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BioAssay: AID 492981

SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD Assay

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(36)
 
 
Active(7)
 
 
Inactive(26)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(36)
 
 
Active(7)
 
 
Inactive(26)
 
 
Inconclusive(3)
 
 
AID: 492981
Data Source: Johns Hopkins Ion Channel Center (JHICC_C4_Inh_SAR_C6_MPD)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-12-14
Hold-until Date: 2011-06-15
Modify Date: 2011-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 7
Related Experiments
Show more
AIDNameTypeProbeComment
2247Primary cell-based screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4).Screening depositor-specified cross reference: HTS assay for TRPC4 inhibitors with 304007 test compounds, and among them 1189 are active
2256Summary of efforts to identify compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Summary3 depositor-specified cross reference: Summary assay for TRPC4 inhibitors.
2636Confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
2637Second confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
434942Confirmation dose response assay for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Confirmatory same project related to Summary assay
434948Specificity screen against TRPC6 for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
434978Second counter screen for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Screening same project related to Summary assay
492983SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Manual ElectrophysiologyOther same project related to Summary assay
492987SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD Assay CRCConfirmatory same project related to Summary assay
492988SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Primary HTS assayScreening same project related to Summary assay
492991SAR Analysis for the identification of selective inhibits of the transient receptor potential cation channel C4 (TRPC4): Automated ElectrophysiologyConfirmatory same project related to Summary assay
492994SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Primary HTS assay CRCConfirmatory same project related to Summary assay
492995SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): TRPC4 Serotonin Receptor CRC MPD assayConfirmatory same project related to Summary assay
492996SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): ACh assay CRCConfirmatory same project related to Summary assay
504554SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6) in cells expressing TRPC4: C4 assay CRCConfirmatory same project related to Summary assay
504589SAR Analysis for the identification of selective inhibits of the transient receptor potential cation channel C4 (TRPC4): Automated Electrophysiology_2Confirmatory same project related to Summary assay
504866SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6) in cells expressing TRPC4: C4 assay CRC 2Confirmatory same project related to Summary assay
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC_TRPC4_Inh_Val)
BioAssay Type: Other, Duplicate, Single 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: Michael Zhu, Ph.D., Ohio State University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS056942-01
Grant Proposal PI: Michael Zhu, Ph.D., Ohio State University
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 transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD Assay


Description:

See the related assay (PubChem AID: 2247).
Protocol
Assay overview:

The purpose of this assay is to assess the specificity of compounds identified as inhibitors of the transient receptor potential cation channel C4 (TRPC4). This specific counter screen is to eliminate any compounds that interact with TRPC6, rather than TRPC4 mu-opioid receptor expressing HEK293 cells. The protocol as in AID 2553 is used for this assay. Compounds were tested in at least duplicate and their effects were evaluated by the calculated integrated fluorescence ratio percentage, normalized with positive controls.

Protocol:

1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, and 400 ug/mL G418
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS, 50 IU/ml penicillin, and 50 ug/mL streptomycin
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of Membrane Potential Dye
5. Incubate 45 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 (EC0), ECmax of Acetylcholine
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Measure fluorescence for 5 seconds at 1Hz to establish baseline
9. Add 4ul of 7.5x compound stock into the cell plates.
10. Incubate plates for 110 seconds
11. Add 6ul maximally activating concentration of Acetylcholine (Acetylcholine ECmax) and read for another 110 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors
14. Outcome assignment: The integrated ratio for each compound was averaged over the replicas and those that resulted in a normalized integrated ratio less than the mean normalized integrated ratio of the ECMax control minus 5SD were considered active (Outcome=2). Otherwise, it is designated as inactive (Outcome=1).

15. Score assignment: An inactive test compound is assigned the score of 0. An active test compound is assigned a score between 5 and 100 by calculating Int((Lg(Abs(avPercentInhibition))-1.41)*263.7)

List of reagents
1. TRPC6-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)
2. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma, Cat# D5796-500ML)
3. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)
4. 100x Penicillin-Streptomycin (Mediatech, Cat# 30-001-CI)
5. CellStripper (Mediatech, Cat# 25-056-Cl)
6. G418: (Invitrogen, Cat# 11811-031)
7. HEPES (Sigma, Cat# H4034)
8. 10XHBSS (#Invitrogen Cat# 14065056)
9. Acetylcholine chloride (Sigma, Cat# A6625)
10. 2-APB (Sigma, Cat# D9754)
11. Membrane Potential Assay Kit, Blue (Molecular Devices, Cat# R8034)
12. Triple-layer flask (VWR, Cat# 62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot# 8281903)
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; the specific concentration for a particular test compound may vary, based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1avPercentInhibition (10μM**)Percentage of the test compound readout over those of controls at a test compound concentration of 10uM.Float

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS056942-01

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