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
BioActive Compounds: 8
Depositor Specified Assays
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.
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).
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.
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.
** Test Concentration.
Data Table (Concise)