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BioAssay: AID 2820

SAR Analysis of Selective Antagonists of GPR55 using an Image-Based Assay - Set 2

Assay Provider: Dr. Mary Abood, (Temple University, formerly at California Pacific Medical Center Research Institute) ..more
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 Tested Compounds
 Tested Compounds
All(38)
 
 
Active(38)
 
 
 Tested Substances
 Tested Substances
All(38)
 
 
Active(38)
 
 
AID: 2820
Data Source: Burnham Center for Chemical Genomics (BCCG-A353-GPR55-Antagonist-DryPowder-Assay-2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-05-07
Hold-until Date: 2011-04-30
Modify Date: 2011-04-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 38
Related Experiments
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AIDNameTypeProbeComment
2013Image-Based HTS for Selective Antagonists for GPR55Confirmatory depositor-specified cross reference
2026Summary of Image-based HTS for Selective Antagonists of GPR55Summary3 depositor-specified cross reference: Summary AID.
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
2815SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 3Confirmatory 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
2835SAR analysis of agonists of the Cannabinoid Receptor 2 using an Image-Based AssayConfirmatory same project related to Summary assay
2836SAR analysis of antagonists of the Cannabinoid Receptor 2 using an Image-Based AssayConfirmatory same project related to Summary assay
485277SAR analysis of agonists of the Cannabinoid Receptor 2 using an Image-Based Assay - Set 3Confirmatory same project related to Summary assay
485278SAR analysis of antagonists of the Cannabinoid Receptor 2 using an Image-Based Assay - Set 3Confirmatory same project related to Summary assay
485282SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 4Confirmatory same project related to Summary assay
485283SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 6Confirmatory same project related to Summary assay
485285SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 4Confirmatory same project related to Summary assay
485286SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assay - Set 4Confirmatory same project related to Summary assay
485287SAR Analysis of Selective Antagonists of GPR55 using an Image-Based Assay - Set 4Confirmatory same project related to Summary assay
488947SAR Analysis for the identification of Selective Antagonists of GPR55 using an Image-Based ScreenOther same project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics(SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1X01 DA026205-01
Assay Provider: Dr. Mary Abood, (Temple University, formerly at California Pacific Medical Center Research Institute)

The cannabinoid and endocannabinoid system has been implicated in the pathophysiology of drug dependence and addiction disorders. GPR55, an orphan G-Protein Coupled Receptor, has been reported to be a cannabinoid receptor, but its status as such remains unresolved due to conflicting results from pharmacological studies. The goal of the project is to identify small molecule antagonists of GPR55, which may aid in the deorphanization efforts of this receptor and ultimately further the understanding of the role of GPR55 in drug addiction.

This high content imaging assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and an enhanced GPR55 receptor. Upon agonist-mediated GPR55 activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane to coated pits and further into endosomal vesicles. This arrestin-GFP redistribution is measured as increased local concentrations of fluorescent arrestins.

This dose response assay is developed and performed to confirm hits originally identified in "Image-based HTS for Selective Antagonists of GPR55" (AID 2013) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
Protocol
Assay Materials:
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and GPR55 receptor
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 100ug/ml G418 and 50ug/ml Zeocin
4) Agonist Working Solution: Lysophosphatidylinositol (Avanti Polar #850090P, 5mM stock in DMSO) diluted to 50uM in water.
5) Control Working Solution: 100% DMSO (for positive control wells: no agonist will be added to mimic 100% inhibition of agonist-mediated response; for negative control wells: LPI agonist will be added to mimic 0% inhibition of agonist-mediated response).
6) Test Compounds Working Solution: 10mM in 100% DMSO
7) Fixative Working Solution: 6% Paraformaldehyde (PFA) diluted in PBS.
8) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).
Assay Procedure:
1) 45 ul of cell suspension (200,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
2) Plates were incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.
3) Serum was removed by media aspiration and replaced with 45 ul serum-free MEM prior to addition of compounds.
4) Compound addition was done on an ECHO550 Liquid Handler. The "dose response protocol" was used to dispense corresponding volumes of each 10 mM compound on the assay plate.
a. Compounds were added to columns 3 to 22. Final concentration ranged from 62nM to 32uM (ten doses), in duplicate.
b. Control working solution was added to columns 1 and 24 representing positive control wells by using 0 uM agonist to mimick 100% inhibition.
c. Control working solution was added to columns 2 and 23 representing negative control wells.
d. DMSO was back-filled to each well to achieve 0.3% final concentration.
5) Plates were pre-incubated with compounds for 30 minutes at 37 degrees C and 5% CO2.
6) After 30 minutes, 5 ul of the agonist (50 uM LPI) working solution was added to the entire plate except column 1 and 24 for a final agonist concentration of 5 uM.
7) Plates were incubated for 75 minutes at 37 degrees C and 5% CO2.
8) Media was aspirated leaving 20 ul liquid in each well using a Titertek plate washer.
9) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.
10) Plates were incubated for 40 minutes at room temperature.
11) Fixative was aspirated and plates were washed twice with 50 ul PBS leaving 20 ul liquid in each well using a Titertek plate washer.
12) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100 ng/ml. Aluminum plate sealers were applied to each plate.
Image Acquisition and Analysis:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
20x 0.45 NA air objective
Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
2 channels acquired sequentially: Exp1Cam1 = Beta-arrestin GFP using 488 nm laser excitation and 540/70 nm emission filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
4 fields per well
2) Image analysis was performed using the Acapella Spot Detection Algorithm with the following analysis settings:
NUCLEI DETECTION
Threshold Adjustment: 1.5
Nuclear Splitting Adjustment: 7
Minimum Nuclear Area: 0
Minimum Nuclear Contrast: 0.1
CYTOPLASM DETECTION
Cytoplasm Threshold Adjustment: 0.45
Cytoplasm Individual Threshold Adjustment: 0.15
SPOT DETECTION
Spot Minimum Distance 3
Spot Peak Radius 0
Spot Reference Radius 2
Spot Minimum Contrast 0.25
Spot Minimum to Cell Intensity 1
3) Metrics calculated from
NUCLEI IMAGES: Cell Count (NumberofCellsAnalyzed), Nuclei Area (AreaoftheNucleus), Integrated Intensity of the Nuclei (TotalIntegratedIntensityoftheNucleus), Average Intensity of the Nuclei (AverageIntensityoftheNucleus)
GFP IMAGES: Integrated Intensity of the Cytoplasm (TotalCytoplasmIntensity), Integrated Intensity of the Detected Spots (TotalSpotIntensity), Ratio of the Integrated Spot to Integrated Cytoplasm Intensities (RatioofSpotIntensitytoCytoplasmintensity), Number of Spots per Cell (AverageSpotsPerCell), Percentage of Cells Positive for Spot Formation (PercentagePositiveCells)
4) The "AverageSpotsPerCell" metric was used to calculate the dose response curves and parameters. IC50 values were calculated using CBIS (ChemInnovations) software employing a sigmoidal dose-response equation through non-linear regression.
Comment
Compounds with an IC50 < 10 uM are defined as actives in the dose response confirmation.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable to this assay
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to 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. For the remaining compounds the score is linearly correlated with a compound's inhibitory 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
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3IC50_Qualifier_1_Range1This qualifier is to be used with the next TID, EC5_1_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1_Range1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1_Range1Standard Error of IC50 valueFloatμM
6nH_1_Range1Standard Error of IC50 valueFloat
7Excluded_Points_first_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% inhibition at 32 uM_first_point_Range1 (32μM**)% inhibition at a given concentrationFloat%
9% inhibition at 16 uM_first_point_Range1 (16μM**)% inhibition at a given concentrationFloat%
10% inhibition at 8 uM_first_point_Range1 (8μM**)% inhibition at a given concentrationFloat%
11% inhibition at 4 uM_first_point_Range1 (4μM**)% inhibition at a given concentrationFloat%
12% inhibition at 2 uM_first_point_Range1 (2μM**)% inhibition at a given concentrationFloat%
13% inhibition at 1 uM_first_point_Range1 (1μM**)% inhibition at a given concentrationFloat%
14% inhibition at 0.5 uM_first_point_Range1 (0.5μM**)% inhibition at a given concentrationFloat%
15% inhibition at 0.25 uM_first_point_Range1 (0.25μM**)% inhibition at a given concentrationFloat%
16% inhibition at 0.125 uM_first_point_Range1 (0.125μM**)% inhibition at a given concentrationFloat%
17% inhibition at 0.0625 uM_first_point_Range1 (0.0625μM**)% inhibition at a given concentrationFloat%
18Excluded_Points_second_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% inhibition at 32 uM_second_point_Range1 (32μM**)% inhibition at a given concentrationFloat%
20% inhibition at 16 uM_second_point_Range1 (16μM**)% inhibition at a given concentrationFloat%
21% inhibition at 8 uM_second_point_Range1 (8μM**)% inhibition at a given concentrationFloat%
22% inhibition at 4 uM_second_point_Range1 (4μM**)% inhibition at a given concentrationFloat%
23% inhibition at 2 uM_second_point_Range1 (2μM**)% inhibition at a given concentrationFloat%
24% inhibition at 1 uM_second_point_Range1 (1μM**)% inhibition at a given concentrationFloat%
25% inhibition at 0.5 uM_second_point_Range1 (0.5μM**)% inhibition at a given concentrationFloat%
26% inhibition at 0.25 uM_second_point_Range1 (0.25μM**)% inhibition at a given concentrationFloat%
27% inhibition at 0.125 uM_second_point_Range1 (0.125μM**)% inhibition at a given concentrationFloat%
28% inhibition at 0.0625 uM_second_point_Range1 (0.0625μM**)% inhibition at a given concentrationFloat%
29IC50_Qualifier_2_Range1This qualifier is to be used with the next TID, IC5_1_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2_Range1IC50 value determined using sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2_Range1Standard Error of IC50 valueFloatμM
32nH_2_Range1Standard Error of IC50 valueFloat
33Excluded_Points_third_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% inhibition at 32 uM_third_point_Range1 (32μM**)% inhibition at a given concentrationFloat%
35% inhibition at 16 uM_third_point_Range1 (16μM**)% inhibition at a given concentrationFloat%
36% inhibition at 8 uM_third_point_Range1 (8μM**)% inhibition at a given concentrationFloat%
37% inhibition at 4 uM_third_point_Range1 (4μM**)% inhibition at a given concentrationFloat%
38% inhibition at 2 uM_third_point_Range1 (2μM**)% inhibition at a given concentrationFloat%
39% inhibition at 1 uM_third_point_Range1 (1μM**)% inhibition at a given concentrationFloat%
40% inhibition at 0.5 uM_third_point_Range1 (0.5μM**)% inhibition at a given concentrationFloat%
41% inhibition at 0.25 uM_third_point_Range1 (0.25μM**)% inhibition at a given concentrationFloat%
42% inhibition at 0.125 uM_third_point_Range1 (0.125μM**)% inhibition at a given concentrationFloat%
43% inhibition at 0.0625 uM_third_point_Range1 (0.0625μM**)% inhibition at a given concentrationFloat%
44Excluded_Points_fourth_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% inhibition at 32 uM_fourth_point_Range1 (32μM**)% inhibition at a given concentrationFloat%
46% inhibition at 16 uM_fourth_point_Range1 (16μM**)% inhibition at a given concentrationFloat%
47% inhibition at 8 uM_fourth_point_Range1 (8μM**)% inhibition at a given concentrationFloat%
48% inhibition at 4 uM_fourth_point_Range1 (4μM**)Float%
49% inhibition at 2 uM_fourth_point_Range1 (2μM**)% inhibition at a given concentrationFloat%
50% inhibition at 1 uM_fourth_point_Range1 (1μM**)% inhibition at a given concentrationFloat%
51% inhibition at 0.5 uM_fourth_point_Range1 (0.5μM**)% inhibition at a given concentrationFloat%
52% inhibition at 0.25 uM_fourth_point_Range1 (0.25μM**)% inhibition at a given concentrationFloat%
53% inhibition at 0.125 uM_fourth_point_Range1 (0.125μM**)% inhibition at a given concentrationFloat%
54% inhibition at 0.0625 uM_fourth_point_Range1 (0.0625μM**)% inhibition at a given concentrationFloat%

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

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