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BioAssay: AID 651764

Late-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 2

Name: Late-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 2. ..more
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
Inactive(27)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Active(1)
 
 
Inactive(27)
 
 
 Related BioAssays
 Related BioAssays
AID: 651764
Data Source: The Scripps Research Institute Molecular Screening Center (OPRD1_AG_LUMI_384_4XEC50_SET2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-07
Hold-until Date: 2013-06-19
Modify Date: 2013-06-19

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
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AIDNameTypeComment
504355Summary of the probe development efforts to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorssummarySummary (OPRM1 and OPRD1 agonists)
504326Luminescence-based cell-based primary high throughput screening assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsscreeningPrimary screen (OPRM1 and OPRD1 agonists in singlicate).
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 receptorsscreeningPrimary screen (OPRM1 and OPRD1 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 receptorsscreeningConfirmation screen (OPRM1-OPRD1 agonists in triplicate)
504900Luminescence-based cell-based high throughput confirmation assay for inverse agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsscreeningConfirmation screen (OPRM1-OPRD1 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 receptorsconfirmatoryDose response assay (OPRM1-OPRD1 agonists in triplicate)
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)screeningCounterscreen (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)screeningCounterscreen (HTR5A agonists in singlicate)
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)screeningCounterscreen (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)screeningCounterscreen (HTR5A inverse 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)confirmatoryDose 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 homodimerizationconfirmatoryDose 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 homodimerizationconfirmatoryDose response counterscreen assay (OPRM1 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 assayconfirmatoryLate-stage dose response (OPRM1 and OPRD1 heterodimer formation activators in triplicate or quadruplicate)
651689Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRM1 counterscreenconfirmatoryLate-stage dose response counterscreen (OPRM1 activators in triplicate or quadruplicate)
651686Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response OPRD1 counterscreenconfirmatoryLate-stage dose response counterscreen (OPRD1 activators in triplicate or quadruplicate)
651688Late-stage results from the probe development effort to identify activators of OPRM1 and OPRD1 heterodimer formation: luminescence-based cell-based dose response HTR5A counterscreenconfirmatoryLate-stage dose response counterscreen (HTR5A activators in triplicate or quadruplicate)
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 compoundsconfirmatory
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_AG_LUMI_384_4XEC50_SET2

Name: Late-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 2.

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, OPRD1, delta, homodimer, opioid, receptor, GPCR, beta-arrestin, fragment complementation, enzyme donor, enzyme acceptor, PathHunter, U2OS, beta-galactosidase, beta-arrestin, luminescence, holoenzyme, agonist, activator, pain, Deltorphin B, 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 OPRD1 homodimerization, resulting in membrane recruitment of beta-arrestin. This assay employs U2OS cells which express OPRD1 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 OPRD1 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. Deltorphin B is used as the high control for agonists, and wells containing cells treated with DMSO are 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-OPRD1 (Delta) 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, 500 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, Deltorphin B (1 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, Deltorphin B 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 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 100-100, and for inactive compounds 0-0.

List of Reagents:

PathHunter TradeMark OPRD1-Beta-Arrestin U20S Cell Line (Discover X part 93-0400C3)
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)
Deltorphin B (Sigma, T0675)
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: = 100 / ( 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
3LogEC50The Log of EC50Float
4Hill SlopeThe Hill SlopeFloat
5R squaredThe value of R squaredFloat
6Activation at 50 uM [1] (50μM**)Activation at 50 uM compound concentration; replicate [1]Float%
7Activation at 50 uM [2] (50μM**)Activation at 50 uM compound concentration; replicate [2]Float%
8Activation at 50 uM [3] (50μM**)Activation at 50 uM compound concentration; replicate [3]Float%
9Activation at 50 uM [4] (50μM**)Activation at 50 uM compound concentration; replicate [4]Float%
10Activation at 16.6 uM [1] (16.6μM**)Activation at 16.6 uM compound concentration; replicate [1]Float%
11Activation at 16.6 uM [2] (16.6μM**)Activation at 16.6 uM compound concentration; replicate [2]Float%
12Activation at 16.6 uM [3] (16.6μM**)Activation at 16.6 uM compound concentration; replicate [3]Float%
13Activation at 16.6 uM [4] (16.6μM**)Activation at 16.6 uM compound concentration; replicate [4]Float%
14Activation at 5.5 uM [1] (5.5μM**)Activation at 5.5 uM compound concentration; replicate [1]Float%
15Activation at 5.5 uM [2] (5.5μM**)Activation at 5.5 uM compound concentration; replicate [2]Float%
16Activation at 5.5 uM [3] (5.5μM**)Activation at 5.5 uM compound concentration; replicate [3]Float%
17Activation at 5.5 uM [4] (5.5μM**)Activation at 5.5 uM compound concentration; replicate [4]Float%
18Activation at 1.9 uM [1] (1.9μM**)Activation at 1.9 uM compound concentration; replicate [1]Float%
19Activation at 1.9 uM [2] (1.9μM**)Activation at 1.9 uM compound concentration; replicate [2]Float%
20Activation at 1.9 uM [3] (1.9μM**)Activation at 1.9 uM compound concentration; replicate [3]Float%
21Activation at 1.9 uM [4] (1.9μM**)Activation at 1.9 uM compound concentration; replicate [4]Float%
22Activation at 0.616 uM [1] (0.616μM**)Activation at 0.616 uM compound concentration; replicate [1]Float%
23Activation at 0.616 uM [2] (0.616μM**)Activation at 0.616 uM compound concentration; replicate [2]Float%
24Activation at 0.616 uM [3] (0.616μM**)Activation at 0.616 uM compound concentration; replicate [3]Float%
25Activation at 0.616 uM [4] (0.616μM**)Activation at 0.616 uM compound concentration; replicate [4]Float%
26Activation at 0.204 uM [1] (0.204μM**)Activation at 0.204 uM compound concentration; replicate [1]Float%
27Activation at 0.204 uM [2] (0.204μM**)Activation at 0.204 uM compound concentration; replicate [2]Float%
28Activation at 0.204 uM [3] (0.204μM**)Activation at 0.204 uM compound concentration; replicate [3]Float%
29Activation at 0.204 uM [4] (0.204μM**)Activation at 0.204 uM compound concentration; replicate [4]Float%
30Activation at 0.0692 uM [1] (0.0692μM**)Activation at 0.0692 uM compound concentration; replicate [1]Float%
31Activation at 0.0692 uM [2] (0.0692μM**)Activation at 0.0692 uM compound concentration; replicate [2]Float%
32Activation at 0.0692 uM [3] (0.0692μM**)Activation at 0.0692 uM compound concentration; replicate [3]Float%
33Activation at 0.0692 uM [4] (0.0692μM**)Activation at 0.0692 uM compound concentration; replicate [4]Float%
34Activation at 0.0229 uM [1] (0.0229μM**)Activation at 0.0229 uM compound concentration; replicate [1]Float%
35Activation at 0.0229 uM [2] (0.0229μM**)Activation at 0.0229 uM compound concentration; replicate [2]Float%
36Activation at 0.0229 uM [3] (0.0229μM**)Activation at 0.0229 uM compound concentration; replicate [3]Float%
37Activation at 0.0229 uM [4] (0.0229μM**)Activation at 0.0229 uM compound concentration; replicate [4]Float%
38Activation at 0.0076 uM [1] (0.0076μM**)Activation at 0.0076 uM compound concentration; replicate [1]Float%
39Activation at 0.0076 uM [2] (0.0076μM**)Activation at 0.0076 uM compound concentration; replicate [2]Float%
40Activation at 0.0076 uM [3] (0.0076μM**)Activation at 0.0076 uM compound concentration; replicate [3]Float%
41Activation at 0.0076 uM [4] (0.0076μM**)Activation at 0.0076 uM compound concentration; replicate [4]Float%
42Activation at 0.0025 uM [1] (0.0025μM**)Activation at 0.0025 uM compound concentration; replicate [1]Float%
43Activation at 0.0025 uM [2] (0.0025μM**)Activation at 0.0025 uM compound concentration; replicate [2]Float%
44Activation at 0.0025 uM [3] (0.0025μM**)Activation at 0.0025 uM compound concentration; replicate [3]Float%
45Activation at 0.0025 uM [4] (0.0025μM**)Activation at 0.0025 uM compound concentration; replicate [4]Float%

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

Data Table (Concise)
Classification
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