AID	Name	Type	Comment
488927	Specificity screen against TRPC4 for compounds that modulate transient receptor potential cation channel C6 (TRPC6)	screening
488926	Second counter screen for compounds that modulate transient receptor potential cation channel C6 (TRPC6)	screening
488961	Confirmatory screen for identification of compounds that activate transient receptor potential cation channel C6 (TRPC6)	screening
2602	Summary of efforts in the identification of compounds that activate the transient receptor potential cation channel C6 (TRPC6)	summary
488924	Counter screen for compounds that modulate transient receptor potential cation channel C6 (TRPC6)	screening
488928	Second specificity screen against TRPC4 for compounds that modulate transient receptor potential cation channel C6 (TRPC6)	screening

Data Source: Johns Hopkins Ion Channel Center (TRPC6_Activator_MPD)
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 activators of transient receptor potential cation channel C6 (TRPC6)

Description:
The Transient Receptor Potential (TRP) channels are a family of transmembrane proteins that tune responses within sensory cells [1]. One subfamily, the canonical TRP (TRPC) channels, are nonselective cation channels activated by a multitude of biophysical and biochemical stimuli [2-4]. There are currently seven mammalian isoforms within this channel subfamily, six of which encode functional proteins in humans. Genetic analysis has suggested a link between TRPC mutations and many human pathologies including cardiovascular diseases [5], respiratory diseases [6], kidney diseases [7], and cancer [8]. However, validation of TRP channel malfunction as a cause for these diseases has been a slow process due to the paucity of specific modulators of this channel family. To date there are no known isoform specific activators, nor are there small molecules that specifically target TRP channels as a whole [9].

It is therefore the goal of this screen to identify small molecule probes that activate TRPC6 heterologously expressed in HEK293 cells. Those compounds selected as hits will later be counter screened for selectivity.



Principle of the assay

To screen for small molecule activators of the canonical transient receptor potential cation channel 6, a HEK293 cell line stably expressing TRPC6 is employed and channel activity is monitored through a commercially available membrane potential dye (MPD). The activation of TRPC6 will cause influx of calcium ions into the cell resulting in a depolarization of plasma membrane potential [10]. Compounds that cause depolarization and therefore, increase MPD fluorescence are considered TRPC6 activator hits. Those small molecules that evoke membrane depolarization through auxiliary mechanisms will be excluded through later counter-screening using HEK293 cells that do not express TRPC6.

Keywords:
TRPC6, Non-selective cation channel, Membrane Potential HTS assay, 384, primary, agonist, activator, FDSS, fluorescence, MPD, Kinetic, 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. Dietrich A, et al. In vivo TRPC functions in the cardiopulmonary vasculature. Cell Calcium. 42(2):233-44 (2007) PMID: 17433435
6. Li S, Westwick J, Poll C. Transient receptor potential (TRP) channels as potential drug targets in respiratory disease. Cell Calcium. 33(5-6):551-8 (2003) PMID: 12765700
7. Winn MP, et al. A Mutation in the TRPC6 Cation Channel Causes Familial Focal Segmental Glomerulosclerosis. Science. 308:1801-4 (2005) PMID:15879175
8. Gkika D, Prevarskaya N. Molecular mechanisms of TRP regulation in tumor growth and metastasis. Biochim Biophys Acta. 1793(6):953-8 (2009) PMID: 19103233
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. 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
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.

Assay overview:
The objective of this assay is to identify compounds that activate 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 activate TRC6 result in membrane depolarization. This change in membrane potential will result in an increased 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 MPD. Cell plates were then loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader, where test compounds were added real-time fluorescence was measured for 110 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 (B score activator 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 greater than three times the standard deviation plus the mean of the B score Activator Ratio of all library members were considered to be activators 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), and ECmax of Acetylcholine
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 4 ul of 7.5x compound stock into the cell plates and read for 110 seconds.
10. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
11. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [11]
12. Calculate B scores [12] for test compounds using integrated ratios calculated in Step 10
13. Outcome assignment: If the B score of the test compound is greater than 3 times the standard deviation (SD) of the B scores of all library compounds (B score Activator Ratio>X+3*SD), AND the ratio of initial fluorescence intensity is within 5 times the standard deviation plus the mean of the ratios of all library compounds, the compound is designated in the Outcome as active (value=2) as an activator of the TRPC6 channel. 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 Int(30.0*Log(abs([B score Ratio]))-68.0)). 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