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BioAssay: AID 651689

Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRM1 counterscreen

Name: Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRM1 counterscreen. ..more
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 Tested Compounds
 Tested Compounds
All(12)
 
 
Active(1)
 
 
Inactive(11)
 
 
 Tested Substances
 Tested Substances
All(12)
 
 
Active(1)
 
 
Inactive(11)
 
 
AID: 651689
Data Source: The Scripps Research Institute Molecular Screening Center (OPRM1_AG_LUMI_384_3XEC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-10-23
Hold-until Date: 2013-06-19
Modify Date: 2013-06-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
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)
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 receptorsScreeningdepositor-specified cross reference: Primary screen (OPRM1 and OPRD1 agonists in singlicate).
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)Screeningdepositor-specified cross reference: Counterscreen (HTR5A agonists in singlicate)
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)
504900Luminescence-based cell-based high throughput confirmation assay for inverse agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsScreeningdepositor-specified cross reference: Confirmation screen (OPRM1-OPRD1 inverse agonists in triplicate)
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-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)
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)Screeningdepositor-specified cross reference: Counterscreen (HTR5A inverse agonists in triplicate)
588407Luminescence-based cell-based high throughput dose response assay for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsConfirmatorydepositor-specified cross reference: Dose response assay (OPRM1-OPRD1 agonists in triplicate)
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)Confirmatorydepositor-specified cross reference: Dose response counterscreen assay (HTR5A agonists in triplicate)
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 homodimerizationConfirmatorydepositor-specified cross reference: Dose response counterscreen assay (OPRD1 agonists in triplicate)
588435Counterscreen 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 homodimerizationConfirmatorydepositor-specified cross reference: Dose response counterscreen assay (OPRM1 agonists in triplicate)
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
651685Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response assayConfirmatorysame project related to Summary assay
651686Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRD1 counterscreenConfirmatorysame project related to Summary assay
651688Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response HTR5A 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: OPRM1_AG_LUMI_384_3XEC50

Name: Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRM1 counterscreen.

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:

Late stage, late stage AID, powders, OPRM1, mu, homodimer, opioid, receptor, GPCR, beta-arrestin, fragment complementation, enzyme donor, enzyme acceptor, PathHunter, U2OS, beta-galactosidase, beta-arrestin, luminescence, holoenzyme, agonist, activator, pain, DAMGO, 384, dose response, EC50, 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 powder samples of compounds that are agonists of OPRM1-OPRD1 heterodimerization for agonist activity of OPRM1 homodimerization, resulting in membrane recruitment of beta-arrestin. 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. DAMGO is used as the high control for agonists, and wells containing cells treated with DMSO is used as the low control. Compounds are tested in triplicate in a 10-point dilution series starting at a nominal concentration of 40 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, 5000 cells in 20 uL of cell plating media were seeded into each well of 384 well plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 100 nL of test compound in DMSO, DAMGO (10 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 10 uL of PathHunter trademark reagent (prepared according to the manufacturer's protocol); followed by 1 hour incubation at room temperature. Then well luminescence was measured on the Perkin Elmer's Envision 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 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 EC50 values were reported. In cases where the highest concentration tested (i.e. 40 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 40 uM.

PubChem Activity Outcome and Score:

Compounds with an EC50 of 10 uM or less were considered active. Compounds with an EC50 of more 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 100-100 and for inactive compounds 66-0.

List of Reagents:

PathHuntertrade mark OPRM1-Beta-Arrestin U20S Cell Line (Discover X part 93-0213C3)
DMEM Medium (Invitrogen, 11965)
F12 Medium (Invitrogen, 11765)
Heat Inactivated Fetal Bovine Serum (Invitrogen, 10082147)
Non Essential Amino Acids 100X ( Invitrogen, 11140-050)
HEPES (Invitrogen, 15630-080)
Sodium Pyruvate 100X (Invitrogen, 11360-070)
Penicillin Streptomycin (Invitrogen, 15640)
Geneticin (Invitrogen ,10131-027)
Hygromycin B (Invitrogen, 10687-010)
Puromycin (Invitrogen, A1113802)
Detachin (Genlantis, T100100)
DPBS without Calcium /Magnesium (Invitrogen, 14190-136)
DMSO Dry (Sigma, D2650)
PathHunter Cell Plating 5 Reagent (Discover X, 93-0563R5A)
Standard 384 well white plate with lid (Corning, 3750)
DAMGO (Sigma, part E7384)
PathHunter Detection Mix (DiscoverX, 93-0001)
Comment
This assay was performed by the SRIMSC with powder samples of test compounds.
Categorized Comment
Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: = [Baseline Response] + ( [Maximal Response] - [Baseline Response] ) / ( 1 + 10 ^ ( ( [LogEC50] - Log( [Concentration] * 10^-6) ) * [Hill Slope] ) )

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
2EC50*The concentration at which 50 percent of the activity in the agonist assay is observed; EC50 shown in micromolar.FloatμM
3Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
4Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
5LogEC50The Log of EC50Float
6Hill 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
7R squaredThis 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
8Activation at 40 uM [1] (40μM**)Value of % activation at 40 uM compound concentration; replicate [1]Float%
9Activation at 40 uM [2] (40μM**)Value of % activation at 40 uM compound concentration; replicate [2]Float%
10Activation at 40 uM [3] (40μM**)Value of % activation at 40 uM compound concentration; replicate [3]Float%
11Activation at 40 uM [4] (40μM**)Value of % activation at 40 uM compound concentration; replicate [4]Float%
12Activation at 12.6 uM [1] (12.6μM**)Value of % activation at 12.6 uM compound concentration; replicate [1]Float%
13Activation at 12.6 uM [2] (12.6μM**)Value of % activation at 12.6 uM compound concentration; replicate [2]Float%
14Activation at 12.6 uM [3] (12.6μM**)Value of % activation at 12.6 uM compound concentration; replicate [3]Float%
15Activation at 12.6 uM [4] (12.6μM**)Value of % activation at 12.6 uM compound concentration; replicate [4]Float%
16Activation at 4 uM [1] (4μM**)Value of % activation at 4 uM compound concentration; replicate [1]Float%
17Activation at 4 uM [2] (4μM**)Value of % activation at 4 uM compound concentration; replicate [2]Float%
18Activation at 4 uM [3] (4μM**)Value of % activation at 4 uM compound concentration; replicate [3]Float%
19Activation at 4 uM [4] (4μM**)Value of % activation at 4 uM compound concentration; replicate [4]Float%
20Activation at 1.6 uM [1] (1.6μM**)Value of % activation at 1.6 uM compound concentration; replicate [1]Float%
21Activation at 1.6 uM [2] (1.6μM**)Value of % activation at 1.6 uM compound concentration; replicate [2]Float%
22Activation at 1.6 uM [3] (1.6μM**)Value of % activation at 1.6 uM compound concentration; replicate [3]Float%
23Activation at 1.6 uM [4] (1.6μM**)Value of % activation at 1.6 uM compound concentration; replicate [4]Float%
24Activation at 0.501 uM [1] (0.501μM**)Value of % activation at 0.501 uM compound concentration; replicate [1]Float%
25Activation at 0.501 uM [2] (0.501μM**)Value of % activation at 0.501 uM compound concentration; replicate [2]Float%
26Activation at 0.501 uM [3] (0.501μM**)Value of % activation at 0.501 uM compound concentration; replicate [3]Float%
27Activation at 0.501 uM [4] (0.501μM**)Value of % activation at 0.501 uM compound concentration; replicate [4]Float%
28Activation at 0.159 uM [1] (0.159μM**)Value of % activation at 0.159 uM compound concentration; replicate [1]Float%
29Activation at 0.159 uM [2] (0.159μM**)Value of % activation at 0.159 uM compound concentration; replicate [2]Float%
30Activation at 0.159 uM [3] (0.159μM**)Value of % activation at 0.159 uM compound concentration; replicate [3]Float%
31Activation at 0.159 uM [4] (0.159μM**)Value of % activation at 0.159 uM compound concentration; replicate [4]Float%
32Activation at 0.05 uM [1] (0.05μM**)Value of % activation at 0.05 uM compound concentration; replicate [1]Float%
33Activation at 0.05 uM [2] (0.05μM**)Value of % activation at 0.05 uM compound concentration; replicate [2]Float%
34Activation at 0.05 uM [3] (0.05μM**)Value of % activation at 0.05 uM compound concentration; replicate [3]Float%
35Activation at 0.05 uM [4] (0.05μM**)Value of % activation at 0.05 uM compound concentration; replicate [4]Float%
36Activation at 0.02 uM [1] (0.02μM**)Value of % activation at 0.02 uM compound concentration; replicate [1]Float%
37Activation at 0.02 uM [2] (0.02μM**)Value of % activation at 0.02 uM compound concentration; replicate [2]Float%
38Activation at 0.02 uM [3] (0.02μM**)Value of % activation at 0.02 uM compound concentration; replicate [3]Float%
39Activation at 0.02 uM [4] (0.02μM**)Value of % activation at 0.02 uM compound concentration; replicate [4]Float%
40Activation at 0.0063 uM [1] (0.0063μM**)Value of % activation at 0.0063 uM compound concentration; replicate [1]Float%
41Activation at 0.0063 uM [2] (0.0063μM**)Value of % activation at 0.0063 uM compound concentration; replicate [2]Float%
42Activation at 0.0063 uM [3] (0.0063μM**)Value of % activation at 0.0063 uM compound concentration; replicate [3]Float%
43Activation at 0.0063 uM [4] (0.0063μM**)Value of % activation at 0.0063 uM compound concentration; replicate [4]Float%
44Activation at 0.002 uM [1] (0.002μM**)Value of % activation at 0.002 uM compound concentration; replicate [1]Float%
45Activation at 0.002 uM [2] (0.002μM**)Value of % activation at 0.002 uM compound concentration; replicate [2]Float%
46Activation at 0.002 uM [3] (0.002μM**)Value of % activation at 0.002 uM compound concentration; replicate [3]Float%
47Activation at 0.002 uM [4] (0.002μM**)Value of % activation at 0.002 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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