SAR Analysis for the identification of Selective Antagonists of ERK1/2 Activity in GPR35-Overexpressing U2OS Cells
The aim of this assay was to characterize downstream ERK phosphorylation activity of compounds originally identified in "Image-based HTS for Selective Antagonists of GPR35" (AID 2058). Componds were either acquired from commercial sources or synthesized by the Sanford-Burnham Center for Chemical Genomics. ..more
BioActive Compounds: 20
Data Source: Dr. Mary Abood
Source Affiliation: Temple University
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1X01MH085708-01
Assay Provider: Dr. Lawrence Barak, Duke University
The aim of this assay was to characterize downstream ERK phosphorylation activity of compounds originally identified in "Image-based HTS for Selective Antagonists of GPR35" (AID 2058). Componds were either acquired from commercial sources or synthesized by the Sanford-Burnham Center for Chemical Genomics.
This In-Cell Western assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and human GPR35 receptor. Upon agonist-mediated GPCR activation, ERK1/2 phosphorylation occurs as measured by pERK1/2 antibodies.
1) 96-well plates (FALCON 353075)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and human GPR35 receptor
3) Culture Media: DMEM with 10% Fetal Bovine Serum and selection antibiotics - 200ug/ml G418 and 100ug/ml Zeocin
4) EC80 concentration of Agonist: 10 uM Zaprinast
5) DMSO solution
6) Test compounds Working Solution: 10mM Stock in 100% DMSO, diluted in assay buffer (HBSS Cellgro #21-023-CV)
7) Fixative Solution: 4% Paraformaldehyde (PFA) diluted in PBS
8) Permeabilization Solution: 0.1% Triton X-100 in PBS
9) Primary phosphor-ERK1/2 antibody (Cell Signaling Technology, diluted 1:100)
10) Goat anti-rabbit 800CW secondary antibody diluted 1:800 in Licor blocking buffer together with Sapphire700 (diluted 1:1000) and DRAQ5 (diluted 1:2000) for normalization purpose
1) Cells were grown to confluence in 96-well plates and serum-starved overnight prior to assay.
2) Prior to drug treatment cells were washed once with HBSS at room temperature.
3) Compounds were added at varying dose response concentrations together with the agonist (10uM Zaprinast) to compound and negative control wells.
4) DMSO was added to all wells to achieve a final concentration of 0.1%.
5) Plates were incubated for 15 minutes at room temperature.
6) Media was aspirated from all wells.
7) Fixative solution was added to each well for a final concentration of 4% PFA and plates were incubated for 60 minutes at room temperature.
8) Fixative was aspirated and cells were permeabilized with 0.1% Trion X-100 in PBS for 5 washes at 10 minutes per wash.
9) LI-COR blocking buffer was added and samples were shaken on a rotator for 1 hour.
10) Primary antibodies were applied overnight at 4 degree C with rotation, followed by washing 4 times for 5 minutes each in PBS+ 0.1% Tween-20 with gentle shaking at room temperature.
11) Cells were incubated with secondary antibodies for 2 hours at room temperature. Sapphire700 and DRAQ5 solutions were added together with the secondary antibodies for normalization. Plates were protected from light.
12) Plates 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.
13) Plates were dried and scanned using a LI-COR Odyssey Infrared Imager.
14) IC50 values were calculated employing a sigmoidal dose-response equation through non-linear regression in Prism 4.0 software.
Compounds with an IC50 <10uM were 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
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3),
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)