Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries 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 is a luminescence assay based off of a kappa opioid cell line and beta-arrestin technology from DiscoveRx.

The dose response assay is developed and performed to confirm hits originally identified in the uHTS luminescent, beta-arrestin assay for antagonists of the KOR receptor.

Assay materials:

1) OPRK1 beta-Arrestin (DiscoveRx)
2) Assay Medium: Opti-MEM Medium supplemented with 1% hiFBS, 1X Pen/Strep/Glu, 125 ug/mL Hygromycin (1/2 recommended), 250 ug/mL Geneticin (1/2 recommended)
3) Growth Medium: MEM supplemented with 10% hiFBS, 1X Pen/Strep/Glu, 125 ug/mL Hygromycin (1/2 recommended), 250 ug/mL Geneticin (1/2 recommended)

uHTS protocol:

Day 1
1) Harvest cells using Enzyme-Free Dissociation Buffer (Invitrogen Cat#13151-14). Add 500 cells/well in 5 uL of media to each well of a white, 1536 well plate.
2) Spin cells at 500 rpm for 1 min, then wrap plates in Saran Wrap.
3) Incubate overnight at 37C with 5% CO2.

Day 2
1) Using a Highres Biosolutions pintool pin 30 nL to wells. Columns 1-4 should be DMSO only (Control wells), Columns 5-48 contain test compounds (10uM final in well concentration).
2) Immediately following pintool addition, add 1.0 uL of assay media to columns 1-2 and 1.0 uL of assay media containing 240 nM dynorphin A for a final assay concentration of 40 nM. Centrifuge plates at 500 rpm for 1 min immediately following additions.
3) Incubate for 1hr and 30 minutes.
4) During test incubation, prepare Detection Reagent Solution from DiscoveRx (1 part Galacton Star: 5 parts Emerald II and 19 parts Cell Assay Buffer)
5) Add 2.5ul of detection reagent solution to each well.
6) Incubate at room temperature for 60 min in the dark
7) Read plates in a Perkin Elmer Envision using a luminescence protocol

Dose Response protocol:
Day 1
1) Harvest cells using Enzyme-Free Dissociation Buffer (Invitrogen Cat#13151-14). Add 500 cells/well in 5 uL of media to each well of a white, 1536 well plate.
2) Spin cells at 500 rpm for 1 min, then wrap plates in Saran Wrap.
3) Incubate overnight at 37C with 5% CO2.

Day 2
1) Using a Labcyte Echo, DMSO and test compounds are transferred to wells. DMSO only is transferred to columns 1-3 and 46-48(Control wells), while varying volumes of test compounds are transferred to columns 4-45 to achieve the desired test concentrations. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2) Immediately following Echo transfer, 1.0 uL of assay media is added to columns 1-3 and 1.0 uL of assay media containing 240 nM dynorphin A is added to columns 4-48 for a final assay concentration of 40 nM. Centrifuge plates at 500 rpm for 1 min immediately following additions.
3) Incubate for 1hr and 30 minutes.
4) During test incubation, prepare Detection Reagent Solution from DiscoveRx (1 part Galacton Star: 5 parts Emerald II and 19 parts Cell Assay Buffer)
5) Add 2.5ul of detection reagent solution to each well.
6) Incubate at room temperature for 60 min in the dark
7) Read plates in a Perkin Elmer Envision using a luminescence protocol

Compounds with greater than 50% activity at 10 or 20 uM concentration.

Compounds with greater than 50% activity for %Response of Repeats at 10 uM are defined as actives in the primary assay.

Compounds with an IC50 < 20 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. 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 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. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 44 + 6*(pIC50 - 3)*QC,
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 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

Grant Number: 1X01DA026208-01