HTS Image-Based Screen for Agonists of the DOR Receptor
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 opioid receptors are composed of multiple of multiple receptor subtypes whose contribution the addictive behaviors are not fully delineated. ..more
BioActive Compound: 1
Data Source: Sanford-Burnham Center for Chemical Genomics
Source Affiliation: Sanford-Burnham Medical Research Institute
Network: NIH Molecular Libraries Probe Production Centers Network
Grant Number: 1X01DA026208-01
Assay Provider: Dr. Lawrance Barak, Duke University, Durham, NC
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 opioid receptors are composed of multiple of multiple receptor subtypes whose contribution the addictive behaviors are not fully delineated.
The aim of this assay is to identify small molecule agonists of the human delta opioid receptor (DOR). The dose response assay is developed and performed to evaluate selectivity of hits originally identified in a uHTS luminescent beta-arrestin assay for agonists of the KOR receptor. This image-based DOR agonist assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and the delta opioid receptor (DOR). 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.
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and the DOR1 receptor
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 200ug/ml G418 and 50ug/ml Zeocin
4) Positive Control Working Solution: SNC80 (Tocris #0764-10mg - 5mM stock in DMSO) diluted in water to 1uM.
5) Negative Control Working Solution: 100% DMSO in water
6) Test compounds from dry powder 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).
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 oC and 5% CO2.
3) Serum is removed by media aspiration and replaced with 45ul 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 10mM 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. Positive control was added to column 1. SNC80 final concentration was 100nM.
c. Negative control was added to column 2. DMSO final concentration was 0.31%.
d. DMSO was back-filled to each well to achieve a 0.31% final concentration.
5) Plates were incubated for 45 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.
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.
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 Threshold Adjustment: 0.2
Cytoplasm Individual Threshold Adjustment: 0.1
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.
EC50 values were calculated using CBIS software (ChemInnovations) employing a sigmoidal dose-response equation through non-linear regression.
Compounds with an EC50 of <10uM were considered "confirmed actives".
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 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 in this assay.
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 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. Equation that takes into account the items discussed above is
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 the 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 are 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 in this assay
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)