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BioAssay: AID 602194

Counterscreen for biased ligands (agonists) of the melanocortin 4 receptor (MC4R): Luminescence-based cell-based high throughput assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors

Name: Counterscreen for biased ligands (agonists) of the melanocortin 4 receptor (MC4R): Luminescence-based cell-based high throughput assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors. ..more
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 Tested Compounds
 Tested Compounds
All(2396)
 
 
Active(140)
 
 
Inactive(2257)
 
 
 Tested Substances
 Tested Substances
All(2399)
 
 
Active(140)
 
 
Inactive(2259)
 
 
AID: 602194
Data Source: The Scripps Research Institute Molecular Screening Center (OPRM1-OPRD1_AG_LUMI_1536_3X%ACT CSRUN for MC4R AG)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-01-17

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 140
Related Experiments
Show more
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-OPRD1 agonists in singlicate)
540308Luminescence-based cell-based primary high throughput screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4RScreeningdepositor-specified cross reference: Primary screen (MC4R agonists in singlicate)
540319Summary of the probe development efforts to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4RSummarydepositor-specified cross reference: Summary (MC4R agonists)
602263Luminescence-based cell-based high throughput dose response assay for biased ligands (agonists) of the melanocortin 4 receptor (MC4R)Confirmatorydepositor-specified cross reference
602264Counterscreen for biased ligands (agonists) of the melanocortin 4 receptor (MC4R): Luminescence-based cell-based high throughput dose response assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptorsConfirmatorydepositor-specified cross reference
651547Late-stage FRET-based cell-based primary screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4RConfirmatorydepositor-specified cross reference
651554Late-stage FRET-based cell-based primary screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): antagonists of MC4ROtherdepositor-specified cross reference
651555Late-stage luminescence-based cell-based screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4RConfirmatorydepositor-specified cross reference
651557Late-stage luminescence-based cell-based primary screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): antogonists of MC4RConfirmatorydepositor-specified cross reference
602192Luminescence-based cell-based high throughput confirmation assay for biased ligands (agonists) of the melanocortin 4 receptor (MC4R)Screeningsame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Scott DeWire, Trevena Inc
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 RC2 MH090877-01
Grant Proposal PI: Scott DeWire, Trevena Inc
External Assay ID: OPRM1-OPRD1_AG_LUMI_1536_3X%ACT CSRUN for MC4R AG

Name: Counterscreen for biased ligands (agonists) of the melanocortin 4 receptor (MC4R): Luminescence-based cell-based high throughput assay to identify agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors.

Description:

Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, and disease-associated mutations (1-4). However, it has recently been demonstrated that GPCRs do not only signal in this simplistic fashion, but rather activate a network of downstream effects comprised of parallel signal transduction pathways. GPCR ligands biased towards induction or blockade of specific signaling pathways may have different physiology compared with unbiased molecules, through selective engagement of a desired subset of signal cascades. For example, the melanocortin 4 receptor (MC4R) transduces its signal via coupling to Gs and adenylyl cyclase activation, and is involved in the regulation of energy homeostasis and chronic disease-associated cachexia. Recent studies indicate that classical antagonists do not mimic MC4R regulation by its endogenous ligand, Agouti-Related Protein (AgRP). Indeed, AgRP has several actions including antagonizing Gs-mediated adenylyl cyclase activation, inducing beta-arrestin recruitment and MC4R endocytosis (5), as well as stimulating Gi-mediated inhibition of adenylyl cyclase (6). As a result, the identification of small molecules that act as biased MC4R ligands, by blocking Gs protein coupling but stimulating beta-arrestin functions and/or Gi protein coupling, may lead to a better understanding of this receptor and its role in metabolic/wasting diseases.

References:

1. Pan, H.L., Wu, Z.Z., Zhou, H.Y., Chen, S.R., Zhang, H.M., and Li, D.P., Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.
2. Lagerstrom, M.C. and Schioth, H.B., Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov, 2008. 7(4): p. 339-57.
3. Thompson, M.D., Cole, D.E., and Jose, P.A., Pharmacogenomics of G protein-coupled receptor signaling: insights from health and disease. Methods Mol Biol, 2008. 448: p. 77-107.
4. Bosier, B. and Hermans, E., Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci, 2007. 28(8): p. 438-46.
5. Breit, A., Wolff, K., Kalwa, H., Jarry, H., Buch, T., and Gudermann, T. 2006. The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors. J Biol Chem 281:37447-37456.
6. Buch, T.R., Heling, D., Damm, E., Gudermann, T., and Breit, A. 2009. Pertussis toxin-sensitive signaling of melanocortin-4 receptors in hypothalamic GT1-7 cells defines agouti-related protein as a biased agonist. J Biol Chem 284:26411-26420.

Keywords:

counterscreen, OPRM1, MOR-1, mu, delta, OPRD1, opioid, receptor, GPCR, beta-arrestin, U2OS, PathHunter, complementation, reconstitution, enzyme, beta-gal, beta-galactosidase, triplicate, MC4R, melanocortin, melanocortin 4 receptor, receptor, GPCR, biased ligand, lumi, luminescence, agonist, agonism, activate, activator, activation, increase, cachexia, biased, ligands, 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 assay is to determine whether compounds identified as active in a set of previous experiments entitled, "Luminescence-based cell-based primary high throughput screening assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4R" (AID 540308) are nonselective due to activation of heterodimerization between mu (OPRM1) and delta (OPRD1) opioid receptors.

This assay measures heterodimer formation between the OPRM1 and OPRD1 opioid receptors through 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 OPRD1, 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 OPRD1 homodimers or OPRM1-OPRD1 (Mu-Delta) heterodimers 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 will be used as the high (100% RLU) control for agonists, and wells containing cells treated with DMSO will be used as the low (0% RLU) control. Compounds were tested in triplicate at a final nominal concentration of 9.2 uM.

Protocol Summary:

The U2OS-OPRM1-OPRD1 (Mu-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, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B, 0.25 ug/mL Puromycin 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, Deltorphin B (0.9 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, 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.

PubChem Activity Outcome and Score:

The average percent activation and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent activation greater than the hit cutoff calculated for the primary screen (AID 504326) was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 10-0.

List of Reagents:

PathHunter TM beta-arrestin recruitment assay, containing the U2OS OPRM1 OPRD1 Beta-arrestin cell line; PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
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)
Puromycin (Invitrogen, part A11138-02)
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: Deltorphin B (Sigma-Aldrich, part A6559)
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. In this case the results of each separate campaign were assigned 'Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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 quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment
BAO: version: 1.4b1090

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

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: cell-based format

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-small molecule

BAO: assay design: redistribution reporter: second messenger: cyclic amp

BAO: detection technology: fluorescence: fret: tr-fret

BAO: bioassay specification: bioassay type: binding

BAO: bioassay specification: assay footprint: microplate: 1536 well plate

BAO: bioassay specification: assay measurement throughput quality: single concentration multiple replicates

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Activation at 9.2 uM (9.2μM**)Normalized percent activation of the counterscreen at a compound concentration of 9.2 uM.Float%
2Standard DeviationStandard deviation derived from the normalized percent activation of the triplicate data for each compound.Float
3Activation at 9.2 uM [1] (9.2μM**)Percent activation of the counterscreen at a compound concentration of 9.2 uM; replicate 1.Float%
4Activation at 9.2 uM [2] (9.2μM**)Percent activation of the counterscreen at a compound concentration of 9.2 uM; replicate 2.Float%
5Activation at 9.2 uM [3] (9.2μM**)Percent activation of the counterscreen at a compound concentration of 9.2 uM; replicate 3.Float%

** Test Concentration.
Additional Information
Grant Number: 1 RC2 MH090877-01

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