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BioAssay: AID 720709

qHTS for Antagonist of cAMP-regulated guanine nucleotide exchange factor 3 (EPAC1): primary screen

In multi-cellular eukaryotic organisms, the effects of cAMP are transduced by two ubiquitously-expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) [1, 2]. Both PKA and EPAC are ubiquitously more ..
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 Tested Compounds
 Tested Compounds
All(364602)
 
 
Active(517)
 
 
Inactive(352864)
 
 
Inconclusive(11244)
 
 
 Tested Substances
 Tested Substances
All(364940)
 
 
Active(517)
 
 
Inactive(353176)
 
 
Inconclusive(11247)
 
 
 Related BioAssays
 Related BioAssays
AID: 720709
Data Source: NCGC (EPAC1-antagonist-p1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-10-29
Modify Date: 2013-10-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 517
Related Experiments
AIDNameTypeComment
720698qHTS for cAMP-regulated guanine nucleotide exchange factor (EPAC): SummarySummarydepositor-specified cross reference
720708qHTS for Antagonist of cAMP-regulated guanine nucleotide exchange factor 2 (EPAC2): primary screenConfirmatorydepositor-specified cross reference
720720qHTS for Antagonist of cAMP-regulated guanine nucleotide exchange factor 3 (EPAC1): SummarySummarydepositor-specified cross reference
720721qHTS for Antagonist of cAMP-regulated guanine nucleotide exchange factor 4 (EPAC2): SummarySummarydepositor-specified cross reference
720707qHTS for Agonist of cAMP-regulated guanine nucleotide exchange factor 3 (EPAC1): primary screenConfirmatorysame project related to Summary assay
720711qHTS for Agonist of cAMP-regulated guanine nucleotide exchange factor 4 (EPAC2): primary screenConfirmatorysame project related to Summary assay
Description:
In multi-cellular eukaryotic organisms, the effects of cAMP are transduced by two ubiquitously-expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) [1, 2]. Both PKA and EPAC are ubiquitously expressed in all tissues, and an increase in intracellular cAMP levels will lead to the activation of both PKA and EPAC. Therefore, careful dissections of the individual role and relative contribution of EPAC and PKA within the overall cAMP signaling in various model systems are critical for further elucidating the mechanism of cAMP signaling. To date no EPAC-specific antagonists and agonist, as well as isoform-specific agonists have been reported, and developing EPAC-specific pharmacological probes to determine the physiological functions that EPAC play in the overall cAMP-mediated signaling remains a major challenge within the research field. Furthermore, identified regulators of EPAC may lead to new mechanism-based therapeutic strategies specifically targeting the EPAC / cAMP-signaling components as EPAC has been implicated to play important roles in major human pathological conditions such as diabetes, heart disease, and cancer.

This qHTS will be used to screen the Molecular Libraries Small Molecule Repository (MLSMR) as a concentration-titration series using the NCGC unique quantitative screening (qHTS) platform to find inhibitors for EPAC1. In addition, this assay will also be used as a counterscreen for the EPAC2 qHTS. This fluorescent based assay is developed and validated for EPAC1 that measures exchange using EPAC effector Rap1 complex with BODIPY-GDP, a GDP analog with a tethered fluorophores. Inhibitors are identified by monitoring the change in fluorescence signal as the exchanged free BODIPY-GDP has a reduced fluorescence compared to Rap1-bound BODIPY-GDP. Decrease in signal (inhibition) is the desired outcome for the EPAC1 antagonist while no activity in this assay is desired for the EPAC2 antagonist counterscreen.

[1] de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J.L. (1998) Nature 396, 474-477

[2] Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Science 282, 2275-2279

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

MLPCN Grant: NS066510
Assay Submitter (PI): Xiaodong Cheng, The University of Texas, Medical Branch
Protocol
Briefly, three uL of reagents (100 nM EPAC1, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control ATA and DMSO.
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 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.
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 0.0009732759 uM (0.000973276μM**)% Activity at given concentration.Float%
16Activity at 0.00195 uM (0.00194655μM**)% Activity at given concentration.Float%
17Activity at 0.00292 uM (0.00291983μM**)% Activity at given concentration.Float%
18Activity at 0.00584 uM (0.00583966μM**)% Activity at given concentration.Float%
19Activity at 0.00876 uM (0.00875954μM**)% Activity at given concentration.Float%
20Activity at 0.018 uM (0.0175191μM**)% Activity at given concentration.Float%
21Activity at 0.026 uM (0.0262786μM**)% Activity at given concentration.Float%
22Activity at 0.053 uM (0.0525571μM**)% Activity at given concentration.Float%
23Activity at 0.079 uM (0.0790424μM**)% Activity at given concentration.Float%
24Activity at 0.158 uM (0.157671μM**)% Activity at given concentration.Float%
25Activity at 0.237 uM (0.236507μM**)% Activity at given concentration.Float%
26Activity at 0.473 uM (0.473014μM**)% Activity at given concentration.Float%
27Activity at 0.710 uM (0.709522μM**)% Activity at given concentration.Float%
28Activity at 1.285 uM (1.2848μM**)% Activity at given concentration.Float%
29Activity at 2.271 uM (2.27067μM**)% Activity at given concentration.Float%
30Activity at 4.257 uM (4.25713μM**)% Activity at given concentration.Float%
31Activity at 6.386 uM (6.3857μM**)% Activity at given concentration.Float%
32Activity at 11.67 uM (11.6744μM**)% Activity at given concentration.Float%
33Activity at 19.16 uM (19.1571μM**)% Activity at given concentration.Float%
34Activity at 38.31 uM (38.3142μM**)% Activity at given concentration.Float%
35Activity at 57.50 uM (57.4959μM**)% Activity at given concentration.Float%
36Activity at 114.9 uM (114.943μM**)% Activity at given concentration.Float%
37Activity at 115.0 uM (115μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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