250)), where avPercentage is the average of the duplicates of the test compound.

List of reagents

1. hERG stably expressed CHO cells (provided by JHICC)
2. Others (See PubChem AID 1672)
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, 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. The condition is optimal for screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1, NOT for the assay of hERG stably expressed CHO cell modulators. Normalization is to this set of data and cannot be used for comparison with other counter screens.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1avPercentage (10μM**)Calculated results of the tested compounds of Integer (100-[AveragePercentage]) of the duplicates of the test compound readout (Percentage (%)) at a concentration of 10 uM.Float

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084087-01

Data Table (Concise)
Classification
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