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BioAssay: AID 624166

Counter screen dose-response assay for SAR compounds that potentiate the regulator of G-protein signaling 4 (RGS4) in non-induced RGS4 cells

Assay Implementation: Zhihong Lin Ph.D., Joseph Babcock, Shunyou Long M.S., Alison Neal, B.S., Owen McManus Ph.D., Meng Wu, Ph.D. ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 624166
Data Source: Johns Hopkins Ion Channel Center (JHICC_RGS4_POT_CounterCRC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-05-22

Data Table ( Complete ):           Active    All
Sequence: regulator of G-protein signaling 4 isoform 2 [Homo sapiens]
Description ..   
Protein Family: Regulator of G protein signaling (RGS) domain found in the RGS4 protein

Gene:RGS4          More BioActivity Data..
BioActive Compounds: 9
Depositor Specified Assays
485274Summary of probe development for potentiators of the regulator of G-protein signaling 4 (RGS4)summary
463111Primary cell-based high-throughput screening assay for identification of compounds that potentiate/activate regulator of G-protein signaling 4 (RGS4)screening
602282Validation assay for identification of compounds that activate the regulator of G-protein signaling 4 (RGS4)screening
602283Counter screen for identification of compounds that activate the regulator of G-protein signaling 4 (RGS4): Non-induced cells with the primary screen assayscreening
Data 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: Richard Neubig, M.D., Ph.D., University of Michigan
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH087441-01A1
Grant Proposal PI: Richard Neubig, M.D., Ph.D.
Assay Implementation: Zhihong Lin Ph.D., Joseph Babcock, Shunyou Long M.S., Alison Neal, B.S., Owen McManus Ph.D., Meng Wu, Ph.D.


Signal transduction pathways mediated by G protein-coupled receptors (GPCRs) are major therapeutic targets with many known pharmacologic modulators. The regulators of G protein signaling (RGS proteins) are a key family of modulators and scaffolds for GPCRs which have been identified by many authors as appealing drug targets [1-5]. RGS proteins bind to activated G proteins and strongly inhibit their signals by catalyzing the hydrolysis of bound GTP [6-7]. Consistent with this mode of action, genetic deletion of RGS greatly enhances receptor signaling [8-10]. Furthermore, RGS proteins display isoform-specific tissue distribution [11-14] and their expression is altered in pathophysiological states including Parkinson's disease [15], neuropathic pain [16-17], schizophrenia, hypertension, and congestive heart failure [18]. An activator of RGS action would be expected to suppress the function of GPCR agonists such as vasoconstrictors, inflammatory mediators, endogenous neurotransmitters, or catecholamines such as L-DOPA. It has recently been established that loss of function of RGS2 causes hypertensive symptoms in mice [19] and may contribute to vasoactive disorders in humans as well [20-22]. Also, reduced expression of RGS4 has been correlated with schizophrenia [23-24]. Consequently, compounds that could either enhance the function of RGS proteins or could increase their expression might be of therapeutic benefit. The identification of selective modulators of RGS proteins would also greatly facilitate experimental studies of this sizeable protein family (with 20-30 members) by permitting rapid and specific chemical potentiation. Such RGS modulators would be particularly useful to determine which RGS proteins modulate particular receptor signaling pathways.

The purpose of the assay is to determine the dose-response relations of the compounds identified as potentiators in the primary screen (PubChem AID: 463111) and confirmatory screens (i.e PubChem AID: 485274) using a receptor-stimulated cell-based intracellular Ca++ mobilization assay. Compounds were tested in triplicate at varying concentrations.

[1]. Zhong, H., and Neubig, R.R. 2001. Regulator of G protein signaling proteins: novel multifunctional drug targets. J Pharmacol Exp Ther 297:837-845.
[2]. Neubig, R.R., and Siderovski, D.P. 2002. Regulators of G-protein signalling as new central nervous system drug targets. Nat Rev Drug Discov 1:187-197.
[3]. Druey, K.M. 2003. Regulators of G protein signalling: potential targets for treatment of allergic inflammatory diseases such as asthma. Expert Opin Ther Targets 7:475-484.
[4]. Cho, H., Harrison, K., and Kehrl, J.H. 2004. Regulators of G protein signaling: potential drug targets for controlling cardiovascular and immune function. Curr Drug Targets Immune Endocr Metabol Disord 4:107-118.
[5]. Riddle, E.L., Schwartzman, R.A., Bond, M., and Insel, P.A. 2005. Multi-tasking RGS proteins in the heart: the next therapeutic target? Circ Res 96:401-411.
[6]. Druey, K.M., Blumer, K.J., Kang, V.H., and Kehrl, J.H. 1996. Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family. Nature 379:742-746.
[7]. Yan, Y., Chi, P.P., and Bourne, H.R. 1997. RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis. J Biol Chem 272:11924-11927.
[8]. Jeong, S.W., and Ikeda, S.R. 2000. Endogenous regulator of G-protein signaling proteins modify N-type calcium channel modulation in rat sympathetic neurons. J Neurosci 20:4489-4496.
[9]. Clark, M.J., Neubig, R.R., and Traynor, J.R. 2004. Endogenous regulator of G protein signaling proteins suppress Galphao-dependent, mu-opioid agonist-mediated adenylyl cyclase supersensitization. J Pharmacol Exp Ther 310:215-222.
[10]. Fu, Y., Zhong, H., Nanamori, M., Mortensen, R.M., Huang, X., Lan, K., and Neubig, R.R. 2004. RGS-insensitive G-protein mutations to study the role of endogenous RGS proteins. Methods Enzymol 389:229-243.
[11]. Gold, S.J., Ni, Y.G., Dohlman, H.G., and Nestler, E.J. 1997. Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain. J Neurosci 17:8024-8037.
[12]. Nomoto, S., Adachi, K., Yang, L.X., Hirata, Y., Muraguchi, S., and Kiuchi, K. 1997. Distribution of RGS4 mRNA in mouse brain shown by in situ hybridization. Biochem Biophys Res Commun 241:281-287.
[13]. Kurrasch, D.M., Huang, J., Wilkie, T.M., and Repa, J.J. 2004. Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues. Methods Enzymol 389:3-15.
[14]. Doupnik, C.A., Xu, T., and Shinaman, J.M. 2001. Profile of RGS expression in single rat atrial myocytes. Biochim Biophys Acta 1522:97-107.
[15]. Tekumalla, P.K., Calon, F., Rahman, Z., Birdi, S., Rajput, A.H., Hornykiewicz, O., Di Paolo, T., Bedard, P.J., and Nestler, E.J. 2001. Elevated levels of DeltaFosB and RGS9 in striatum in Parkinson's disease. Biol Psychiatry 50:813-816.
[16]. Garnier, M., Zaratin, P.F., Ficalora, G., Valente, M., Fontanella, L., Rhee, M.H., Blumer, K.J., and Scheideler, M.A. 2003. Up-regulation of regulator of G protein signaling 4 expression in a model of neuropathic pain and insensitivity to morphine. J Pharmacol Exp Ther 304:1299-1306.
[17]. Xie, G.X., and Palmer, P.P. 2005. RGS proteins: new players in the field of opioid signaling and tolerance mechanisms. Anesth Analg 100:1034-1042.
[18]. Mittmann, C., Chung, C.H., Hoppner, G., Michalek, C., Nose, M., Schuler, C., Schuh, A., Eschenhagen, T., Weil, J., Pieske, B., et al. 2002. Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure. Cardiovasc Res 55:778-786.
[19].Heximer, S.P., Knutsen, R.H., Sun, X., Kaltenbronn, K.M., Rhee, M.H., Peng, N., Oliveira-dos-Santos, A., Penninger, J.M., Muslin, A.J., Steinberg, T.H., et al. 2003. Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. J Clin Invest 111:445-452.
[20]. Bodenstein, J., Sunahara, R.K., and Neubig, R.R. 2007. N-terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol Pharmacol 71:1040-1050.
[21]. Gu, S., Tirgari, S., and Heximer, S.P. 2008. The RGS2 gene product from a candidate hypertension allele shows decreased plasma membrane association and inhibition of Gq. Mol Pharmacol 73:1037-1043.
[22]. Yang, J., Kamide, K., Kokubo, Y., Takiuchi, S., Tanaka, C., Banno, M., Miwa, Y., Yoshii, M., Horio,T., Okayama, A., et al. 2005. Genetic variations of regulator of G-protein signaling 2 in hypertensive patients and in the general population. J Hypertens 23:1497-1505.
[23]. Chowdari, K.V., Mirnics, K., Semwal, P., Wood, J., Lawrence, E., Bhatia, T., Deshpande, S.N., B, K.T., Ferrell, R.E., Middleton, F.A., et al. 2002. Association and linkage analyses of RGS4 polymorphisms in schizophrenia. Hum Mol Genet 11:1373-1380.
[24]. Mirnics, K., Middleton, F.A., Stanwood, G.D., Lewis, D.A., and Levitt, P. 2001. Disease-specific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Mol Psychiatry 6:293-301.
[25]. Miret, Juan J., et al., Multiplexed G-Protein-Coupled Receptor Ca2+ Flux Assays for High-Throughput Screening. J Biomol Screen 10: 780-787 (2005) PMID: 16234348.
[26]. 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, 4(2),67-73 (1999) PMID: 10838414.
[27]. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 24(2), 167-175 (2006). PMID: 16465162.


RGS4, parental cell, M3 receptor, dose response assay, 384, agonist, activator, potentiator, allosteric, FDSS, calcium, fluorescence, Kinetic, Fluo4-AM, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN
Principle of the assay
The objective of the current screen is to determine the dose-response relations of compounds (re-synthesized or purchased by the Vanderbilt Specialized Chemistry Center) that activate RGS4 using a similar assay as was used in the primary screen, but using parental cells not expressing RGS4 (Non-induced RGS4). Compounds were tested in triplicate at varying concentrations.

Protocol for dose response of RGS4 Screen:
1. Cell culture: Cells (HEK293-FlpIn-TREx/M3R/RGS4) are routinely cultured in DMEM (high glucose, w/ glutamine), 10%FBS, 1%Pen/Strep, 15ug/ml Blasticidin, 400ug/ml G418, 200ug/ml Hygromycin.
2. Cell plating: Add 50 ul/well of 200,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS, 1% Pen/Strep. NOT include 10 ng/ml Doxycyclin (DOX) to induce RGS4 expression.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 20 ul /well of 2uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37C in incubator.
6. Prepare 6x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer (HBSS-HEPES pH 7.4).
7. Remove Fluo4-AM dye solution and add 20 ul/well of assay buffer to cells.
8. Incubate 30 minutes at room temperature (RT).
9. Add 6x compounds in cell plates and incubate 20 minutes at RT.
9. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 10 seconds at 1Hz to establish baseline.
11. Add 4 ul of EC20 (carbachol) into the cell plates and record fluorescence for 100 seconds.
12. After 100 seconds, add 4 ul of ECmax (carbachol) into the cell plates and record fluorescence for 100 seconds.
13. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout at Step 12.
14. Outcome assignment:
If the test compound causes an activation effect in RGS4 parental cells (Non-induced RGS4) in any concentration tested and a dose-response is generated, the compound is considered to be active.
If the test compound does not cause an activation effect on in RGS4 parental cells (Non-induced RGS4) in any concentration tested or a dose-response is not generated, the compound is designated as inactive.
15. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100

List of reagents

1. HEK293-FlpIn-TREx/M3R/RGS4 cell lines (provided by assay provider)
2. PBS: pH7.4 (Invitrogen, Cat #10010049)
3. Medium: DMEM (Sigma, Cat #D5796)
4. Fetal Bovine Serum (Gemini, Cat #100106)
5. Hygromycin (Mediatech, Cat #30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Cat #30-001-CI)
7. Cell/stripper (Mediatech, Cat #25-056-Cl)
8. G418: (Invitrogen, Cat #11811-031)
9. Blasticidin (Sigma, Cat #R21001)
10. Doxycycline hyclate (Sigma, Cat #D9891)
11. HEPES (Sigma, Cat #H4034)
12. Fluo-4 (Invitrogen, Cat #F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Cat #P-3000MP)
14. Atropine (Sigma, Cat #A0132)
15. Carbachol (Sigma, Cat #C4382)
16. Triple-layer flask (VWR, Cat #62407-082)
17. BD Biocoat 384-well plates (BD, Cat #354663 and Lot #7346273)
To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1NNumber of repeatInteger
2NonRGS-EC50 (uM)*EC50FloatμM
3NonRGS-EC50 (SD)EC50_SDFloat
4NonRGS-HillHill constantFloat
5NonRGS-Hill(SD)Hill constant_SDFloat
6NonRGS-Resp(33.3uM) (33.3μM**)At 33.3uM-Resp: Integ Ratio treated with the specified concentration Float
7NonRGS-Resp(11.1uM) (11.1μM**)At 11.1uM-Resp: Integ Ratio treated with the specified concentrationFloat
8NonRGS-Resp(3.7uM) (3.7μM**)At 3.7uM-Resp: Integ Ratio treated with the specified concentrationFloat
9NonRGS-Resp(1.2uM) (1.2μM**)At 1.2uM-Resp: Integ Ratio treated with the specified concentrationFloat
10NonRGS-Resp(0.4uM) (0.4μM**)At 0.4uM-Resp: Integ Ratio treated with the specified concentrationFloat
11NonRGS-Resp(0.13uM) (0.13μM**)At 0.13uM-Resp: Integ Ratio treated with the specified concentrationFloat
12NonRGS-Resp(0.05uM) (0.05μM**)At 0.05uM-Resp: Integ Ratio treated with the specified concentrationFloat
13NonRGS-Resp(0.015uM) (0.015μM**)At 0.015uM-Resp: Integ Ratio treated with the specified concentrationFloat
14NonRGS-Resp(0.005uM) (0.005μM**)At 0.005uM-Resp: Integ Ratio treated with the specified concentrationFloat
15NonRGS-Resp(0.002uM) (0.002μM**)At 0.002uM-Resp: Integ Ratio treated with the specified concentrationFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH087441-01A1

Data Table (Concise)