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BioAssay: AID 652077

Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1)

Name: Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1). ..more
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 Tested Compounds
 Tested Compounds
All(11)
 
 
Active(7)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(11)
 
 
Active(7)
 
 
Inactive(4)
 
 
AID: 652077
Data Source: The Scripps Research Institute Molecular Screening Center (OPRK1_ANT_FRET_384_3X%IC50)
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 ):           Active    All
Target
BioActive Compounds: 7
Depositor Specified Assays
AIDNameTypeComment
652031Maybridge screen to identify antagonists of kappa opioid receptor 1 (OPRK1): fluorescence-based cell-based assayscreeningMaybridge screen (OPRK1 inhibitors in singlicate)
652032Late-stage results from the probe development effort to identify antagonists of OPRK1: fluorescence-based cell-based dose response assayconfirmatoryLate-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 counterscreenconfirmatoryLate-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 counterscreenconfirmatoryLate-stage dose response counterscreen (OPRM1 antagonists in quadruplicate)
652045Summary of the probe development efforts to identify antagonists of the kappa 1 (OPRK1) opioid receptorsummarySummary (OPRK1 antagonists)
652108Late-stage results from the probe development effort to identify antagonists of OPRK1: In vivo tail flick assayother
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: OPRK1_ANT_FRET_384_3X%IC50

Name: Late-stage fluorescence-based cell-based dose response assay for antagonists of kappa opioid receptor 1 (OPRK1).

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:

Maybridge Library, Maybridge, OPRK1, kappa, opioid, receptor, GPCR, beta-arrestin, U2OS, Tangotrade mark, beta-lactamase, FRET, FRET-enabled substrate, TEV, TEV protease, beta-arrestin, EC80 challenge, U-50488, antagonist, inhibitor, dose response, inhibit, pain, analgesic, dynorphin, neuropathic pain, drug addiction, addiction, 384, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to confirm activity of compounds from the Maybridge Library that act as antagonists of OPRK1. This assay uses Tango OPRK1-BLA U2OS cells which contain the human opiod kappa receptor (OPRK1) 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 OPRK1 receptor by agonist causes migration of the 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 OPRK1 antagonists will inhibit OPRK1 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 triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.

Protocol Summary:

U2OS cells were cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, 200 ug/mL Zeocin, 50 ug/mL Hygromycin, and 100 ug/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (McCoy's 5A Medium supplemented with 10% v/v charcoal dextran stripped fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomyci). The assay was started by dispensing 10 ul of cell suspension to each well, followed by overnight incubation at 37 C in 5% CO2 and 95% RH. The next day, 50 nL of test compound (9 uM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, U-50488 in Assay Medium (8 nM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37 C in 5% CO2 for 4 hrs. After the incubation, 2.2 ul/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm.

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 and U-50488
Low_Control is defined as wells containing U-50488
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.

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 100-80, and for inactive compounds 58-0.

List of Reagents:

Tango trade mark OPRK1-bla U2OS cells (Invitrogen, part K1576)
GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (Invitrogen, part K1025)
Probenecid (Sigma, part P8761)
McCoy's 5A Medium (modified) (1X) (Invitrogen, 16600-082)
Fetal Bovine Serum, dialyzed (Invitrogen, part 26400-036)
Fetal Bovine Serum, charcoal (Invitrogen, part 12676-029)
NEAA (Invitrogen, part 1114-050)
Penicillin-Streptomycin antibiotic mix (Invitrogen, part 15140-122)
Sodium Pyruvate (Invitrogen, part 11360-070)
PBS without calcium or magnesium (Invitrogen, part 14190-136)
HEPES (Invitrogen, part 15630-080)
Trypsin/EDTA (Invitrogen, part 25300-054)
U-50488 (Sigma, D8040)
Zeocin (Invitrogen, part R250-01)
Hygromycin (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
384-well plates (Greiner, part 788092)
T175 tissue culture flasks (Corning, part 431080)
Comment
This assay was performed by the SRIMSC with powder samples of purchased test compounds.
Categorized Comment
Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: = 100 / ( 1 + 10^( ( [LogIC50] - Log( [Concentration] * 10^-6 ) * [Hill Slope] ) )

Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
2Log IC50The Log of IC50Float
3Hill SlopeThe Hill SlopeFloat
4R squaredThe value of R squaredFloat
5Inhibition at 50 uM [1] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [1]Float%
6Inhibition at 50 uM [2] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [2]Float%
7Inhibition at 50 uM [3] (50μM**)Value of % inhibition at 50 uM compound concentration; replicate [3]Float%
8Inhibition at 16.6 uM [1] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [1]Float%
9Inhibition at 16.6 uM [2] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [2]Float%
10Inhibition at 16.6 uM [3] (16.6μM**)Value of % inhibition at 16.6 uM compound concentration; replicate [3]Float%
11Inhibition at 5.5 uM [1] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [1]Float%
12Inhibition at 5.5 uM [2] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [2]Float%
13Inhibition at 5.5 uM [3] (5.5μM**)Value of % inhibition at 5.5 uM compound concentration; replicate [3]Float%
14Inhibition at 1.9 uM [1] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [1]Float%
15Inhibition at 1.9 uM [2] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [2]Float%
16Inhibition at 1.9 uM [3] (1.9μM**)Value of % inhibition at 1.9 uM compound concentration; replicate [3]Float%
17Inhibition at 0.616 uM [1] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [1]Float%
18Inhibition at 0.616 uM [2] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [2]Float%
19Inhibition at 0.616 uM [3] (0.616μM**)Value of % inhibition at 0.616 uM compound concentration; replicate [3]Float%
20Inhibition at 0.204 uM [1] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [1]Float%
21Inhibition at 0.204 uM [2] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [2]Float%
22Inhibition at 0.204 uM [3] (0.204μM**)Value of % inhibition at 0.204 uM compound concentration; replicate [3]Float%
23Inhibition at 0.0692 uM [1] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [1]Float%
24Inhibition at 0.0692 uM [2] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [2]Float%
25Inhibition at 0.0692 uM [3] (0.0692μM**)Value of % inhibition at 0.0692 uM compound concentration; replicate [3]Float%
26Inhibition at 0.0229 uM [1] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [1]Float%
27Inhibition at 0.0229 uM [2] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [2]Float%
28Inhibition at 0.0229 uM [3] (0.0229μM**)Value of % inhibition at 0.0229 uM compound concentration; replicate [3]Float%
29Inhibition at 0.0076 uM [1] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [1]Float%
30Inhibition at 0.0076 uM [2] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [2]Float%
31Inhibition at 0.0076 uM [3] (0.0076μM**)Value of % inhibition at 0.0076 uM compound concentration; replicate [3]Float%
32Inhibition at 0.0025 uM [1] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [1]Float%
33Inhibition at 0.0025 uM [2] (0.0025μM**)Value of % inhibition at 0.0025 uM compound concentration; replicate [2]Float%
34Inhibition 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)
Classification
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