Second confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4)
Assay Implementation: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Kaiping Xu M.S., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 334
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Primary, Primary Screening, Confirmatory, 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: Meng Wu Ph.D., Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Kaiping Xu M.S., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
HTS execution: Melissa Miller, Amy Scott M.S., Shunyou Long M.S., Kaiping Xu M.S., Meng Wu Ph.D.
Name: Second confirmatory screen for identification of compounds that inhibit transient receptor potential cation channel C4 (TRPC4).
See the related assay (PubChem AID: 2247).
The objective of this assay is to validate compounds that inhibit the TRPC4 cation channel. 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.
The HEK293 stable cell line was seeded into 384-well plates. After overnight incubation, the cells were loaded with a calcium-sensitive dye, Fluo4, followed by an assay buffer wash. Cell plates were then loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader, where compounds were added and incubated for 110 seconds, before application of a submaximal concentration of DAMGO. Plates were again incubated for 110 seconds before application of DAMGO at a maximal activating concentration. Real-time fluorescence was then measured for 100 seconds. Library compound effect was evaluated by computing the integrated ratio of each well used for calculation of fluorescence ratio percentage, normalized with negative controls without test compound addition. Compounds are considered active if they cause a decrease of the normalized ratio greater than the mean normalized ratio of the ECMax control minus 3SD. If the compound is active in both duplicates and is active in the primary screen, it is considered to be active as an inhibitor of the TRPC4 cation channel.
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 at 7.5uM 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 7.5uM test compound stock into the cell plates (final concentration 1uM).
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: If the compound causes a decrease of the normalized ratio greater than the mean normalized ratio of the ECMax control minus 3SD, and retains a positive integrated ratio, the compounds is considered active. If the compound is active in both duplicates and active in the primary screen, the compound is confirmed as an inhibitor hit of the TRPC4 cation channel in the outcome. Otherwise, it is designated as inactive.
18. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned a score between 0 and 100 by calculation of (abs(avPercent-min/max-min)*100) where avPercentage is the average of the duplicates of the test compound, max is the avPercentage of the most active test compound, and min is defined as the avPercentage of the least active test compound.
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)