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BioAssay: AID 2227

Primary cell-based high-throughput screening assay for identification of compounds that allosterically potentiate 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
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 Tested Compounds
 Tested Compounds
All(305604)
 
 
Active(1291)
 
 
Inactive(304313)
 
 
 Tested Substances
 Tested Substances
All(305669)
 
 
Active(1291)
 
 
Inactive(304378)
 
 
AID: 2227
Data Source: Johns Hopkins Ion Channel Center (JHICC_TRPC4_Allo)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-12-28
Modify Date: 2010-03-30

Data Table ( Complete ):           Active    All
Target
Sequence: alternatively spliced Trp4 [Mus musculus]
Description ..   
Protein Family: Transient receptor ion channel II

Gene:TRPC4     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 1291
Depositor Specified Assays
AIDNameTypeComment
2246Summary of compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4).summarySummary assay for TRPC4 allosteric potentiator assays.
2554Second confirmatory screen for identification of compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4)screeningConfirmatory assay for TRPC4 allosteric potentiators where 2267 compounds were tested in duplicates at the concentration of 1 uM, and 152 were active.
2555Confirmatory screen for identification of compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4)screeningConfirmatory assay for TRPC4 allosteric potentiators where 2267 compounds were tested in duplicates at the concentration of 10 uM, and 324 were active.
434952Specificity screen against TRPC6 for compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4)screening
434941Confirmation dose response assay for compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4)confirmatory
493223SAR analysis for compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4): Primary HTS assay CRCconfirmatory
434979Second counter screen for compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4)screening
449724Counter screen for compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4)screening
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Primary, Primary Screening, 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: Primary cell-based high-throughput screening assay for identification of compounds that allosterically potentiate transient receptor potential cation channel C4 (TRPC4).

Description:

Transient receptor potential (TRP) channels comprise a large family of cation channels, and have emerged as important sensors of cellular environment, including those for vision, taste, olfaction, hearing, touch, and thermo- and osmosensation [1]. TRP cationic channels are key molecules that are involved in a variety of diverse biological and pathological processes, ranging from neurological diseases [2], i.e. neuropathic pain [3], and cardiovascular diseases [4], to other diseases involving sensory physiology [5, 6].

Among the seven main subfamilies of TRP channels, separated according to structural homology, the TRPC (canonical) channels are among the first founding members of the TRP family [7, 8]. The TRPC subfamily is composed of seven members that are nonselective, calcium-permeable cation channels, which cause both a depolarization of the cell membrane potential and entry of calcium. Disruption of TRPC function usually results in a change of risk factors in complex diseases, rather than an apparent phenotype. The modulation of intracellular calcium as well as the membrane potential contributes to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy, as well as normal and abnormal cell proliferation. Currently there are no specific modulators available for TRPC isoforms, nor are there good common modulators available for all TRPC members [9].

The objective of the current screen is to identify compounds that potentiate Ca2+ flux [10] in the presence of DAMGO at an EC20 concentration which, itself, minimally activates TRPC4 cation channels. These compounds are considered, potentially, as allosteric potentiators/activators for TRPC4 cation channels.


Principle of the assay

To screen the allosteric activator/potentiator of the TRPC4 cation channels, a HEK293 cell line which stably expresses both TRPC4beta and mu-Opioid receptor was employed for the specific activation of TRPC4 cation channel. Approximate 20% of activation is obtained by the application of DAMGO in an EC20 concentration. The TRPC4 cation channel activity was monitored by calcium flux with a commercial Fluo 4 kit [10]. Any compounds shown to increase the Fluo 4 fluorescence in the presence of DAMGO at EC20 concentrations were considered as allosteric hits for TRPC4. Allosteric mu-Opioid receptor activators/potentiators will may be excluded through the counter- screen against HEK293 stable cells expressing the mu-Opioid receptor alone.

Keywords:

TRPC4, mu-Opioid receptor, HTS assay, 384, primary, agonist, activator, potentiator, allosteric, 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. 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
3. 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
4. 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
5. Nilius, B. TRP channels in disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1772(8), 805-812 (2007) PMID: 17368864
6. 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
7. 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
8. Abramowitz, J., and Birnbaumer, L. Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J. 23(2), 297-328 (2009) PMID: 18940894
9. 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
10. 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.
11. 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.
12. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175 (2006). PMID: 16465162.
Protocol
Assay overview:

The objective of the current assay is to identify compounds that potentiate Ca2+ flux in the presence of DAMGO at EC20 concentration, which, itself, minimally activates TRPC4 cation channels. These compounds, with no activity in the absence of DAMGO, are considered, potentially, as allosteric potentiators/activators for TRPC4 cation channels. A HEK293 cell line which stably expresses both TRPC4beta and mu-Opioid receptor is employed for the specific activation of the TRPC4 channel. The activation of the mu-Opioid receptor by DAMGO, and, consequentially, the TRPC4 cation channel has been monitored with a Fluo 4 calcium flux kit.

The stable HEK293 cells were plated into 384-well plates. After overnight incubation, the cells were loaded with a calcium-sensitive dye, Fluo 4, followed by a washing. Then the cells were mounted on a Hamamatsu FDSS 6000 kinetic imaging plate reader, and where compounds were added and incubated for 110 seconds, and their fluorescence were was measured for another 110 seconds upon the addition of a stimulus solution of DAMGO at EC20 concentration for another 110 seconds. Compound effect was evaluated by the calculatedcalculating the Fluo 4 fluorescence ratio (Initial Ratio, see Result Definitions, 2) to remove the fluorescent test compounds, and the integrated ratio (an indicator of area under the kinetic calcium response curve) for B score calculation (B score AlloActivator Ratio , see Result Definitions, 1). If the compound has an Initial Ratio of 0, AND causes more than 5 times the standard deviation of the B score AlloActivator RatioB-scores of the integrated ratios of the library compounds, the compound is then considered to be active as an allosteric potentiator/activator of the TRPC4 cation channels.


Protocol for the TRPC4 project:

1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, and 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% FBS.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of 1x Fluo 4 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 Fluo 4 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. Add 4 ul of 7.5x compound stock into the cell plates.
11. Incubate all cell plates for 110 seconds at RT in the dark
12. Prepare EC20 DAMGO stimulus solution
13. Measure fluorescence for 5 seconds at 1Hz to establish baseline
14. Add 6 ul/well of EC20 DAMGO stimulus solution and continue measuring fluorescence for 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 [11].
17. Calculate B scores [12] for test compounds using integrated ratios calculated in Step 15.
18. Outcome assignment: If the B score of the test compound is more than 5 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the ratio of initial fluorescence intensity is within 5 times the standard deviation of the ratios of the library compounds, the compound is designated in the Outcome as active (Value=2) as an allosteric potentiator of the TRPC4 channels. Otherwise, it is designated as inactive (value=1).
19. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Integer((Log10(abs([B score AlloActivator Ratio]))-1.1)*100);, they are normalized to the smallest and largest LOG(B score AlloActivator Ratio), B score AlloActivator Ratio, as in the result definition. The inactive test compounds are assigned 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. 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)
Comment
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1B score AlloActivator Ratio (10μM**)B score calculated based on the ratio of the test compound of the primary screen at a compound concentration of 10 uMFloat
2Initial Ratio (10μM**)Initial Ratio upon the addition of the compounds, as an indicator of compound fluorescence at a compound concentration of 10 uMFloat

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS056942-01

Data Table (Concise)
Classification
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