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BioAssay: AID 485366

qHTS validation assay of beta-arrestin-biased ligands of beta2-adrenergic receptor

The concept of biased agonism [1] is an emerging concept of molecular pharmacology which proposes that seven transmembrane receptors (7TMRs) or G protein-coupled receptors (GPCRs), exist in multiple signaling conformations and complexes and that ligands (either agonists or antagonists) can induce signaling biased responses for distinct signal transduction cascades. ..more
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 Tested Compounds
 Tested Compounds
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Inactive(1252)
 
 
Inconclusive(14)
 
 
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 Tested Substances
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Inactive(1265)
 
 
Inconclusive(15)
 
 
AID: 485366
Data Source: NCGC (TANGO001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-06
Modify Date: 2010-11-04

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeComment
485386qHTS assay of beta-arrestin-biased ligands of beta2-adrenergic receptor: SummarySummarydepositor-specified cross reference
492947qHTS assay of beta-arrestin-biased ligands of beta2-adrenergic receptorConfirmatorydepositor-specified cross reference
504448Summary of HTS for Non-Canonical Ligands for Beta 2 Adrenergic Receptor InternalizationSummarydepositor-specified cross reference
504454HTS for Beta-2AR agonists via FAP methodScreeningdepositor-specified cross reference
504459HTS for Beta-2AR agonists via FAP method from Validation SetScreeningdepositor-specified cross reference
540358HTS for Beta-2AR agonists via FAP method, Single Point, Cherry Pick 1Otherdepositor-specified cross reference
588463qHTS assay of beta-arrestin-biased ligands of beta2-adrenergic receptor: Hit ValidationConfirmatorydepositor-specified cross reference
588763Dose response for HTS for Beta-2AR agonists via FAP method from CP1Confirmatorydepositor-specified cross reference
588765HTS for Beta-2AR agonists via FAP method, Counterscreen, Cherry Pick 1Otherdepositor-specified cross reference
588775Dose response for HTS for Beta-2AR agonists via FAP method from Powderset1Confirmatorydepositor-specified cross reference
588790qHTS assay of beta-arrestin-biased ligands of beta2-adrenergic receptor: Hit Validation with GloSensorConfirmatorysame project related to Summary assay
623882Dose response for HTS for Beta-2AR agonists via FAP method from Powderset2Confirmatorysame project related to Summary assay
623947Dose response for HTS for Beta-2AR agonists via FAP method from Powderset3Confirmatorysame project related to Summary assay
651694Cytotoxicity Dose Response with AM2.2-beta2AR cells for PowderSet01Confirmatorysame project related to Summary assay
651698Counter screen for HTS for Beta-2AR agonists with FAP-tagged mouse CCR5 with Powderset1Confirmatorysame project related to Summary assay
651701Summary of HTS Screening Project for Inhibitors of fluorogen-FAP tag interactionsSummarysame project related to Summary assay
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: Fast-track/HHSN271700800025C
PI: Bryan Roth and Bob Lefkowitz

The concept of biased agonism [1] is an emerging concept of molecular pharmacology which proposes that seven transmembrane receptors (7TMRs) or G protein-coupled receptors (GPCRs), exist in multiple signaling conformations and complexes and that ligands (either agonists or antagonists) can induce signaling biased responses for distinct signal transduction cascades.

Recently, this concept has been highlighted by beta-arrestin-biased signaling through 7TMRs [1]. beta-arrestins have been known to interact with agonist-occupied 7TMRs and thus to sterically inhibit coupling of the receptor to G proteins, leading to termination of signaling. Furthermore, accumulating evidence over the last decade has drawn attention to a novel function of beta-arrestins as signal transducers that scaffold various signaling molecules upon activation of 7TMRs. In particular, beta-arrestins have been shown to mediate activation of several signaling pathways upon stimulation of 7TM receptors even in the absence of G protein activation [2]. This beta-arrestin-biased signaling presumably leads to distinct physiological outcomes from those by G protein activation.
Additionally, the active receptor conformations which prefer on this beta-arrestin signaling are likely different from those activating G protein signaling

One of the systems in which biased agonism has been thoroughly elucidated is beta2-adrenergic receptors (beta2-ARs), where the 'antagonist' ligand carvedilol has been demonstrated to selectively activate beta-arrestin-mediated signaling pathways [3].

The goal of this screening project will be to discover and optimize novel beta-arrestin-biased ligands for beta2-AR signaling. A luminescent cell-based assay for beta2-AR-mediated agonism, known as the Tango assay [4], has been validated in which ligand binding of a recombinant beta2-AR and subsequent recruitment of a recombinant beta-arrestin lead to the release of the transcriptional activator tTA, which upregulates transcription of a tTA-responsive luciferase gene. Compounds with beta2-AR agonist activity lead to an increase in luciferase expression, and upon addition of a luciferase detection reagent, demonstrate an increase in luminescence. Compounds were screened as a concentration-titration series that ranged from 46 microM to 0.6 nM.
Protocol
NCGC Assay Protocol Summary:
Tango cells were dispensed into white, solid, tissue-culture-treated 1536-well plates at 1500 cells/well in DMEM, containing 1% fetal bovine serum (FBS). Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. Plates were incubated at 37 degrees C, 5% CO2 for 18 hr. Then 2.5 uL luminescent substrate mix (in-house prepared: 150 mM Tris-Cl, pH 7.6, 3 mM MgCl2, 1% Triton X-100, 5 mM DTT, 0.5 mM CoA, 0.15 mM ATP, 0.44 mM D-luciferin, final concentrations) was added to each well. Plates were then incubated for 15 minutes at room temperature, and subsequently measured on a ViewLux plate reader for luminescent signal using a clear filter with a 10 second exposure and 2x binning. The %Activity was determined from the corrected luminescence values. Isoproterenol, a well-characterized beta2-AR full agonist, and metaproterenol, a less potent beta2-AR agonist, were included both in 16-pt titration and at full response concentration to normalize %Activity of identified agonists; DMSO-pinned controls were used to normalize 0%Activity (zero agonism), while EC100 isoproterenol and/or EC100 metaproterenol were used to normalize 100%Activity (full agonism).
Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active agonists showed concentration-dependent increases in luminescence, concordant with an increase in bound beta2-AR and luciferase expression. Inactive compounds showed no effect on luminescence signal.
Keywords: biased agonism, beta2-AR, beta-arrestin, GPCR, 7TMR, Tango, luminescence, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.00295 uM (0.00295μM**)% Activity at given concentration.Float%
15Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
16Activity at 0.074 uM (0.0737μM**)% Activity at given concentration.Float%
17Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
18Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
19Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
20Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
21Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: Fast-track/HHSN271700800025C

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