SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): Primary HTS assay CRC
Assay Implementation: Melissa Miller, Shunyou Long M.S., David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
BioActive Compounds: 23
Depositor Specified Assays
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): Primary HTS assay CRC
Transient receptor potential (TRP) channels act as key regulators of many sensory systems including those for thermo-, photo-, and osmosensation [1-3]. A diversity of biological disorders has been linked to TRP channel malfunction ranging from neuropathic pain , cardiovascular diseases , to other diseases involving sensory physiology [6,7].
The TRP family of cation channels can be separated into seven subtypes according to structural similarity. Members of this family are nonselective cation channels that allow entry of extracellular calcium into cells resulting in a depolarization of membrane potential . The mammalian TRPC (canonical) subfamily is proposed to function as store and second-messenger operated cation channels . Disruption of TRPC may cause aberrant modulation of intracellular calcium and changes in membrane potential leading to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy. Currently there are few pharmacological modulators of the TRPC family and no compounds known to target specific TRPC isoforms .
Principle of the assay
To screen for compounds that inhibit the TRPC4 cation channel, a HEK293 cell line which stably expresses both the TRPC4beta and the mu-Opioid receptor is employed. Stimulation of the mu-Opioid receptor by DAMGO induces activation of TRPC4 through the G alpha signaling pathway. Channel activity is monitored by calcium flux with a commercial Fluo4 kit . Compounds that show decreased Fluo4 fluorescence in the presence of the mu-Opioid receptor activator (DAMGO, in ECmax concentration) are considered inhibitor hits. mu-Opioid receptor inhibitors will be excluded through later counter-screening against HEK293 stable cells expressing the mu-Opioid receptor alone.
The objective of this assay is to confirm dose dependent inhibition of calcium flux through the TRPC4 cation channel. The same protocol is employed for this assay as the primary screen, except that each compound is tested at ten concentrations. Compound inhibition of TRPC4 was tested in at least duplicate using a calcium-sensitive fluorescent dye.
TRPC4, mu-Opioid receptor, HTS assay, 384, confirmatory, CRC, inhibitor, blocker, FDSS, Calcium, fluorescence, Kinetic, Fluo 4, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
1. Venkatachalam, K., and Montell, C. TRP Channels. Annual Review of Biochemistry 76(1), 387-417 (2007) PMID: 17579562
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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' factors
17. IC50 and Hill Constant calculation from replicates was generated using Microcol Origin 6.0
18. Outcome assignment: If the test compound causes a maximum inhibition of TRPC4 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 TRPC4 at any concentration tested or a dose response is not generated, the compound is designated as inactive (outcome=1).
19. 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. 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)