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BioAssay: AID 492996

SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4): ACh 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(31)
 
 
Active(9)
 
 
Inactive(22)
 
 
 Tested Substances
 Tested Substances
All(31)
 
 
Active(9)
 
 
Inactive(22)
 
 
AID: 492996
Data Source: Johns Hopkins Ion Channel Center (JHICC_C4_Inh_SAR_ACh_CRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-12-15
Hold-until Date: 2011-06-15
Modify Date: 2011-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 9
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
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
492987SAR Analysis for the identification of selective inhibitors of the transient receptor potential cation channel C4 (TRPC4) in TRPC6 expressing cells: MPD Assay CRCConfirmatory 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
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
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): ACh assay CRC


Description:

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 [4], cardiovascular diseases [5], 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 [8]. The mammalian TRPC (canonical) subfamily is proposed to function as store and second-messenger operated cation channels [9]. 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 [10].

The objective of this assay is to confirm dose dependent inhibition of calcium flux through the TRPC4 cation channel. An alternative activation mechanism is employed in this assay to confirm the activity of each test compound is indeed on the TRPC4 channel and not on the activation pathway. In this case, acetylcholine (AChCl) is used to induce a Gq coupled response from which TRPC4 may be activated. Compound inhibition of TRPC4 was tested in at least duplicate using a calcium-sensitive fluorescent dye and each compound was tested at ten concentrations.


Keywords:

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.


References:
1. Venkatachalam, K., and Montell, C. TRP Channels. Annual Review of Biochemistry 76(1), 387-417 (2007) PMID: 17579562
2. Clapham, D., Julius, D., Montell, C. and Schultz, G. International Union of Pharmacology. XLIX. Nomenclature and structure-function relationships of transient receptor potential channels. Pharmacological Reviews 57(4), 427-450 (2005) PMID: 16382100
3. Reaves, B. J., and Wolstenholme, A. J. The TRP channel superfamily: insights into how structure, protein-lipid interactions and localization influence function. Biochemical Society Transactions 035(1), 77-80 (2007) PMID: 17233605
4. Patapoutian, A., Tate, S., and Woolf, C. J. Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov 8(1), 55-68 (2009) PMID: 19116627
5. Vassort G, Alvarez J. Transient receptor potential: a large family of new channels of which several are involved in cardiac arrhythmia. Can J Physiol Pharmacol. 2009 Feb;87(2):100-7.PMID: 19234573
6. Nilius, B. TRP channels in disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1772(8), 805-812 (2007) PMID: 17368864
7. Woudenberg-Vrenken, T. E., Bindels, R. J. M., and Hoenderop, J. G. J. The role of transient receptor potential channels in kidney disease. Nat Rev Nephrol 5(8), 441-449 (2009) PMID: 19546862
8. Zhu, X., Jiang, M., Peyton, M., et al. trp, a Novel Mammalian Gene Family Essential for Agonist-Activated Capacitative Ca2+ Entry. Cell 85(5), 661-671 (1996) PMID: 8646775
9. Abramowitz, J., and Birnbaumer, L. Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J. 23(2), 297-328 (2009) PMID: 18940894
10. Okuhara, D. Y., Hsia, A. Y., and Xie, M. Transient receptor potential channels as drug targets. Expert Opinion on Therapeutic Targets 11(3), 391-401 (2007) PMID: 17298296
11. Miret, Juan J., et al., Multiplexed G-Protein-Coupled Receptor Ca2+ Flux Assays for High-Throughput Screening. J Biomol Screen 2005 10: 780-787 (2005) PMID: 16234348.
12. Zhang, J.-H., T.D.Y. Chung, and K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 1999. 4(2),67-73. (1999) PMID: 10838414.
13. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175 (2006). PMID: 16465162.
Protocol
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.
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 AChCl
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 maximally activating concentration of AChCl (AChCl ECmax) and read for another 110 seconds.
14. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z' factors
16. IC50 and Hill Constant calculation from replicates was generated using Microcol Origin 6.0
17. 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).
18. 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. Acetylcholine chloride (Sigma, Cat# A6625)
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)
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.
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
Classification
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