SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Primary HTS assay
Assay Implementation: Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
BioActive Compounds: 19
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): Primary HTS assay
See the related assay (PubChem AID: 2247).
The objective of this assay is to confirm the activity of compounds that inhibit the TRPC4 cation channel using the same assay as the primary HTS screen. A HEK293 cell line which stably expresses both the TRPC4beta and mu-Opioid receptor was employed, and channel activity was monitored by use of Fluo4, a cell-permeable calcium-sensitive dye. Compounds that inhibit TRPC4 result in a decrease in calcium flux in the presence of a mu-Opioid receptor agonist, DAMGO. This reduction in Ca2+ will result in a decreased fluorescence of Fluo4, as compared to control wells.
Protocol for the TRPC4 Validation project:
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.
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. 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).
18. 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 calculation of INT((Lg (avPercentInhibition)-1.61)*280) where avPercentInhibition as in Result Definitions
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 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.
** Test Concentration.
Data Table (Concise)