AID	Name	Type	Comment
2247	Primary cell-based screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4).	screening	Primary HTS assay for TRPC4 inhibitors with 304007 compounds where 1189 were identified as active.
2256	Summary of efforts to identify compounds that inhibit transient receptor potential cation channel C4 (TRPC4)	summary

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, Amy Scott, Shunyou Long M.S., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D., Meng Wu Ph.D.


Name: Confirmation dose response assay for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)

Description:
See the related assay (PubChem AID: 2247)

The purpose of this assay is to confirm dose dependent responses for compounds that inhibit the transient receptor potential cation channel C4 (TRPC4) as identified in the primary screen (AID: 2247). The same protocol is employed for this assay as the primary screen, except that each compound is tested at multiple concentrations. Compound inhibition of TRPC4 is tested in duplicate using a calcium-sensitive fluorescent dye.

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, 500 ug/mL G418 and 40 ug/mL hygromycin
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, 50 ug/mL streptomycin
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of 1x Fluo4 solution to cells
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), and ECmax of DAMGO
7. Remove Fluo4 dye solution and add 40 ul/well of assay buffer to cells
8. Remove 40 ul solution and add 20 ul/well of assay buffer to cells
9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 5 seconds at 1Hz to establish baseline
11. Add 4 ul of 7.5x compound stock into the cell plates.
12. Incubate plates for 110 seconds
13. Add submaximal concentration of DAMGO and incubate for 110 seconds
14. Add maximally activating concentration of DAMGO (DAMGO ECmax) and read for another 110 seconds.
15. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
16. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors
17. The National Center for Chemical Genomics CurveFit program was used to generate EC50 and Hill Constant
18. Outcome assignment: If the test compound causes inhibition of TRPC4 in any concentration tested, a dose response is generated, AND the compound was picked up as an inhibitor of TRPC4 in the primary screen the compound is considered to be active (outcome=2).
If the test compound does not cause inhibition of TRPC4 at any concentration tested, a dose response is not generated, OR the compounds was not an inhibitor hit in the primary screen, the compound is designated as inactive (outcome=1).
19. Score assignment: An inactive test compound is assigned a score of 0. An active test compound is assigned a score of 100.

List of reagents
1. TRPC4 and mu-Opioid Receptor-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)
2. PBS: pH7.4 (Invitrogen Cat#10010023)
3. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma D5796-500ML)
4. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)
5. L-Glutamine (Invitrogen, Cat#25030081)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. CellStripper (Mediatech 25-056-Cl)
8. G418: (Invitrogen, Cat#11811-031)
9. Hygromycin#(Mediatech, Cat#30-240-CR)
10. HEPES (Sigma, Cat#H4034)
11. 10XHBSS (#Invitrogen Cat#14065056)
12. Pluronic F-127 (20% solution in DMSO) (Invitrogen Cat#P3000MP)
13. [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) (Sigma Cat#E7384-10mg10MG)
14. Fluo-4 Calcium Assay Kit (Invitrogen, Cat # F14202)
15. Triple-layer flask (VWR, Cat #62407-082)
16. 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, 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.

Grant Number: 1 R21 NS056942-01