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BioAssay: AID 463165

Primary cell-based high-throughput screening assay for identification of compounds that inhibit regulator of G-protein signaling 4 (RGS4)

Assay Implementation: Zhihong Lin Ph.D., Joseph Babcock, Shunyou Long M.S., Bill Shi Ph.D., David Meyers Ph.D., Meng Wu Ph.D. ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 463165
Data Source: Johns Hopkins Ion Channel Center (JHICC_RGS_Inh_HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-08-27

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 1365
Related Experiments
485289Summary of probe development for inhibitors of the regulator of G-protein signaling 4 (RGS4)Summarydepositor-specified cross reference
492999Validation assay for identification of compounds that inhibit the regulator of G-protein signaling 4 (RGS4)Screeningdepositor-specified cross reference
493000Counter screen for identification of compounds that inhibit regulator of G-protein signaling 4 (RGS4): Non-induced cells with the primary screen assayScreeningdepositor-specified cross reference
493001Second counter screen for identification of compounds that inhibit regulator of G-protein signaling 4 (RGS4): Non-induced cells with the primary screen assay without carbachol activationScreeningdepositor-specified cross reference
588523Biochemical assay for compounds that inhibit AlF4- anion-dependent RGS4 binding to Galpha-oConfirmatorydepositor-specified cross reference
588552Biochemical assay for compounds that inhibit RGS4 stimulation of Galpha1 nucleotide hydrolysis under receptor-independent steady-state conditionsConfirmatorydepositor-specified cross reference
Data Source: Johns Hopkins Ion Channel Center (JHICC_RGS_Inh_HTS)
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: 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., Bill Shi Ph.D., David Meyers Ph.D., Meng Wu Ph.D.
HTS execution: Joseph Babcock, Zhihong Lin Ph.D., Shunyou Long M.S., Xiaofang Huang M.S., Amy Stevens M.S., Melissa Miller, Meng Wu Ph.D., Owen McManus Ph.D.

Name: Primary cell-based high-throughput screening assay for identification of compounds that inhibit regulator of G-protein signaling 4 (RGS4)


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 vasoactivators, 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 goal of this project is to identify inhibitors of the regulator of G protein signaling 4 (RGS4), using a receptor-stimulated cell-based Ca++ assay.

[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. PMID: 11356902.
[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. PMID: 12120503.
[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. PMID: 12885267.
[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. PMID: 15180451.
[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. PMID: 15746448.
[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. PMID: 8602223.
[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. PMID: 9115254.
[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. PMID: 10844018.
[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. PMID: 15014136.
[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. PMID: 15313569.
[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. PMID: 9315921.
[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. PMID: 9425263.
[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. PMID: 15313556.
[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. PMID: 11750060.
[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. PMID: 11720701.
[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. PMID: 12604710.
[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. PMID: 15781518.
[18]. Mittmann C, Chung CH, Hoppner G, Michalek C, Nose M, Schuler C, Schuh A, Eschenhagen T, Weil J, Pieske B, Hirt S, Wieland T. 2002. Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure. Cardiovasc Res 55:778-786. PMID: 12176127.
[19]. Heximer SP, Knutsen RH, Sun X, Kaltenbronn KM, Rhee MH, Peng N, Oliveira-dos-Santos A, Penninger JM, Muslin AJ, Steinberg TH, Wyss JM, Mecham RP, Blumer KJ. 2003. Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. J Clin Invest 111:445-452. PMID: 12697745.
[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. PMID: 17220356.
[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. PMID: 18230714.
[22]. Yang J, Kamide K, Kokubo Y, Takiuchi S, Tanaka C, Banno M, Miwa Y, Yoshii M, Horio T, Okayama A, Tomoike H, Kawano Y, Miyata T. 2005. Genetic variations of regulator of G-protein signaling 2 in hypertensive patients and in the general population. J Hypertens 23:1497-1505. PMID: 16003176.
[23]. Chowdari KV, Mirnics K, Semwal P, Wood J, Lawrence E, Bhatia T, Deshpande SN, B K T, Ferrell RE, Middleton FA, Devlin B, Levitt P, Lewis DA, Nimgaonkar VL. 2002. Association and linkage analyses of RGS4 polymorphisms in schizophrenia. Hum Mol Genet 11:1373-1380. PMID: 12023979.
[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. PMID: 11326297.
[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, M3 receptor, HTS assay, 384, primary, antagonist, inhibitors, allosteric, FDSS, calcium, fluorescence, Kinetic, Fluo-4-AM, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.
Assay overview:
To screen for compounds that inhibit 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 show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered agonist hits. M3 receptor and other endogenous receptor inhibitors will be excluded through later counter-screening against non-induced parental cells.
Protocol for RGS4 Primary Screen:
1. Cell culture: Cells (HEK293-FlpIn-TREx/M3R/RGS4) are routinely cultured in DMEM (high glucose, w/ glutamine), 10% FBS, 1%Pen/Strep, 15 ug/ml Blasticidin, 400 ug/ml G418, 200 ug/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. Include 10 ng/ml Doxycyclin (DOX) to induce RGS4 expression.
3. Incubate overnight at 37 degrees C and 5% CO2.
4. Remove medium and add 20 ul /well of 2 uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37 degrees C 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 7x EC20 (carbachol) into the cell plates and record fluorescence for 100 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 prime factors [26]
14. Calculate B scores [27] for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds above the mean (B score ratio>3*SD+mean), AND the ratio of initial fluorescence intensity is within 5 times the standard deviation plus or minus the mean of the ratios of the library compounds, the compound is designated in the Outcome as active (value=2) as an inhibitor of RGS4. Otherwise, it is designated as inactive (value=1).
16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(B score ratio))-0.6)/1.25)*100), they are normalized to the smallest and largest LOG(B score ratio), B score ratio, as in the result definition. The inactive test compounds are assigned a score of 0.
List of reagents
1. HEK293-FlpIn-TREx/M3R/RGS4 cell lines (provided by assay provider)
2. PBS: pH7.4 (Invitrogen Catalog number 10010049)
3. Medium: DMEM (Sigma, Catalog number D5796)
4. Fetal Bovine Serum (Gemini, Catalog number 100-106)
5. Hygromycin (Mediatech, Catalog number 30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Catalog number 30-001-CI)
7. Cell/stripper (Mediatech, Catalog number 25-056-Cl)
8. G418: (Invitrogen, Catalog number 11811-031)
9. Blasticidin (Sigma, Catalog number R21001)
10. Doxycycline hyclate (Sigma, Catalog number D9891)
11. HEPES (Sigma, Catalog number H4034)
12. Fluo-4 (Invitrogen, Catalog number F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Catalog number P-3000MP)
14. Atropine (Sigma, Catalog number A0132)
15. Carbachol (Sigma, Catalog number C4382)
16. Triple-layer flask (VWR, Catalog number 62407-082)
17. BD Biocoat 384-well plates (BD, Catalog number (35)4663 and Lot number 7346273)
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK293
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1B Score (10μM**)B score calculated based on the ratio of the test compound of the primary screen at a compound concentration of 10 uM.Float

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data