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BioAssay: AID 652033

Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response OPRD1 counterscreen

Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response OPRD1 counterscreen. ..more
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 Tested Compounds
 Tested Compounds
All(14)
 
 
Active(5)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(14)
 
 
Active(5)
 
 
Inactive(9)
 
 
 Related BioAssays
 Related BioAssays
AID: 652033
Data Source: The Scripps Research Institute Molecular Screening Center (OPRD1_ANT_FRET_384_3XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-02-12
Hold-until Date: 2013-10-21
Modify Date: 2013-10-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5
Related Experiments
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AIDNameTypeComment
652045Summary of the probe development efforts to identify antagonists of the kappa 1 (OPRK1) opioid receptorSummarydepositor-specified cross reference
652075Late-stage results from the probe development effort to identify antagonists of OPRK1: radiometric-based biochemical hERG counterscreen assayOtherdepositor-specified cross reference
652076Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based biochemical cytochrome P450 inhibition assayOtherdepositor-specified cross reference
652077Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1)Confirmatorydepositor-specified cross reference
652078Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based pharmacokinetic plasma protein binding assayOtherdepositor-specified cross reference
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)Confirmatorydepositor-specified cross reference
652080Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response OPRD1 counterscreen, Set 2Confirmatorydepositor-specified cross reference
652081Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreen, Set 2Confirmatorydepositor-specified cross reference
652082Fluorescence-based cell-based confirmation assay for antagonists of kappa opioid receptor 1 (OPRK1)Otherdepositor-specified cross reference
652083Late-stage results from the probe development effort to identify antagonists of OPRK1: CEREP radiometric-based biochemical counterscreen panel assayOtherdepositor-specified cross reference
652084Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assay, Set 2.Confirmatorydepositor-specified cross reference
652085Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based in vivo plasma and brain levelsOtherdepositor-specified cross reference
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 compoundsConfirmatorydepositor-specified cross reference
652087Counterscreen for antagonists of kappa opioid receptor 1 (OPRK1): fluorescence-based cell-based assay to identify antagonists of Sphingosine 1-Phosphate Receptor 1 (S1P1)Otherdepositor-specified cross reference
652088Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based biochemical hepatic microsome stability assayOtherdepositor-specified cross reference
652108Late-stage results from the probe development effort to identify antagonists of OPRK1: In vivo tail flick assayOtherdepositor-specified cross reference
652113Late-stage results from the probe development effort to identify antagonists of OPRK1: LCMS-based parallel artificial membrane permeability (PAMPA) assayOtherdepositor-specified cross reference
652031Maybridge screen to identify antagonists of kappa opioid receptor 1 (OPRK1): fluorescence-based cell-based assayScreeningsame project related to Summary assay
652032Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assayConfirmatorysame project related to Summary assay
652034Late-stage results from the probe development effort to identify antagonists of OPRK1: luminescence-based cell-based dose response OPRM1 counterscreenConfirmatorysame 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: OPRD1_ANT_FRET_384_3XIC50

Name: Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response OPRD1 counterscreen.

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, OPRD1, delta, opioid, receptor, GPCR, beta-arrestin, U2OS, Tangotrade mark, beta-lactamase, FRET, FRET-enabled substrate, TEV, TEV protease, beta-arrestin, EC80 challenge, SNC80, antagonist, inhibitor, inhibit, 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 OPRD1 receptor. This assay uses Tango OPRD1-bla U2OS cells which express OPRD1 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a Beta-arrestin/TEV protease fusion protein and a Beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the OPRD1 receptor by agonist causes migration of the Beta-arrestin fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as OPRD1 antagonists will inhibit agonist activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in quadruplicate (except for SID 144087319, which was tested in triplicate) using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.

Protocol Summary:

The Tango OPRD1-U20S dividing cell line was routinely cultured in 150 mm dishes at 37 C, 5% CO2 and 95% relative humidity (RH). The growth medium consisted of McCoys 5A Media supplemented with 10% v/v dialyzed fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 1X antibiotic mix (penicillin streptomycin). On day 1 of the assay, 16,000 cells in 10 uL of assay media (DMEM-Glutamax with sodium pyruvate, 10% fetal bovine serum stripped with charcoal-dextran, 25 mM HEPES, 0.1 mM non-essential amino acids, and antibiotic mix (penicillin streptomycin) were seeded into each well of a 384-well plate. 50 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, 1.1 uL of 3.7 uM SNC80 OPRD1 Agonist (EC80 Challenge;final concentration 370 nM) or DMSO in assay medium was added to appropriate wells and incubated 16-24 hours at 37 C ,5% CO2 and 95% RH. On day 2, 2.5 uL of LiveBLazer (trade mark) FRET B/G (CCF4-AM) loading mix (prepared according to manufacturer's instructions; 6 uL solution A, 60 uL Solution B, 904 uL Solution C, and 30 uL Solution D) were added to each well, and plates incubated at room temperature in the dark for 2 hours. Well fluorescence was measured on Perkin Elmer's Envision using an Excitation filter 405 nm, Emission filters at 460 nm and 590 nm, bottom read.

Percent Inhibition was calculated from the median ratio as follows:

%_Inhibition = 1 - ( ( FI_Test_Compound - Median_FI_High_Control ) / ( 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 SNC80 EC80 challenge
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.

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 100-87, and for inactive compounds 75-0.

List of Reagents:

Tango (trade mark) OPRD1-bla U20S Dividing Cells (Invitrogen, part K1778)
McCoy's 5A Medium, (Invitrogen, part 16600-082)
Dialyzed Fetal Bovine Serum, (Invitrogen, part 26400-036)
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)
DMEM, High Glucose, GlutaMAX (Invitrogen, part 10569-010)
Fetal Bovine Serum, Charcoal Stripped (Invitrogen, part 12676-011)
DMSO Dry (Sigma, part D2650)
SNC80 OPRD1 Agonist MW449.63 (Sigma, part S2812)
SDM25N hydrochloride OPRD1 Antagonist MW468.98 (Tocris, part 1410)
384 black low profile, clear bottom, low volume plate (Greiner, part 788092)
Low profile plate lids (Greiner, part 656190)
LiveBLAzer (trade mark)-FRET/BG Loading Mix (Invitrogen, part K1030)
Comment
This assay was performed by the SRIMSC with powder samples of synthesized test compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: = [Baseline Response] + ( [Maximal Response] - [Baseline Response] ) / ( 1 + 10 ^ ( ( [LogEC50] - 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
5Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
6Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
7R squaredThe value of R squaredFloat
8Inhibition at 50 uM [1] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [1]Float%
9Inhibition at 50 uM [2] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [2]Float%
10Inhibition at 50 uM [3] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [3]Float%
11Inhibition at 50 uM [4] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [4]Float%
12Inhibition at 16.6 uM [1] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [1]Float%
13Inhibition at 16.6 uM [2] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [2]Float%
14Inhibition at 16.6 uM [3] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [3]Float%
15Inhibition at 16.6 uM [4] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [4]Float%
16Inhibition at 5.5 uM [1] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [1]Float%
17Inhibition at 5.5 uM [2] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [2]Float%
18Inhibition at 5.5 uM [3] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [3]Float%
19Inhibition at 5.5 uM [4] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [4]Float%
20Inhibition at 1.9 uM [1] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [1]Float%
21Inhibition at 1.9 uM [2] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [2]Float%
22Inhibition at 1.9 uM [3] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [3]Float%
23Inhibition at 1.9 uM [4] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [4]Float%
24Inhibition at 0.616 uM [1] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [1]Float%
25Inhibition at 0.616 uM [2] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [2]Float%
26Inhibition at 0.616 uM [3] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [3]Float%
27Inhibition at 0.616 uM [4] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [4]Float%
28Inhibition at 0.204 uM [1] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [1]Float%
29Inhibition at 0.204 uM [2] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [2]Float%
30Inhibition at 0.204 uM [3] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [3]Float%
31Inhibition at 0.204 uM [4] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [4]Float%
32Inhibition at 0.0692 uM [1] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [1]Float%
33Inhibition at 0.0692 uM [2] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [2]Float%
34Inhibition at 0.0692 uM [3] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [3]Float%
35Inhibition at 0.0692 uM [4] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [4]Float%
36Inhibition at 0.0229 uM [1] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [1]Float%
37Inhibition at 0.0229 uM [2] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [2]Float%
38Inhibition at 0.0229 uM [3] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [3]Float%
39Inhibition at 0.0229 uM [4] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [4]Float%
40Inhibition at 0.0076 uM [1] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [1]Float%
41Inhibition at 0.0076 uM [2] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [2]Float%
42Inhibition at 0.0076 uM [3] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [3]Float%
43Inhibition at 0.0076 uM [4] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [4]Float%
44Inhibition at 0.0025 uM [1] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [1]Float%
45Inhibition at 0.0025 uM [2] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [2]Float%
46Inhibition at 0.0025 uM [3] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [3]Float%
47Inhibition at 0.0025 uM [4] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [4]Float%

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

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