Name: High throughput screening of inhibitors of transient receptor potential cation channel C6 (TRPC6)
Data Source: Johns Hopkins Ion Channel Center
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: 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: Meng Wu Ph.D., Melissa Miller, Amy Scott, Shunyou Long M.S., Joseph Babcock, 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: High throughput screening of inhibitors of transient receptor potential cation channel C6 (TRPC6)

Description:

The Transient Receptor Potential (TRP) channels are a family of transmembrane proteins that tune the excitable properties of sensory cells [1]. One subfamily, the canonical TRP (TRPC) channels, are nonselective cation channels gated by intracellular calcium stores, ligand binding, or mechanical force [2-4]. TRPC channel genes have been linked to many diseases; however, the only channelopathy associated with a single TRPC channel, TRPC6, is Focal and Segmental Glomerulosclerosis (FSGS) [5]. A gain of function mutation in TRPC6 causes severe proteinuria and disrupted calcium regulation in children and adults [6-7]. As there are no known specific inhibitors of TRPC6, nor are their known specific inhibitors of TRP channels as a whole, it has been difficult to understand the role of TRPC6 in this disease [8]. It is therefore of great interest to find a specific modulator of this TRP isoform.

Principle of the assay
To screen for small molecule inhibitors of TRPC6, a HEK293 cell line stably expressing TRPC6 is employed. Stimulation of endogenous muscarinic receptors by acetylcholine will induce activation of TRPC6 through a Galphaq signaling pathway [3]. Channel activity is monitored through a commercially available FLIPR membrane potential dye [9]. Activation of TRPC6 will cause influx of calcium ions into the cell resulting in a depolarization of plasma membrane potential. Compounds that are shown to inhibit this depolarization in the presence of acetylcholine (ECMax) will be considered inhibitor hits. Compounds that inhibit membrane depolarization through parallel mechanisms will be excluded through later counter-screening using HEK293 cells that do not express TRPC6.

Keywords:

TRPC6, membrane potential HTS assay, Non-selective cation channel, FSGS, 384, primary, antagonist, inhibitor, blocker, FDSS, fluorescence, Kinetic, MPD, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.

References

1. Damann N, Voets T, Nilius B. TRPs in our senses. Curr Biol. 8(18):R880-889 (2008) PMID: 18812089
2. Montell C. New Light on TRP and TRPL. Mol Pharmacol. 52(5): 755-63 (1997) PMID: 9351965
3. Raghu P, Hardie RC. Regulation of Drosophila TRPC channels by lipid messengers. Cell Calcium. Jun;45(6):566-73 (2009) PMID: 19362736
4. Spassova MA, et al. A common mechanism underlies stretch activation and receptor activation of TRPC6 channels. Proc Natl Acad Sci U S A. 03(44):16586-91 (2006 ) PMID: 17056714
5. Nilius B, Owsianik G. Transient receptor potential channelopathies. Pflugers Arch. 2010 PMID: 20127491
6. Winn MP, et al. A Mutation in the TRPC6 Cation Channel Causes Familial Focal Segmental Glomerulosclerosis. Science. 308:1801-4 (2005) PMID:15879175
7. Heeringa SF, et al. A novel TRPC6 mutation that causes childhood FSGS. PLoS One. 4(11): e7771 (2009) PMID: 19936226
8. 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
9. Baxter DF, et al. A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels. J Biomol Screen. 7(1):79-85 (2002) PMID: 11897058
10. 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.
11. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175 (2006). PMID: 16465162.

Assay overview:
The objective of this assay is to identify compounds that inhibit the TRPC6 cation channel. A HEK293 cell line which stably expresses TRPC6 was employed, and channel activity monitored by use of a membrane potential dye (MPD). Compounds that inhibit TRC6 result in a decrease in membrane depolarization in the presence of the muscarinic receptor agonist, acetylcholine. This reduction in membrane potential will result in a decreased fluorescence of MPD 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 membrane potential dye (MPD). Cell plates were then loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader, where compounds were added and incubated for 110seconds before application of acetylcholine at a maximally activating concentration. Real-time fluorescence was then measured for 100 seconds. Library compound effect was evaluated by computing the integrated ratio of each well which was used for calculation and assignment of a B score for that well (B score inhibitor Ratio, see Result Definitions, 1). Compounds that affect the fluorescence ratio within two seconds after application were flagged as fluorescent and removed from further analysis. Remaining compounds that scored less than the mean of the B score Inhibitor Ratio minus three times the standard deviation of all library members were considered to be active as inhibitors of the TRPC6 cation channel.

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, and 400 ug/mL G418
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS, 50 IU/ml penicillin, and 50 ug/mL streptomycin
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of Membrane Potential Dye
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), ECmax of Acetylcholine, and Ecmax 2-APB
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Measure fluorescence for 5 seconds at 1Hz to establish baseline
9. Add 4ul of 7.5x compound stock into the cell plates.
10. Incubate plates for 110 seconds
11. Add 6ul maximally activating concentration of Acetylcholine (Acetylcholine ECmax) and read for another 110 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [10]
14. Calculate B scores [11] for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is less than the mean minus 3 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds (B score Inhibitor Ratio 19. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(63.4*Log(abs([B score Ratio]))-96.0). Inactive test compounds are assigned a score of 0.

List of reagents
1. TRPC6-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)
2. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma, Cat# D5796-500ML)
3. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)
4. 100x Penicillin-Streptomycin (Mediatech, Cat# 30-001-CI)
5. CellStripper (Mediatech, Cat# 25-056-Cl)
6. G418: (Invitrogen, Cat# 11811-031)
7. HEPES (Sigma, Cat# H4034)
8. 10XHBSS (#Invitrogen Cat# 14065056)
9. Acetylcholine chloride (Sigma, Cat# A6625)
10. 2-APB (Sigma, Cat# D9754)
11. Membrane Potential Assay Kit, Blue (Molecular Devices, Cat# R8034)
12. Triple-layer flask (VWR, Cat# 62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot# 8281903)

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.

Grant Number: 1 R21 NS056942-01