Sequence: cannabinoid receptor 2 [Homo sapiens]

Gene:CNR2     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
2013	Image-Based HTS for Selective Antagonists for GPR55	confirmatory
2026	Summary of Image-based HTS for Selective Antagonists of GPR55	summary	Summary AID.

Data Source: Sanford-Burnham Center for Chemical Genomics
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

Drug addiction is a disease that involves the G-protein coupled receptors in the central nervous system resulting in compulsive or abnormal behavior. Recent studies have shown that opioid receptors play a role in regulating other receptors that interact with drug and other substance abuse. The cannabinoid receptors (type 1 and 2) are members of the G-protein coupled receptor family and have been found to be involved in alterations in mood and cognition, as experienced by marijuana users.

The specific aim of this assay is to identify small molecule antagonists of the human cannabinoid receptor type 2 (CB2). The dose response assay is developed and performed to evaluate selectivity of hits originally identified in "Image-Based HTS for Selective Antagonists for GPR55" (AID 2013) and to study the structure-activity relationship. Compounds are either acquired from commercial sources or synthesized internally.

This imaging assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and the cannabinoid receptor type 2. Upon agonist-mediated GPCR activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane and further to coated pits / endosomal vesicles, which can be quantified as increased local aggregation of the GFP-arrestins.

Assay Materials:
1) 384-well plates, black with clear bottom (Aurora Biosciences # 032451)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and the CB2 receptor
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 200 ug/ml G418 and 100 ug/ml Zeocin
4) Positive Control Working Solution: WIN55212 (Sigma W102 25mg) - 5 mM stock in DMSO) diluted to 2.75 uM in water.
5) Negative Control Working Solution: AM251 (Sigma A6226-10 mg) - diluted to 11 uM in water.
6) Test compounds from dry powders working solution: 10 mM 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 (10 mM TRIS, 10 mM EDTA, 100 mM NaCl, pH 7.4).

Hit Confirmation 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 are incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.
3) Serum is removed by media aspiration and replacing with 45 ul serum-free MEM prior to addition of compounds.
4) Compound addition was done on the 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 62 nM to 32 uM (ten doses), in duplicate.
b. Positive control was added to column 1. AM251 final concentration was 1uM.
c. DMSO was added to column 2 and 23. DMSO final concentration was 0.3%.
d. DMSO was back-filled to each well to achieve a 0.31% final concentration.
e. Negative control was WIN55212 at 250 nM, on column 2.
5) Plates were incubated for 30 minutes at 37 degrees C and 5% CO2.
6) WIN55211 was added to the entire plate except column 23, and incubated again for 75 minutes.
7) Media was aspirated leaving 20 ul liquid in each well using a Titertek plate washer.
8) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.
9) Plates were incubated for 40 minutes at room temperature.
10) Fixative was aspirated and plates were washed twice with 50 ul PBS leaving 20 ul liquid in each well using a Titertek plate washer.
11) 40 ul 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:
40x 0.9 NA water 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.

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:
40x 0.6 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.

Analysis settings:
Nuclei Detection
Threshold Adjustment: 1.5
Nuclear Individual Threshold Adjustment: 0.45
Nuclear Splitting Adjustment: 7
Minimum Nuclear Area: 70
Minimum Nuclear Distance: 7
Minimum Nuclear Contrast: 0.1
Cytoplasm Detection
Cytoplasm Threshold Adjustment: 0.2
Cytoplasm Individual Threshold Adjustment: 0.1
Spot Detection
Spot Minimum Distance 3
Spot Peak Radius 0
Spot Reference Radius 3
Spot Minimum Contrast 0.3
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 "RatioofSpotIntensitytoCytoplasmintensity" metric was used to calculate the dose response curves and parameters.

IC50 values were calculated using CBIS software (ChemInnovations) employing a sigmoidal dose-response equation through non-linear regression.

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.

Grant Number: 1X01 DA026205-01