Dose response-assay for SAR compounds that potentiate the regulator of G-protein signaling 4 (RGS4)
Assay Implementation: Zhihong Lin Ph.D., Joseph Babcock, Shunyou Long M.S., Alison Neal, Owen McManus Ph.D., Meng Wu Ph.D. ..more
BioActive Compounds: 13
Data Source: Johns Hopkins Ion Channel Center (JHICC_RGS4_POT_CRC)
BioAssay Type: Concentration-Response Relationship
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, 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 , neuropathic pain [16-17], schizophrenia, hypertension, and congestive heart failure . 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  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 compounds identified as potentiators in the primary screen (PubChem AID: 463111) and confirmatory screens (PubChem AID: 485274) using a receptor-stimulated cell-based intracellular Ca++ mobilization assay. Compounds were tested in triplicate at varying concentrations.
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RGS4, 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 by the Vanderbilt Specialized Chemistry Center) that activate the RGS4 with a similar assay as was used in the primary screen. Compounds that increase RGS4 activity will decrease GPCR-mediated calcium mobilization after carbachol addition. Compounds were tested in triplicate at ten different 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. 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.
10. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
11. Measure fluorescence for 10 seconds at 1Hz to establish baseline.
12. Add 4 ul of EC20 (carbachol) into the cell plates and record fluorescence for 100 seconds.
13. After 100 seconds, add 4 ul of ECmax (carbachol) into the cell plates and record fluorescence for 100 seconds.
14. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout at Step 12.
15. Outcome assignment:
If the test compound causes an activation effect on RGS4 in any concentrations tested and a dose-response is generated, the compound is considered to be active.
If the test compound does not cause activation effect on RGS4 in any concentrations tested or a dose-response is not generated, the compound is designated as inactive.
16. 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)