SAR analysis of Antagonists of the Kappa Opioid Receptor (KOR) using an Image-Based Assay - Set 2
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 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 antagonists of the human kappa opioid receptor (KOR). This dose response assay is developed and performed to confirm hits originally identified in "uHTS identification of small molecule agonists of the kappa opioid receptor via a luminescent beta-arrestin assay" (AID 1777) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
This image-based KOR agonist assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and the kappa opioid receptor (KOR). 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 KOR1 receptor
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 200ug/ml G418 and 100ug/ml Zeocin
4) Positive Control Working Solution: (Dynorphin-A, American Peptide #24450 - 5mM stock in DMSO) diluted in water to 10uM.
5) Negative Control Working Solution: Naloxone hydrochloride dehydrate (Sigma N7758)- 5mM stock diluted to 5uM 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 degree C 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. Dynorphin final concentration was 1uM.
c. Negative control was added to column 2. Naloxone final concentration was 500nM.
d. DMSO was back-filled to each well to achieve a 0.31% final concentration. DMSO final concentration was 0.31%.
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.
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 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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)