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BioAssay: AID 602263

Luminescence-based cell-based high throughput dose response assay for biased ligands (agonists) of the melanocortin 4 receptor (MC4R)

Name: Luminescence-based cell-based high throughput dose response assay for biased ligands (agonists) of the melanocortin 4 receptor (MC4R). ..more
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 Tested Compounds
 Tested Compounds
All(230)
 
 
Active(14)
 
 
Inactive(216)
 
 
 Tested Substances
 Tested Substances
All(231)
 
 
Active(14)
 
 
Inactive(217)
 
 
AID: 602263
Data Source: The Scripps Research Institute Molecular Screening Center (MC4R_AG_LUMI_1536_3XEC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-02-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 14
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-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)
602192Luminescence-based cell-based high throughput confirmation assay for biased ligands (agonists) of the melanocortin 4 receptor (MC4R)Screeningdepositor-specified cross reference: Primary screen (MC4R agonists in triplicate)
602194Counterscreen 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 receptorsScreeningdepositor-specified cross reference: Counterscreen (OPRM1-OPRD1 agonists in triplicate)
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
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 receptorsConfirmatorysame 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: MC4R_AG_LUMI_1536_3XEC50 DRUN

Name: Luminescence-based cell-based high throughput dose response assay for biased ligands (agonists) of the melanocortin 4 receptor (MC4R).

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 activation 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:

dose response, 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 dose response curves for compounds that confirmed MC4R agoinst activity in a set of previous experiments entitled, "Luminescence-based cell-based primary high throughput confirmation assay to identify biased ligands of the melanocortin 4 receptor (MC4R): agonists of MC4R" (AID 602192).

This assay monitors melanocortin 4 receptor (MC4R) activation, 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 MC4R 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 activate MC4R 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. Melanotan II 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 are tested in triplicate using a 10-point, 1:3 dilution series starting at a maximum nominal concentration of 89 uM.

The MC4R 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, 27 nL of test compound 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 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 = ( ( Luminescence_Test_Compound - Median_ Luminescence_Low_Control ) / ( Median_ Luminescence_High_Control - Median_ Luminescence_Low_Control ) ) * 100

Where:

High_Control is defined as wells containing cells, Melanotan II 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:

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. 89 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 89 uM. 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.

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 100-92, and for inactive compounds 89-0.

List of Reagents:

U2OS MC4R cell line (DiscoveRx, part 93-0211C3)
PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
Melanotan II (control agonist, Anaspec, part 61188)
Ham's F-12 media (Invitrogen, part 11765)
DMEM media (Invitrogen, part 11995)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082)
Hygromycin B (Invitrogen, part 10687)
Geneticin (Invitrogen, part 10131)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640)
T-175 tissue culture flasks (Nunc, part 159910)
1536-well plates (Corning, part 7298)
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: confirmatory

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: luminescence: chemiluminescence

BAO: bioassay specification: bioassay type: binding

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

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

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 uM 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
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
7Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
8Response RangeThe range of Y.Float
9Chi 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
10RsquareA 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
11Excluded 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
12Number of DataPointsOverall number of data points of normalized percent inhibition 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 percent activation at 0.005 uM compound conecntration; replicate [1]Float%
14Activation at 0.005 uM [2] (0.005μM**)Value of percent activation at 0.005 uM compound conecntration; replicate [2]Float%
15Activation at 0.005 uM [3] (0.005μM**)Value of percent activation at 0.005 uM compound conecntration; replicate [3]Float%
16Activation at 0.014 uM [1] (0.014μM**)Value of percent activation at 0.014 uM compound conecntration; replicate [1]Float%
17Activation at 0.014 uM [2] (0.014μM**)Value of percent activation at 0.014 uM compound concentration; replicate [2]Float%
18Activation at 0.014 uM [3] (0.014μM**)Value of percent activation at 0.014 uM compound concentration; replicate [3]Float%
19Activation at 0.041 uM [1] (0.041μM**)Value of percent activation at 0.041 uM compound concentration; replicate [1]Float%
20Activation at 0.041 uM [2] (0.041μM**)Value of percent activation at 0.041 uM compound concentration; replicate [2]Float%
21Activation at 0.041 uM [3] (0.041μM**)Value of percent activation at 0.041 uM compound concentration; replicate [3]Float%
22Activation at 0.122 uM [1] (0.122μM**)Value of percent activation at 0.122 uM compound concentration; replicate [1]Float%
23Activation at 0.122 uM [2] (0.122μM**)Value of percent activation at 0.122 uM compound concentration; replicate [2]Float%
24Activation at 0.122 uM [3] (0.122μM**)Value of percent activation at 0.122 uM compound concentration; replicate [3]Float%
25Activation at 0.367 uM [1] (0.367μM**)Value of percent activation at 0.367 uM compound concentration; replicate [1]Float%
26Activation at 0.367 uM [2] (0.367μM**)Value of percent activation at 0.367 uM compound conecntration; replicate [2]Float%
27Activation at 0.367 uM [3] (0.367μM**)Value of percent activation at 0.367 uM compound concentration; replicate [3]Float%
28Activation at 1.1 uM [1] (1.1μM**)Value of percent activation at 1.1 uM compound concentration; replicate [1]Float%
29Activation at 1.1 uM [2] (1.1μM**)Value of percent activation at 1.1 uM compound concentration; replicate [2]Float%
30Activation at 1.1 uM [3] (1.1μM**)Value of percent activation at 1.1 uM compound concentration; replicate [3]Float%
31Activation at 3.3 uM [1] (3.3μM**)Value of percent activation at 3.3 uM compound concentration; replicate [1]Float%
32Activation at 3.3 uM [2] (3.3μM**)Value of percent activation at 3.3 uM compound concentration; replicate [2]Float%
33Activation at 3.3 uM [3] (3.3μM**)Value of percent activation at 3.3 uM compound concentration; replicate [3]Float%
34Activation at 9.9 uM [1] (9.9μM**)Value of percent activation at 9.9 uM compound concentration; replicate [1]Float%
35Activation at 9.9 uM [2] (9.9μM**)Value of percent activation at 9.9 uM compound concentration; replicate [2]Float%
36Activation at 9.9 uM [3] (9.9μM**)Value of percent activation at 9.9 uM compound concentration; replicate [3]Float%
37Activation at 29.7 uM [1] (29.7μM**)Value of percent activation at 29.7 uM compound concentration; replicate [1]Float%
38Activation at 29.7 uM [2] (29.7μM**)Value of percent activation at 29.7 uM compound concentration; replicate [2]Float%
39Activation at 29.7 uM [3] (29.7μM**)Value of percent activation at 29.7 uM compound concentration; replicate [3]Float%
40Activation at 89.2 uM [1] (89.2μM**)Value of percent activation at 89.2 uM compound concentration; replicate [1]Float%
41Activation at 89.2 uM [2] (89.2μM**)Value of percent activation at 89.2 uM compound concentration; replicate [2]Float%
42Activation at 89.2 uM [3] (89.2μM**)Value of percent activation at 89.2 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** 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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