HTS Image-Based Screen for Selective Agonists of the KOR Receptor
Drug addiction is a disease originating in the central nervous system that produces compulsive behaviors despite the negative consequences that may result. Major addictive drugs of abuse include components of tobacco, opiates, marijuana, ethanol, cocaine, and derivatives of amphetamines. While the addictive behaviors produced by these substances may be generally similar, the drugs act at more ..
BioActive Compounds: 24
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: 1X01DA026208-01
Assay Provider: Dr. Lawrance Barak, Duke University, Durham, NC
Drug addiction is a disease originating in the central nervous system that produces compulsive behaviors despite the negative consequences that may result. Major addictive drugs of abuse include components of tobacco, opiates, marijuana, ethanol, cocaine, and derivatives of amphetamines. While the addictive behaviors produced by these substances may be generally similar, the drugs act at different receptor sites in the brain. Recent studies have shown that opioid receptors play a role regulating the addictive behaviors of other receptors that interact with illicit and legal substances of abuse. Opioid receptors are composed of multiple subtypes whose contributions to addictive behaviors are not fully delineated. Moreover, different compounds acting at a single receptor type may produce dissimilar behaviors due to variations in their chemical structures. Thus, both for delineating the biology and signaling ability of each receptor and for developing novel therapies, it would be desirable to identify multiple small molecule ligands that target a given receptor subtype. This entails expanding the chemical space about each of the receptors as variations in receptor conformations due to distinct ligand structures affect receptor signaling behaviors, and consequently physiological responsiveness. Morphine is a prime example of an opioid ligand with distinctive signaling properties.
The specific aim of this project is to identify subtype specific small molecule agonists of the human kappa opioid receptor (KOR). These compounds can be optimized to provide the pharmacological means to precisely control the signaling of their specific target receptor subtype. This work thus provides the addiction field both novel research tools and potential therapeutic scaffolds.
This image-based fluorescent beta-arrestin assay was performed to confirm hits originally identified in the uHTS luminescent beta-arrestin assay for agonists of the KOR receptor.
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and the KOR 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 (American Peptide #24-4-50, 5mM stock in DMSO) diluted in water to 10uM.
5) Negative Control Working Solution: 3.1% DMSO 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).
Dose Response Assay 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 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 500nM to 32uM (seven doses), in duplicate.
b. Positive control was added to column 1. Dynorphin final concentration was 1uM.
c. Negative control was added to column 2. DMSO final concentration was 0.3%.
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 emisssion filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
4 fields per well for Hit Confirmation
2) Image analysis was performed using the Acapella Spot Detection Algorithm.
3) For the dose response hit confirmation, compounds with an EC50 of <10uM were considered "confirmed actives". 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)