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BioAssay: AID 492987

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

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(45)
 
 
Active(16)
 
 
Inactive(29)
 
 
 Tested Substances
 Tested Substances
All(45)
 
 
Active(16)
 
 
Inactive(29)
 
 
AID: 492987
Data Source: Johns Hopkins Ion Channel Center (JHICC_C4_Inh_SAR_C6_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-12-14
Hold-until Date: 2011-06-15
Modify Date: 2011-06-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 16
Related Experiments
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AIDNameTypeProbeComment
2247Primary cell-based screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4).Screening depositor-specified cross reference: Primary HTS assay for TRPC4 inhibitors with 304007 compounds where 1189 were identified as active.
2256Summary of efforts to identify compounds that inhibit transient receptor potential cation channel C4 (TRPC4)Summary3 depositor-specified cross reference
2601Summary of efforts in the identification of inhibitors of transient receptor potential cation channel C6 (TRPC6)Summary depositor-specified cross reference: Summary of efforts in the identification of inhibitors of transient receptor potential cation channe
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
492981SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD AssayScreening 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
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
2553High throughput screening of inhibitors of transient receptor potential cation channel C6 (TRPC6)Screening same project related to Summary assay
2696Confirmation dose response assay for compounds that inhibit transient receptor potential cation channel C6 (TRPC6).Confirmatory same project related to Summary assay
2776Second specificity screen against TRPC4 for compounds that inhibit transient receptor potential cation channel C6 (TRPC6)Screening same project related to Summary assay
2777Specificity screen against TRPC4 for compounds that inhibit transient receptor potential cation channel C6 (TRPC6)Screening same project related to Summary assay
2779Second counter screen for compounds that inhibit transient receptor potential cation channel C6 (TRPC6)Screening same project related to Summary assay
2780Counter screen for compounds that inhibit transient receptor potential cation channel C6 (TRPC6)Screening same project related to Summary assay
488960Confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C6 (TRPC6)Screening 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
504567SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6) in parental cell line: Counter assay CRCConfirmatory same project related to Summary assay
504572SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6): HTS Assay CRCConfirmatory 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
504885SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C6 (TRPC6): HTS Assay CRC 2Confirmatory same project related to Summary assay
Description:
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: Mike Zhu Ph.D., Ohio State University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS056942-01
Grant Proposal PI: Mike Zhu Ph.D.
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 CRC


Description:
See the related assay (PubChem AID: 2247)
Protocol
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 specificity screen is to eliminate any compounds that interact with the TRPC6 channel, rather than TRPC4 mu-opioid receptor expressing HEK293 cells. The protocol as in AID 2553 is used for this assay except each compound is tested at 10 concentrations. Compound inhibition of TRPC6 was tested in at least duplicate using a membrane potential sensitive dye.
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' factors
14. IC50 and Hill Constant calculation from replicates was generated using Microcol Origin 6.0
15. Outcome assignment: If the test compound causes a maximum inhibition of TRPC6 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 TRPC6 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. 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, 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ratio at 20uM (20μM**)Ratio for TRPC4 at the specified compound concentration Float
2SD at 20uM (20μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
3ratio at 6.67uM (6.67μM**)Ratio for TRPC4 at the specified compound concentrationFloat
4SD at 6.67uM (6.67μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
5ratio at 2.22uM (2.22μM**)Ratio for TRPC4 at the specified compound concentrationFloat
6SD at 2.22uM (2.22μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
7ratio at 740.74nM (0.74074μM**)Ratio for TRPC4 at the specified compound concentrationFloat
8SD at 740.74nM (0.74074μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
9ratio at 246.91nM (0.24691μM**)Ratio for TRPC4 at the specified compound concentrationFloat
10SD at 246.91nM (0.24691μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
11ratio at 82.3nM (0.0823μM**)Ratio for TRPC4 at the specified compound concentrationFloat
12SD at 82.3nM (0.0823μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
13ratio at 27.43nM (0.02743μM**)Ratio for TRPC4 at the specified compound concentrationFloat
14SD at 27.43nM (0.02743μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
15ratio at 9.14nM (0.00914μM**)Ratio for TRPC4 at the specified compound concentrationFloat
16SD at 9.14nM (0.00914μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
17ratio at 3.05nM (0.00305μM**)Ratio for TRPC4 at the specified compound concentrationFloat
18SD at 3.05nM (0.00305μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
19ratio at 1.02nM (0.00102μM**)Ratio for TRPC4 at the specified compound concentrationFloat
20SD at 1.02nM (0.00102μM**)Standard Deviation of measurement of TRPC4 ratio at the specified concentrationFloat
21NNumber of replicates for TRPC4 ratio measurementsInteger
22Hill constantHill constant of fittingFloat
23IC50 *IC50 (uM)FloatμM

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

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