Validation assay for identification of compounds that activate the regulator of G-protein signaling 4 (RGS4)
Assay Implementation: Zhihong Lin Ph.D., Kaiping Xu M.S.,Joseph Babcock, Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus Ph.D., Meng Wu Ph.D. ..more
BioActive Compounds: 1022
Data Source: Johns Hopkins Ion Channel Center (JHICC_RGS_Act_Vali)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed, Duplicate
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., Kaiping Xu M.S.,Joseph Babcock, Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus Ph.D., Meng Wu Ph.D.
Name: Validation assay for identification of compounds that activate the regulator of G-protein signaling 4 (RGS4)
See the related assay (PubChem AID: 463111, 485274).
To validate the hit compounds that activate the RGS4 protein from the primary screen, 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 decrease in the Fluo4 fluorescence in induced RGS4 expressed cells upon the addition of EC90 carbachol are considered RGS4 activator/potentiator hits. M3 receptor and other endogenous receptor agonists will be excluded through later counter-screening against non-induced parental cells.
Protocol for RGS4 validation 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.
9. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 10 seconds at 1Hz to establish baseline
11. After 100 seconds, add 4 ul of EC90 (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' factors.
14. Calculate Inhibition (%) as the average of the integrated ratio percentages calculated in Step 12
15. Outcome assignment: If the Inhibition (%) of the test compound is less than 3 times the standard deviation (SD) of the buffer controls, the compound is assigned as active, otherwise inactive.
16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(([Inhibition (%)]-36.5)*1.476+5)), Inhibition (%), 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 Cat#10010049)
3. Medium: DMEM (Sigma, Cat#D5796)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
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# (35)4663 and Lot #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.
** Test Concentration.
Data Table (Concise)