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BioAssay: AID 624148

qHTS of GLP-1 Receptor Agonists: LOPAC Validation

The overall goal of this project was to develop a novel assay for discovering small molecule ligands for class B1 G protein-coupled receptors (GPCRs). Very few agonists (or inverse agonists) with generally weak activity for this entire group of physiologically important receptors are known to date. To establish proof-of-principle that this critical bottleneck can be overcome with a suitable screening approach, our studies focused on the class B1 receptor for glucagon-like peptide-1 (GLP-1R), a potential therapeutic target for diabetes and neurodegenerative disease. ..more
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 Tested Compounds
 Tested Compounds
All(1265)
 
 
Active(2)
 
 
Inactive(1219)
 
 
Inconclusive(45)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(2)
 
 
Inactive(1231)
 
 
Inconclusive(47)
 
 
AID: 624148
Data Source: NCGC (GLPA1001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-05-15
Modify Date: 2012-05-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
AIDNameTypeComment
624152qHTS of GLP-1 Receptor Agonists: SummarySummarydepositor-specified cross reference
624172qHTS of GLP-1 Receptor AgonistsConfirmatorysame project related to Summary assay
624421qHTS of GLP-1 Receptor Inverse Agonists: SummarySummarysame project related to Summary assay
743262qHTS of GLP-1 Receptor Agonists: Hit ValidationConfirmatorysame project related to Summary assay
Description:
The overall goal of this project was to develop a novel assay for discovering small molecule ligands for class B1 G protein-coupled receptors (GPCRs). Very few agonists (or inverse agonists) with generally weak activity for this entire group of physiologically important receptors are known to date. To establish proof-of-principle that this critical bottleneck can be overcome with a suitable screening approach, our studies focused on the class B1 receptor for glucagon-like peptide-1 (GLP-1R), a potential therapeutic target for diabetes and neurodegenerative disease.

The assay was developed in a "mix and measure" format using a cell-based functional readout. Toward this goal, stably transfected HEK293 cell clones were established that co-express (i) a cAMP-responsive luciferase reporter gene, and (ii) GLP-1Rs that upon ligand interaction either stimulate (agonists) or inhibit (inverse agonists) luciferase activity. In different cell lines to be used for screening, expressed GLP-1Rs are specifically optimized for high sensitivity detection of either weak agonists (hypersensitive receptor) or inverse agonists (constitutively active receptor mutant). In addition, control cell lines were developed that express either the cAMP-responsive reporter gene without recombinant GLP-1Rs (enabling initial exclusion of GLP-1R independent effects of candidate probes), or the reporter gene together with wild type GLP-1Rs (for confirmation of probe activity at this receptor), or the reporter gene in combination with the constitutively active orphan receptor GPR119 (enabling initial exclusion of GLP-1R independent inhibitory effects of candidate inverse agonists).

To that end, this assay was optimized for 1536-well format and screened against the MLSMR.

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

Grant: NS064851
PI Name: Martin Beinborn, Tufts Medical Center
Protocol
In a white solid 1536-well plate, 6 uL of cells are dispensed in DMEM without phenol red, 10% Nu serum, and 1% pen-strep medium. The plates are then incubated for 12 hours at 37 deg C at 5% CO2 before 23 nL of compounds are added. After a 4-6 hr incubation at37 deg C and 5% CO2, 3 uL of detection reagent are added and after a 12 min incubation at RT, a luminescent read is obtained on the ViewLux.
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". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
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 PubChem:
Assay Cell Type: HEK293
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.00295 uM (0.00295μM**)% Activity at given concentration.Float%
16Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
17Activity at 0.074 uM (0.0737μM**)% Activity at given concentration.Float%
18Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
19Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
20Activity at 9.220 uM (9.22μM**)% Activity at given concentration.Float%
21Activity at 46.10 uM (46.1μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: NS064851

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