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BioAssay: AID 1961

Image-based HTS for Selective Agonists of GPR55

Assay Provider: Dr. Mary Abood, California Pacific Medical Center Research Institute (currently Temple University) ..more
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 Tested Compounds
 Tested Compounds
All(291988)
 
 
Active(139)
 
 
Inactive(291508)
 
 
Inconclusive(342)
 
 
 Tested Substances
 Tested Substances
All(292143)
 
 
Active(140)
 
 
Inactive(291661)
 
 
Inconclusive(342)
 
 
AID: 1961
Data Source: Burnham Center for Chemical Genomics (BCCG-A221-GPR55-Agonist-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-09-29
Modify Date: 2010-12-30

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 139
Depositor Specified Assays
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AIDNameTypeProbeComment
1965Summary of Image-based HTS for Selective Agonists of GPR55summary3
2347SAR analysis of selective Agonists of GPR55 using an Image-Based Assayconfirmatory
434922SAR analysis of antagonists of the Cannabinoid Receptor 2 using an Image-Based Assay - Set 2confirmatory
2843SAR analysis of selective Agonists of GPR55 using an Image-Based Assay - Set 2confirmatory
2844SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 3confirmatory
2341SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based Assayconfirmatory
434929SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based Assay - Set 3confirmatory
2338SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assayconfirmatory
434925SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 4confirmatory
434928SAR analysis of agonists of the Cannabinoid Receptor 2 using an Image-Based Assay - Set 2confirmatory
463192SAR Analysis for the identification of Selective Agonists of GPR55 using an Image-Based Screenother
485279SAR analysis of Agonists of GPR55 using MAPK Activation Assayother
2339SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based Assayconfirmatory
2340SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assayconfirmatory
434924SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assay - Set 3confirmatory
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 Proposal Number: 1X01 DA026205-01
Assay Provider: Dr. Mary Abood, California Pacific Medical Center Research Institute (currently Temple University)

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 agonists 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.
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) Positive Control Working Solution: Lysophosphatidylinositol (Sigma L7635, 5mM stock in Methanol water) diluted in water to 250uM. Additional DMSO is added to achieve a DMSO concentration of 2.5%.
5) Negative Control Working Solution: 2.5% DMSO diluted in water.
6) Fixative Working Solution: 6% Paraformaldehyde (PFA) diluted in PBS.
7) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).

Primary Screen Procedure:
1) 45ul 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 are incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.
3) Serum is removed by media aspiration and replacing with 45ul serum-free MEM prior to addition of compounds.
4) Compound addition was done on a Biomek FX with 384-head dispenser (Beckman):
a) 5ul of 100uM compound solution was added to columns 3 through 24 of the assay plates for a final assay compound concentration of 10uM and 0.25% DMSO.
b) 5ul of negative control (2.5% DMSO) working solution was added to column 2.
c) 5ul of the positive control (250uM LPI) working solution was added to column 1.
5) Plates were incubated for 75 minutes at 37 degrees C and 5% CO2.
6) Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.
7) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.
8) Plates were incubated for 40 minutes at room temperature.
9) Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
10) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml. Aluminum plate sealers were applied to each plate.

Hit Confirmation Procedure:
1) The same procedure as used for primary screen (above) steps 1 to 3.
2) Compound addition was done on the ECHO550 Liquid Handler. The 'dose response protocol' was used to dispense corresponding volumes of each 10mM compound on the assay plate.
a. Compounds were added to columns 3 to 22. Final concentration ranged from 32uM to 500nM, in duplicate (seven doses).
b. Positive control was added to column 1. LPI final concentration was 25uM.
c. Negative control was added to column 2. DMSO final concentration was 0.3%.
3) Primary screen procedure (above) steps 5 to 10 were followed.

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 emisssion filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
2 fields per well for Primary screen
4 fields per well for Hit Confirmation

2) Image analysis was performed using the Acapella Spot Detection Algorithm.

Analysis settings:
Nuclei Detection
Threshold Adjustment: 1.5
Nuclear Splitting Adjustment: 7
Minimum Nuclear Area: 70
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

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")

3) Actives from the primary screen were determined using CBIS software (ChemInnovations) by calculating the %activation of the "PercentagePositiveCells" metric and using a hit criteria of Activity > 40% and "NumberofCellsAnalyzed" > 50. Wells with cell counts lower than 50 in the 2 acquired images were flagged "cytotoxic / low cell count" and were excluded from hit selection.
Comment
In the primary screen compounds were "active" if Activity > 40% and "NumberofCellsAnalyzed" > 50. Wells with cell counts lower than 50 in the 2 acquired images were flagged "cytotoxic / low cell count". Flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".

The primary screen "actives" were then tested for dose-response hit confirmation. Compounds with an IC50 <10uM were considered "confirmed actives".

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 screening data. The score is correlated with % activity in the assay demonstrated by a compound at 10 uM concentration:
a. If primary % activity is less than 0%, then the assigned score is 0
b. If primary % activity is greater than 100%, then the assigned score is 40
c. If primary % activity is between 0% and 100%, then the calculated score is (%activity)*0.4

2) Second tier (41-80 range) is reserved for dose-response confirmation data.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
Score = 44 + 6*(pEC50 - 3)
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units. This equation results in score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior is likely to be an artifact of that assay will generally have lower score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_QualifierThis qualifier is to be used with the next TID, EC50. If qualifier is "=", EC50 result equals to the value in that column; if qualifier is ">", EC50 result is greater than that value.String
2EC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(EC50)Standard Error of EC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%Activity at 10 uM (10μM**)%Activity at 10 uMFloat%
6Mean_NC (10μM**)Mean luminescence signal of negative controls in the corresponding plateFloatCPS
7StdDev_NC (10μM**)Standard deviation (n=16) of negative controls in the corresponding plateFloatCPS
8Mean_PC (10μM**)Mean luminescence signal of positive controls in the corresponding plateFloatCPS
9StdDev_PC (10μM**)Standard deviation (n=16) of positive controls in the corresponding plateFloatCPS
10PercentagePositiveCells (10μM**)Percentage of Cells Positive for Spot FormationFloat%
11 NumberofCellsAnalyzed (10μM**)Cell countFloatcells
12AreaoftheNucleus (10μM**)Nuclei Area Floatum^2
13TotalIntegratedIntensityoftheNucleus (10μM**)Integrated Intensity of the Nuclei (#FloatRU
14AverageIntegratedIntensityoftheNucleus (10μM**)Average Intensity of the Nuclei FloatRU
15TotalCytoplasmIntensity (10μM**)Integrated Intensity of the Cytoplasm FloatRU
16TotalSpotIntensity (10μM**)Integrated Intensity of the Detected Spots FloatRU
17RatioofSpotintensitytoCytoplasmintensity (10μM**)Ratio of the Integrated Spot to Integrated Cytoplasm Intensities FloatRU
18AverageSpotsPerCell (10μM**)Number of Spots per Cell FloatRU

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

Data Table (Concise)
Classification
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