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BioAssay: AID 652081

Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen, Set 2

Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen, Set 2. ..more
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 Tested Compounds
 Tested Compounds
All(11)
 
 
Active(2)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(11)
 
 
Active(2)
 
 
Inactive(9)
 
 
AID: 652081
Data Source: The Scripps Research Institute Molecular Screening Center (OPRM1_ANT_LUMI_384_3XIC50_SET2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-03-05
Hold-until Date: 2013-10-21
Modify Date: 2013-10-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeComment
652031Maybridge screen to identify antagonists of kappa opioid receptor 1 (OPRK1): fluorescence-based cell-based assayScreeningdepositor-specified cross reference: Maybridge screen (OPRK1 inhibitors in singlicate)
652032Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assayConfirmatorydepositor-specified cross reference: Late-stage dose response (OPRK1 antagonists in quadruplicate)
652033Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response OPRD1 counterscreenConfirmatorydepositor-specified cross reference: Late-stage dose response counterscreen (OPRD1 antagonists in quadruplicate)
652034Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreenConfirmatorydepositor-specified cross reference: Late-stage dose response counterscreen (OPRM1 antagonists in quadruplicate)
652045Summary of the probe development efforts to identify antagonists of the kappa 1 (OPRK1) opioid receptorSummarydepositor-specified cross reference: Summary (OPRK1 antagonists)
652108Late-stage results from the probe development effort to identify antagonists of OPRK1: In vivo tail flick assayOtherdepositor-specified cross reference
652075Late-stage results from the probe development effort to identify antagonists of OPRK1: radiometric-based biochemical hERG counterscreen assayOthersame project related to Summary assay
652076Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based biochemical cytochrome P450 inhibition assayOthersame project related to Summary assay
652077Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1)Confirmatorysame project related to Summary assay
652078Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based pharmacokinetic plasma protein binding assayOthersame project related to Summary assay
652079Late-stage counterscreen for antagonists of kappa opioid receptor 1 (OPRK1): fluorescence-based cell-based dose response assay to identify antagonists of Sphingosine 1-Phosphate Receptor 1 (S1P1)Confirmatorysame project related to Summary assay
652080Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response OPRD1 counterscreen, Set 2Confirmatorysame project related to Summary assay
652082Fluorescence-based cell-based confirmation assay for antagonists of kappa opioid receptor 1 (OPRK1)Othersame project related to Summary assay
652083Late-stage results from the probe development effort to identify antagonists of OPRK1: CEREP radiometric-based biochemical counterscreen panel assayOthersame project related to Summary assay
652084Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assay, Set 2.Confirmatorysame project related to Summary assay
652085Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based in vivo plasma and brain levelsOthersame project related to Summary assay
652086Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of test compoundsConfirmatorysame project related to Summary assay
652087Counterscreen for antagonists of kappa opioid receptor 1 (OPRK1): fluorescence-based cell-based assay to identify antagonists of Sphingosine 1-Phosphate Receptor 1 (S1P1)Othersame project related to Summary assay
652088Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based biochemical hepatic microsome stability assayOthersame project related to Summary assay
652113Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based parallel artificial membrane permeability (PAMPA) assayOthersame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Lakshmi A. Devi, Mount Sinai School of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R03NS053751
Grant Proposal PI: Lakshmi A. Devi, Mount Sinai School of Medicine
External Assay ID: OPRM1_ANT_LUMI_384_3XIC50_SET2

Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen, Set 2.

Description:

Potent and selective OPRK antagonists will be useful for studying the mechanisms involved in OPRK-mediated analgesia and may have therapeutic value as novel analgesics with an improved side effect profile to currently available drugs. Studies have identified a role for dynorphin and OPRK stimulation in neuropathic pain (1). The dynorphins act as endogenous agonists at the opioid receptors, including OPRK (2), and the increased dynorphin expression in neuropathic pain also leads to a sustained activation of OPRK (1, 3). The mechanisms and neural circuits in OPRK-mediated analgesia are active areas of study; it is hoped those studies will assist in the development of novel analgesics that bypass OPRK-mediated depression (4-5). A role for dynorphin/OPRK in modulating drug addiction has been proposed (for review, see (6-7)). The function of dynorphin/OPRK systems in addiction appears to be diverse, and may modulate drug-seeking behavior depending on factors such as drug history, pattern of intake, and stress (for review, see (6)). The availability of potent and selective OPRK ligands may help unravel these mechanisms, as well as prove to be of therapeutic utility. Evidence from preclinical studies indicates that the dynorphin/OPRK system may be dysregulated in affective psychiatric disorders (for review, see (6, 8)). However, solid evidence from clinical studies is lacking. There is increasing evidence for a potential involvement of dynorphin/OPRK in schizophrenia; OPRK agonists appear to induce symptoms in humans and animals that are present in schizophrenia (8-10). Thus, the availability of new research tools such as potent and selective OPRK antagonists will facilitate understanding the physiological and pathophysiological mechanisms of dynorphin/OPRK systems and their roles in psychiatric disease in humans.

References:

1. Xu, M., et al., Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J Neurosci, 2004. 24(19): p. 4576-84.
2. Chavkin, C., I.F. James, and A. Goldstein, Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science, 1982. 215(4531): p. 413-5.
3. Xu, M., et al., Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase. J Neurosci, 2007. 27(10): p. 2570-81.
4. Al-Hasani, R. and M.R. Bruchas, Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology, 2011. 115(6): p. 1363-81.
5. Muschamp, J.W., A. Van't Veer, and W.A. Carlezon, Jr., Tracking down the molecular substrates of stress: new roles for p38alpha MAPK and kappa-opioid receptors. Neuron, 2011. 71(3): p. 383-5.
6. Tejeda, H.A., T.S. Shippenberg, and R. Henriksson, The dynorphin/kappa-opioid receptor system and its role in psychiatric disorders. Cell Mol Life Sci, 2012. 69(6): p. 857-96.
7. Yoo, J.H., I. Kitchen, and A. Bailey, The endogenous opioid system in cocaine addiction: what lessons have opioid peptide and receptor knockout mice taught us? Br J Pharmacol, 2012. 166(7): p. 1993-2014.
8. Schwarzer, C., 30 years of dynorphins--new insights on their functions in neuropsychiatric diseases. Pharmacol Ther, 2009. 123(3): p. 353-70.
9. Bortolato, M. and M.V. Solbrig, The price of seizure control: dynorphins in interictal and postictal psychosis. Psychiatry Res, 2007. 151(1-2): p. 139-43.
10. Sheffler, D.J. and B.L. Roth, Salvinorin A: the "magic mint" hallucinogen finds a molecular target in the kappa opioid receptor. Trends Pharmacol Sci, 2003. 24(3): p. 107-9.

Keywords:

Late stage, late stage AID, OPRK1, kappa, OPRM1, mu, opioid, receptor, GPCR, beta-arrestin, U2OS, lumi, luminescence, antagonist, antagonism, inhibit, inhibitor, inhibition, decrease, DiscoverRx, beta-arrestin, beta-galactosidase, fragment complementation, EC80 challenge, DAMGO, dose response, pain, analgesic, dynorphin, neuropathic pain, drug addiction, addiction, 384, counterscreen, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this counterscreen assay is to test the selectivity of OPRK1 antagonist compounds against the OPRM1 receptor. The assay monitors GPCR-Beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). The reconstituted holoenzyme catalyzes the hydrolysis of a substrate which yields a chemiluminescent signal. This assay employs U2OS cells which express OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compounds and an agonist DAMGO (EC80 challenge), followed by measurement of well luminescence. As designed, compounds that inhibit OPRM1 will decrease the level of beta-arrestin recruitment elicited by DAMGO, resulting in a decrease in the level of reconstitution of the beta-gal holoenzyme, and decreased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.
Protocol Summary:
The PathHunter(R) DiscoverX OPRM1-U20S cell line was routinely cultured in 150 mm dishes at 37 C, 5% CO2 and 95% relative humidity (RH). The growth medium consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1x antibiotic mix (penicillin streptomycin). On Day 1 of the assay, 5000 cells in 20 uL of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384-well plate, and incubated 16-24 hours at 37 C, 5% CO2 and 95% RH. On Day 2, 100 nL of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37 C, 5% CO2 and 95% RH. Next, 2.2 uL of DAMGO OPRMu1 agonist (EC80 Challenge; 1.8 uL of 3.7 uM DAMGO and 0.4 uL assay buffer; final assay concentration 303 nM) or DMSO in assay media were added. After incubation for 3 hours at 37 C, 5% CO2 and 95% RH, 10 uL of Path Hunter Detection Mix prepared according to manufacturer's protocol; 1 part Galacton Star:5 parts Emerald II:19 parts PH Cell Assay Buffer) was added to each well, and plates were incubated at room temperature in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision.
Percent Inhibition was calculated from the median ratio as follows:
%_Inhibition = 1 - ( ( FI_Test_Compound - Median_FI_HighControl ) / ( Median_FI_Low_Control - Median_FI_High_Control ) ) * 100
Where:
FI is defined as Fluorescence Intensity at 460 nm/Fluorescence Intensity at 530 nm.
Test_Compound is defined as wells containing test compound.
Low_Control (0% inhibition) is defined as wells containing DAMGO challenge (303 nM final).
High Control (100% inhibition) is defined as wells containing DMSO.
For each test compound, percent inhibition was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc) normalized from 0 to 100 for each assay. The software-generated IC50 values were reported. In cases where the highest concentration tested (i.e. 50 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 50 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 of 10 uM or less were considered active. Compounds with an IC50 of greater than 10 uM were considered inactive.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-92, and for inactive compounds 1-0.
List of Reagents:
OPRM1-U20S cell line (PathHunter(R) DiscoverX, part 93-0213E3CPOL))
DMEM Medium (Invitrogen, part 11965)
F12 Medium (Invitrogen, part 11765)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082147)
Non Essential Amino Acids 100X ( Invitrogen, part 11140-050)
HEPES (pH 7.3) 1M (Invitrogen, part 15630-080)
Sodium Pyruvate 100X (Invitrogen, part 11360-070)
Penicillin Streptomycin (Invitrogen, part 15640)
Trypsin 0.25%EDTA ( Invitrogen, part 25200056)
DPBS without Calcium /Magnesium (Invitrogen, part 14190-136)
DMSO Dry (Sigma, part D2650 )
DAMGO OPRMu1 Agonist MW513.19 (Sigma, part E7384-5MG)
B-Funaltrexamine Hydrochloride OPRM1 Antagonist MW (Sigma, part O003-2MG)
PathHunter Cell Plating 5 Reagent (Discover X, part 93-0563R5A)
Standard 384 well white plate with lid (Corning, part 3750)
PathHunter Detection Mix (DiscoverX, part 93-0001)
Comment
This assay was performed by the SRIMSC with powder samples of purchased test compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: = 100 / ( 1 + 10^( ( [LogIC50] - Log( [Concentration] * 10^-6 ) * [Hill Slope] ) )
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Cell-based
Assay Cell Type: U-2 OS
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentrationString
2IC50*The concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
3Log IC50The Log of IC50Float
4Hill SlopeThe Hill SlopeFloat
5R squaredThe value of R squaredFloat
6Inhibition at 50 uM [1] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [1]Float%
7Inhibition at 50 uM [2] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [2]Float%
8Inhibition at 50 uM [3] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [3]Float%
9Inhibition at 16.6 uM [1] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [1]Float%
10Inhibition at 16.6 uM [2] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [2]Float%
11Inhibition at 16.6 uM [3] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [3]Float%
12Inhibition at 5.5 uM [1] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [1]Float%
13Inhibition at 5.5 uM [2] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [2]Float%
14Inhibition at 5.5 uM [3] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [3]Float%
15Inhibition at 1.9 uM [1] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [1]Float%
16Inhibition at 1.9 uM [2] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [2]Float%
17Inhibition at 1.9 uM [3] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [3]Float%
18Inhibition at 0.616 uM [1] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [1]Float%
19Inhibition at 0.616 uM [2] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [2]Float%
20Inhibition at 0.616 uM [3] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [3]Float%
21Inhibition at 0.204 uM [1] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [1]Float%
22Inhibition at 0.204 uM [2] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [2]Float%
23Inhibition at 0.204 uM [3] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [3]Float%
24Inhibition at 0.0692 uM [1] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [1]Float%
25Inhibition at 0.0692 uM [2] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [2]Float%
26Inhibition at 0.0692 uM [3] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [3]Float%
27Inhibition at 0.0229 uM [1] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [1]Float%
28Inhibition at 0.0229 uM [2] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [2]Float%
29Inhibition at 0.0229 uM [3] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [3]Float%
30Inhibition at 0.0076 uM [1] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [1]Float%
31Inhibition at 0.0076 uM [2] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [2]Float%
32Inhibition at 0.0076 uM [3] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [3]Float%
33Inhibition at 0.0025 uM [1] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [1]Float%
34Inhibition at 0.0025 uM [2] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [2]Float%
35Inhibition at 0.0025 uM [3] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03NS053751

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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