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BioAssay: AID 485279

SAR analysis of Agonists of GPR55 using MAPK Activation Assay

Assay Provider: Dr. Mary Abood, California Pacific Medical Center Research Institute (currently Temple University) ..more
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 Tested Compounds
 Tested Compounds
All(9)
 
 
Active(8)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(9)
 
 
Active(8)
 
 
Inactive(1)
 
 
AID: 485279
Data Source: Burnham Center for Chemical Genomics (SBCCG-A437-GPR55-Agonist-pERK-DryPowder-Assay)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-09-27
Hold-until Date: 2011-03-30
Modify Date: 2011-03-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 8
Related Experiments
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AIDNameTypeProbeComment
1961Image-based HTS for Selective Agonists of GPR55Confirmatory depositor-specified cross reference
1965Summary of Image-based HTS for Selective Agonists of GPR55Summary3 depositor-specified cross reference: Summary AID.
2338SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based AssayConfirmatory same project related to Summary assay
2339SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based AssayConfirmatory same project related to Summary assay
2340SAR analysis of Agonists of the GPR35 Receptor using an Image-Based AssayConfirmatory same project related to Summary assay
2341SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based AssayConfirmatory same project related to Summary assay
2347SAR analysis of selective Agonists of GPR55 using an Image-Based AssayConfirmatory same project related to Summary assay
2397SAR Analysis of Selective Antagonists of GPR55 using an Image-Based AssayConfirmatory same project related to Summary assay
2480SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
2809SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
2814SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
2820SAR Analysis of Selective Antagonists of GPR55 using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
2822SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
2843SAR analysis of selective Agonists of GPR55 using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
2844SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 3Confirmatory same project related to Summary assay
434922SAR analysis of antagonists of the Cannabinoid Receptor 2 using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
434924SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assay - Set 3Confirmatory same project related to Summary assay
434925SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 4Confirmatory same project related to Summary assay
434928SAR analysis of agonists of the Cannabinoid Receptor 2 using an Image-Based Assay - Set 2Confirmatory same project related to Summary assay
434929SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 3Confirmatory same project related to Summary assay
463192SAR Analysis for the identification of Selective Agonists of GPR55 using an Image-Based ScreenOther same project related to Summary assay
463227SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 5Confirmatory same project related to Summary assay
Description:
Data Source: Dr. Mary Abood
Source Affiliation: Temple University
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1X01 DA026205-01
Assay Provider: Dr. Mary Abood, California Pacific Medical Center Research Institute (currently Temple University)

Phosphorylation of ERK1/2 occurs as a result of GPCR activation. The western-blot analysis utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and human GPR55 receptor. This western blot assay will test selecteded GPR55 agonist compounds against a known GPR55 agonist, LPI (Lysophosphatidylinositol) to measure activation of ERK 1/2 in U2OS cells expressing the GPR55 receptor.

The aim of this assay is to characterize downstream ERK phosphorylation activity of compounds originally identified in Image-based HTS for Selective Agonists of GPR55 (AID 1961). Compounds were either acquired from commercial sources or synthesized by the Sanford-Burnham Center for Chemical Genomics.
Protocol
Assay Materials:
1) 60 x 15mm Style Tissue Culture Dish (BD Falcon #353004)
2) U2OS (Human Osteosarcoma) cell line stably expressing Beta-arrestin GFP and GPR55E receptor (for ref. see Kapur at al. JBC 284: 29817-29827, 2009).
3) Culture Media: DMEM with 4.5 g/L glucose, L-glutamine and sodium pyruvate, (Cellgro #10-013-CV) supplemented with selection antibiotics - 100microg/ml G418 (AG Scientific, INC # G-1033) and 50microg/ml zeocin (Invitrogen # R25001).
4) Positive control working solution: Lysophosphatidylinositol (Sigma L7635 10mM stock in DMSO) diluted in assay buffer (HBSS, Cellgro #21-023-CV) to a final concentration of 10 microM.
5) Negative control Working Solution: 100% DMSO
6) Test compounds Working Solution: 10mM stock in 100% DMSO, diluted in assay buffer (HBSS to a final concentration of 1microM.
7) Lysis Buffer: (50mM Hepes, 150mM NaCl, 1mM EDTA, 1mM EGTA, 10% Glycerol, 1% Triton X-100, 10microM MgCl2, 20mM p-nitrophenyl phosphate, 1mM Na3VO4, 25mM NaF, and a protease inhibitor mixture (1:25, pH 7.5).
8) Blocking buffer was purchased from Licor (#927-40000)
9) Primary antibodies: M8159 Monoclonal Anti-MAP Kinase, Activated (Diphosphorylated ERK1/2) antibody produced in mouse - clone MAPK-YT, ascites fluid (Sigma) and p44/42 MAPK (ERK1/2) Antibody (Cell Signaling Technology #9102).
10) Secondary antibodies: IR-Dye 680 goat anti-Mouse and IR-Dye 800 goat anti-Rabbit (LI-COR Biosciences # 926-32220, 926-32211).
Assay Procedure:
1) U2OS cells were seeded onto 60mm dishes and incubated overnight at 37 degrees C and 5% CO2.
2) Serum was removed by media aspiration and was replaced with 3mL serum free DMEM containing selection antibiotics.
3) Cells were incubated in serum free DMEM for 48 hours at 37 degrees C and 5% CO2 prior to the assay.
4) Media was removed by aspiration and washed once and replaced with 3ml HBSS and incubated at room temperature for 30 minutes.
5) Compounds were applied directly onto the plates for 10 minutes at room temperature.
6) Following the drug treatment, cells were disrupted in 200microl Lysis Buffer.
7) Cell lysates were immediately placed on ice for 10 minutes and then centrifuged at 16,000 x g for 30 minutes at 4 degrees C.
8) Supernatants, corresponding to the cytosolic fraction were collected, and protein concentrations were determined by the Bradford assay (Bio-Rad) using bovine serum albumin as a standard.
9) Cytosolic fractions (20 microg) were separated on a 10% gel by SDS-PAGE.
10) Blots were washed once with PBS and blocked for 1 hour at room temperature.
11) Primary antibodies were diluted in Odyssey Blocking Buffer (1:500 M8159, 1:1000 9102, LI-COR Biosciences). To lower background, 0.1% Tween-20 was added to the diluted antibodies before incubation. Blots were incubated in primary antibodies overnight at 4 degrees C.
12) Following incubation, membranes were washed 4 times for 5 minutes each in PBS with 0.1% Tween-20 with gentle shaking at room temperature.
13) The fluorescently-labeled secondary antibodies were diluted in Odyssey Blocking Buffer (1:10,000 dilution) containing 0.1% Tween-20. Blots were incubated for 60 min at RT and were protected from light.
14) Membranes were washed 4 times for 5 minutes each in PBS + 0.1% Tween-20 with gentle shaking at room temperature, and were protected from light.
15) Membranes were rinsed once with PBS to remove residual Tween-20.
16) Membranes were scanned using a LI-COR Odyssey Imaging System at both 680 and 800 channels.
17) Densitometric analysis was performed using a LI-COR Odyssey Imaging System. Values obtained for both ERK1 and ERK2 (activated isoforms) was normalized to total ERK1/2 levels. The data were normalized to control and presented as percent stimulation.
Comment
Compounds with average percent efficacy higher than 50% are considered active.
To simplify the distinction between the actives of the primary screen and of the confirmatory dose response, a tiered activity scoring system was devised to take into consideration compound efficacy and potential interference with the assay. The outline of the scoring system utilized for the screen is as follows:
1) First tier (0-40 range) is reserved for primary and confirmation screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Compounds that are inactive in this assay are assigned a score of 81.
Compounds that are active in this assay are assigned a score of 90.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: U-2 OS
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average %Efficacy (1μM**)Average percent Efficacy normalized to the negative controlFloat%
2Std.Err(%Efficacy) (1μM**)Standard error of the Average %Percent Efficacy valueFloat%
3SEM(%Efficacy) (1μM**)Standard error of the Average %Percent Efficacy valueFloat%
4%Efficacy_1 (1μM**)PercPercent Efficacy normalized to the positive and negative controls - first replicateFloat%
5%Efficacy_2 (1μM**)Percent Efficacy normalized to the positive and negative controls - second replicateFloat%
6%Efficacy_3 (1μM**)Percent Efficacy normalized to the positive and negative controls - third replicateFloat%
7Average %Stimulation (1μM**)Average percent stimulation normalized to the negative controlFloat%
8Std.Err(%Stimulation) (1μM**)Standard error of the Average %Percent stimulation valueFloat%
9SEM(%Stimulation) (1μM**)SEM of the Average %Percent stimulation valueFloat%
10%Stimulation_1 (1μM**)Percent stimulation normalized to the negative control- second replicateFloat%
11%Stimulation_2 (1μM**)Percent stimulation normalized to the negative control- second replicateFloat%
12%Stimulation_3 (1μM**)Percent stimulation normalized to the negative control- third replicateFloat%
13Positive Control_1Positive control- first replicate - NormalizedFloat%
14Positive Control_2Positive control- second replicate - NormalizedFloat%
15Positive Control_3Positive control- third replicate - NormalizedFloat%
16Negative Control_1Negative control- first replicate - NormalizedFloat%
17Negative Control_2Negative control- second replicate - NormalizedFloat%
18Negative Control_3Negative control- third replicate - NormalizedFloat%

** Test Concentration.
Additional Information
Grant Number: 1X01 DA026205-01

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