Bookmark and Share
BioAssay: AID 624418

qHTS of GLP-1 Receptor Inverse Agonists: Cytotox Screen

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
_
   
 Tested Compounds
 Tested Compounds
All(405121)
 
 
Active(527)
 
 
Inactive(386140)
 
 
Inconclusive(18592)
 
 
 Tested Substances
 Tested Substances
All(408352)
 
 
Active(527)
 
 
Inactive(389197)
 
 
Inconclusive(18628)
 
 
 Related BioAssays
 Related BioAssays
AID: 624418
Data Source: NCGC (GLPIA10)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-07-26

Data Table ( Complete ):           Active    All
BioActive Compounds: 527
Depositor Specified Assays
AIDNameTypeComment
624421qHTS of GLP-1 Receptor Inverse Agonists: Summarysummary
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 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) constitutively active GLP-1R that upon ligand interaction inhibit (inverse agonists) the luciferase activity. In addition, two control cell lines were developed: (i) one that express only the cAMP-responsive reporter gene to enable initial exclusion of GLP-1R independent effects of candidate probes; and (ii) one with the reported gene cloned with another constitutively active orphan receptor GPR119 to enable exclusion of GLP-1R independent inhibitor effects of candidate inverse agonists.

To that end, this assay was optimized for 1536-well format and screened against the MLSMR. Then the same collection was run in this cytotoxicity assay where inhibition was the undesired outcome. Inactive compounds were prioritized in the selection process.

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

Grant: NS064851
PI Name: Martin Beinborn, Tufts Medical Center
Protocol
Six ul of cell suspension (200 cells/uL in DMEM + 10% Nu serum + 1% pen-strep) is dispensed in a white solid 1536-well plate. The plates are incubated for 12 hours at 37 deg C at 5% CO2 followed by addition of 23 nL compounds. After 24 hr incubation at 37 deg C and 5% CO2, 3 uL of luciferase assay detection reagent is added. A luminescence read is obtained on the PerkinElmer Viewlux after 12 min incubation at room temperature.
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 inhibitors are ranked higher than compounds that showed apparent activation.

2. For all nontoxic (inactive) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all toxic (active) compounds, a score range was given for each curve class type given above. Active (inhibiting) 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.
Result Definitions
Show more
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 0.307 uM (0.307μM**)% Activity at given concentration.Float%
16Activity at 1.530 uM (1.53μM**)% Activity at given concentration.Float%
17Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
18Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
19Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

Data Table (Concise)
PageFrom: