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BioAssay: AID 588435

Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization

Name: Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. ..more
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 Tested Compounds
 Tested Compounds
All(226)
 
 
Active(16)
 
 
Inactive(210)
 
 
 Tested Substances
 Tested Substances
All(229)
 
 
Active(16)
 
 
Inactive(213)
 
 
AID: 588435
Data Source: The Scripps Research Institute Molecular Screening Center (OPRM1_AG_LUMI_1536_3XEC50 DCSRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-04
Modify Date: 2012-06-18

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 16
Related Experiments
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AIDNameTypeComment
504326Luminescence-based cell-based primary high throughput screening assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsScreeningdepositor-specified cross reference: Primary screen (OPRM1 and OPRD1 agonists in singlicate).
504355Summary of the probe development efforts to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsSummarydepositor-specified cross reference: Summary (OPRM1 and OPRD1 agonists)
504692Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)Screeningdepositor-specified cross reference: Counterscreen (HTR5A agonists in singlicate)
504904Luminescence-based cell-based high throughput confirmation assay for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsScreeningdepositor-specified cross reference: Confirmation screen (OPRM1 and OPRD1 agonists in triplicate)
504905Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based high throughput screening assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)Screeningdepositor-specified cross reference: Counterscreen (HTR5A agonists in triplicate)
651685Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response assayConfirmatorydepositor-specified cross reference
651686Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRD1 counterscreenConfirmatorydepositor-specified cross reference
651688Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response HTR5A counterscreenConfirmatorydepositor-specified cross reference
651689Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRM1 counterscreenConfirmatorydepositor-specified cross reference
651762Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response assay, Set 2Confirmatorydepositor-specified cross reference
651764Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRD1 counterscreen, Set 2Confirmatorydepositor-specified cross reference
651765Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response HTR5A counterscreen, Set 2Confirmatorydepositor-specified cross reference
651769Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRM1 counterscreen, Set 2Confirmatorydepositor-specified cross reference
651904Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of test compoundsConfirmatorydepositor-specified cross reference
504357Luminescence-based cell-based primary high throughput screening assay to identify inverse agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsScreeningsame project related to Summary assay
504634Counterscreen for inverse agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based full-deck high throughput screening assay to identify inverse agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)Screeningsame project related to Summary assay
504900Luminescence-based cell-based high throughput confirmation assay for inverse agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsScreeningsame project related to Summary assay
504914Counterscreen for inverse agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based high throughput screening assay to identify inverse agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)Screeningsame project related to Summary assay
588407Luminescence-based cell-based high throughput dose response assay for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsConfirmatorysame project related to Summary assay
588408Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based high throughput dose response assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)Confirmatorysame project related to Summary assay
588411Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRD1 homodimerizationConfirmatorysame 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_AG_LUMI_1536_3XEC50 DCSRUN

Name: Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization.

Description:

Opiates such as morphine are the choice analgesic in the treatment of chronic pain due to their potent and rapid action. However, their long-term use is limited because of the development of tolerance and dependence, as well as respiratory suppression and constipation (1). Due to their clinical importance, various strategies have been considered for making opiates more effective while curbing liabilities such as addiction. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. The OPRM1 gene encodes the mu opioid receptor, which is the primary site of action for morphine (2) and other commonly used opioid such as heroin, fentanyl, and methadone. OPRM1 activation and subsequent dissociation of the Gi/Go G-proteins results in reduction of adenylyl cyclase-mediated cAMP production (3). There are at least two other types of opioid receptors: delta (OPRD1) and kappa (OPRK1), each with a distinct pharmacologic profile. In particular, delta (OPRD1) opioid receptor ligands have been useful in enhancing morphine's potency, but the underlying molecular basis is not understood (4). It has been shown that modulation of receptor function by physical association between mu and delta opioid receptors is a potential mechanism (5). The assay provider has previously found that a combination of OPRM1 agonist with OPRD1 antagonist selectively activates the OPRM1-OPRD1 heteromer (5) and recently showed that this could be blocked by antibodies that selectively recognize the heteromer (6). Since OPRD1 antagonist have anxiogenic effects, these are not ideal as therapies. Hence, the identification of compounds that selectively activate mu-delta opioid receptor heterodimerization may have potential in the treatment of pain and alleviate unwanted effects associated with opiate use.

References:

1. Raehal KM, Bohn LM. Mu opioid receptor regulation and opiate responsiveness. AAPS J. 2005 Oct 19;7(3):E587-91.
2. Matthes H, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques B, Kieffer B (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383:819-823.
3. Maguea SD and Blendy JAOPRM1 SNP (A118G): Involvement in disease development, treatment response, and animal models. Drug and Alcohol Dependence. 2010 May 108 (3): 172-182.
4. Traynor J, Elliot J. Delta-opioid receptor subtypes and cross talk with mu-receptors. Trends Pharmacol Sci 1993 14:84-86.
5. Gomes I, Jordan BA, Gupta A, Trapaidze N, Nagy V, Devi LA. Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci. 2000 Nov 15;20(22):RC110.
6. Gupta, A., Mulder, J., Gomes, I., Rozenfeld, R., Bushlin, I., Ong, E., Lim, M., Maillet, E., Junek, M., Cahill, C.M., Harkany, T. Devi, L.A. Increased abundance of opioid receptor heteromers after chronic morphine administration. Science Signaling 3:ra54, 2010

Keywords:

counterscreen, homodimerization, dose response, titration, OPRM1, MOR-1, mu, delta, OPRD1, opioid, receptor, GPCR, beta-arrestin, U2OS, PathHunter, complementation, reconstitution, enzyme, beta-gal, beta-galactosidase, lumi, luminescence, luminescent, chemiluminescence, agonist, activator, activate, dimer, heterodimer, homodimer, pain, triplicate, HTS, high throughput screen, 1536, Scripps Florida, 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 identify compounds that are agonists of OPRM1 homodimerization, resulting in membrane recruitment of beta-arrestin. This assay will determine dose response curves. Compounds tested in this assay were identified as active in a previous set of experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors" (AID 504326), that confirmed activity in a set of experiments entitled, "Luminescence-based cell-based high throughput confirmation assay for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors" (AID 504904), and that were inactive in a set of experiments entitled, "Counterscreen for agonists of OPRM1-OPRD1 heterodimerization: luminescence-based cell-based high throughput screening assay to identify agonists of 5-hydroxytryptamine (serotonin) 5A receptor (HTR5A)" (AID 504905).

The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). 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 compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRM1 homodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 93 uM.

Protocol Summary:

The U2OS-OPRM1 (Mu) cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham's F-12 Nutrient Media (F-12) and Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

The day before the assay 1000 cells in 3 uL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 28 nL of test compound in DMSO, DAMGO (9.2 uM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 3 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHunter TM reagent (prepared according to the manufacturer's protocol); followed by 1 hour incubation at room temperature. Then, Well Luminescence was read on the ViewLux plate reader.

The percent activation for each compound was calculated as follows:

%_Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100

Where:

High_Control is defined as wells containing cells, DAMGO and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as wells containing cells and DMSO.

For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 93 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 93 uM.

PubChem Activity Outcome and Score:

Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.

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, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-91, and for inactive compounds 88-0.

List of Reagents:

PathHunter TM B-arrestin recruitment assay, containing the U2OS OPRM1 Beta-arrestin cell line; PathHunter Detection Kit (DiscoveRx, part, 93-0213C3)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-073)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: DAMGO (Sigma-Aldrich, part E7384)
1536-well plates (Greiner, part 789173)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was unable to provide all samples requested for testing.
Categorized Comment
Experiment Date: 09/28/2011

BAO: bioassay specification: assay stage: secondary: counter screen

BAO: bioassay specification: assay biosafety level: bsl1

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: receptor: transmembrane receptor: g protein coupled receptor

BAO: meta target: biological process target: regulation of molecular function

BAO: meta target detail: binding reporter specification: interaction: protein-protein

BAO: design: binding reporter: protein fragment complementation assay

BAO: detection technology: luminescence: chemiluminescence

Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]

Assay: CurveFit [1]: Mask: Excluded Points

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data EC50 came from a fitted curve or was determined manually to be less than or greater than its listed EC50 concentration.String
2EC50*The concentration at which 50 percent of the activity in the activator assay is observed; (EC50) shown in micromolar.FloatμM
3LogEC50Log10 of the qualified EC50 (EC50) from the activator assay in M concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
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
7Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
8Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
9Response RangeThe range of Y.Float
10Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
11RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
12Number of DataPointsOverall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
13Activation at 0.005 uM [1] (0.005μM**)Value of % activation at 0.005 uM concentration; replicate [1]Float%
14Activation at 0.005 uM [2] (0.005μM**)Value of % activation at 0.005 uM concentration; replicate [2]Float%
15Activation at 0.005 uM [3] (0.005μM**)Value of % activation at 0.005 uM concentration; replicate [3]Float%
16Activation at 0.014 uM [1] (0.014μM**)Value of % activation at 0.014 uM concentration; replicate [1]Float%
17Activation at 0.014 uM [2] (0.014μM**)Value of % activation at 0.014 uM concentration; replicate [2]Float%
18Activation at 0.014 uM [3] (0.014μM**)Value of % activation at 0.014 uM concentration; replicate [3]Float%
19Activation at 0.042 uM [1] (0.042μM**)Value of % activation at 0.042 uM concentration; replicate [1]Float%
20Activation at 0.042 uM [2] (0.042μM**)Value of % activation at 0.042 uM concentration; replicate [2]Float%
21Activation at 0.042 uM [3] (0.042μM**)Value of % activation at 0.042 uM concentration; replicate [3]Float%
22Activation at 0.127 uM [1] (0.127μM**)Value of % activation at 0.127 uM concentration; replicate [1]Float%
23Activation at 0.127 uM [2] (0.127μM**)Value of % activation at 0.127 uM concentration; replicate [2]Float%
24Activation at 0.127 uM [3] (0.127μM**)Value of % activation at 0.127 uM concentration; replicate [3]Float%
25Activation at 0.380 uM [1] (0.38μM**)Value of % activation at 0.380 uM concentration; replicate [1]Float%
26Activation at 0.380 uM [2] (0.38μM**)Value of % activation at 0.380 uM concentration; replicate [2]Float%
27Activation at 0.380 uM [3] (0.38μM**)Value of % activation at 0.380 uM concentration; replicate [3]Float%
28Activation at 1.1 uM [1] (1.1μM**)Value of % activation at 1.1 uM concentration; replicate [1]Float%
29Activation at 1.1 uM [2] (1.1μM**)Value of % activation at 1.1 uM concentration; replicate [2]Float%
30Activation at 1.1 uM [3] (1.1μM**)Value of % activation at 1.1 uM concentration; replicate [3]Float%
31Activation at 3.4 uM [1] (3.4μM**)Value of % activation at 3.4 uM concentration; replicate [1]Float%
32Activation at 3.4 uM [2] (3.4μM**)Value of % activation at 3.4 uM concentration; replicate [2]Float%
33Activation at 3.4 uM [3] (3.4μM**)Value of % activation at 3.4 uM concentration; replicate [3]Float%
34Activation at 10.3 uM [1] (10.3μM**)Value of % activation at 10.3 uM concentration; replicate [1]Float%
35Activation at 10.3 uM [2] (10.3μM**)Value of % activation at 10.3 uM concentration; replicate [2]Float%
36Activation at 10.3 uM [3] (10.3μM**)Value of % activation at 10.3 uM concentration; replicate [3]Float%
37Activation at 30.8 uM [1] (30.8μM**)Value of % activation at 30.8 uM concentration; replicate [1]Float%
38Activation at 30.8 uM [2] (30.8μM**)Value of % activation at 30.8 uM concentration; replicate [2]Float%
39Activation at 30.8 uM [3] (30.8μM**)Value of % activation at 30.8 uM concentration; replicate [3]Float%
40Activation at 92.5 uM [1] (92.5μM**)Value of % activation at 92.5 uM concentration; replicate [1]Float%
41Activation at 92.5 uM [2] (92.5μM**)Value of % activation at 92.5 uM concentration; replicate [2]Float%
42Activation at 92.5 uM [3] (92.5μM**)Value of % activation at 92.5 uM 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
Classification
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