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BioAssay: AID 1454

qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay; Stimulation with EGF

The Ras/extracellular-signal-regulated kinase (ERK) mitogen activated protein (MAP) kinase signaling pathway (ERK1/2 cascade) plays a key role in transmitting signals from the cell surface to the nucleus (Nishida and Gotoh 1993; Chang and Karin, 2001). The cascade is initiated by the small G-protein Ras, which recruits Raf from the cytosol, where activation occurs. Alternatively, this pathway can be activated by elevating intracellular cAMP, or by activation of a receptor tyrosine kinase as epidermal growth factor receptor. ..more
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 Tested Compounds
 Tested Compounds
All(131326)
 
 
Active(544)
 
 
Inactive(124949)
 
 
Inconclusive(5965)
 
 
 Tested Substances
 Tested Substances
All(133385)
 
 
Active(548)
 
 
Inactive(126845)
 
 
Inconclusive(5992)
 
 
AID: 1454
Data Source: NCGC (ERKS580)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2008-12-19

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 544
Depositor Specified Assays
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AIDNameTypeProbeComment
995qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assayconfirmatory
1724Confirmation Concentration-Response Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assayconfirmatory
1725Confirmation Concentration-Response Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay; Stimulation with EGFconfirmatory
1726Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: EGFR T790M/L858R Kinase Inhibitionconfirmatory
1727Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: EGFR L858R Kinase Inhibitionconfirmatory
1728Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: c-Raf Inhibitionconfirmatory
1729Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: EGFR T790M Kinase Inhibitionconfirmatory
1730Counterscreen Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assayconfirmatory
1731Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: EGFR Kinase Inhibitionconfirmatory
1732Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: MEK Inhibitionconfirmatory
1742Quantitative High-Throughput Screen for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: Summarysummary1
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

MLSCN Grant: 1X01MH082406-01
Assay Submitter (PI): Dr. Wei Zheng, NCGC/NIH

NCGC Assay Overview:

The Ras/extracellular-signal-regulated kinase (ERK) mitogen activated protein (MAP) kinase signaling pathway (ERK1/2 cascade) plays a key role in transmitting signals from the cell surface to the nucleus (Nishida and Gotoh 1993; Chang and Karin, 2001). The cascade is initiated by the small G-protein Ras, which recruits Raf from the cytosol, where activation occurs. Alternatively, this pathway can be activated by elevating intracellular cAMP, or by activation of a receptor tyrosine kinase as epidermal growth factor receptor.

Activation of the ERK pathway is essential in increased cell division and cell survival. Sustained and constitutive activation of the ERK pathway, however, has been linked to uncontrolled cell proliferation, increased cell survival, and tumor progression. Thus, the ERK has been as an attractive target for cancer chemotherapy in the past few years (Sebolt-Leopold and Herrera, 2004). Given its physiological and pathological importance, assessment of ERK phosphorylation has been broadly performed in both basic research and drug discovery. Most assays for the measurement of ERK phosphorylation use the antibody-based detection methods, such as western blot and ELISA. These assays require multiple reagent additions with cell wash steps and are not suitable for high throughout screening.

The current cell-based ERK phosphorylation assay was performed using AlphaScreen platform. AlphaScreen is a two bead-based proximity-dependent chemical energy transfer luminescent assay platform. The assay reagents (ERK SureFire(TM) kit) were supplied from TGR BioSciences (Australia). In the presence of specific antibodies, phosphorylated ERK will bring "donor" and "acceptor" beads to a proximity range (<200 nm), and subsequently lead a chemical luminescent reaction upon exciting donor beads with 680 nm laser. To exclude the assay-related false positives, we used a biotinylated rabbit IgG to perform counter-screen. The biotinylated antibody mimics the phosphorylated ERK1/2 for bringing streptavidin coated beads and protein-A coated beads to a proximity range for the AlphaScreen detection.
Protocol
NCGC Assay Protocol Summary:

HEK293 cells stably expressing type-1 vasopressin receptor (V1b) were maintained in DMEM medium (Invitrogen, Carlsbad, CA) at 37C under a humidified atmosphere containing 5% CO2 and 95% air. The medium contained 10% FBS, 2 mM L-glutamine, non-essential amino acid, pyruvate and 50 U/ml penicillin and 50 ug/ml streptomycin (Invitrogen). The aliquots of frozen cells were stored at -135C and used for the compound screening. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. Epidermal growth factor (EGF) was used to stimulate the ERK phosphorylation pathway. The ERK phosphorylation in cells was determined using an AlphaScreen assay kit provided by PerkinElmer. Data were normalized to the controls for basal activity (DMSO only) and 100% activation (25 nM EGF). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

(1) Seed 4 ul/well of resuspended frozen HEK293 cells containing 2000 cells in white 1536-well plates for overnight with 1% FBS at 37C under a humidified atmosphere containing 5% CO2 and 95% air.
(2) Add 23 nL compounds in DMSO solution. The final titration was 0.6 nM to 46 uM.
(3) Incubate at 37C for 30 min.
(4) Dispense 0.5uL EGF (25 nM in final)
(5) Incubate at 37C for 5 min.
(6) Dispense 0.5uL of 1:2 diluted 5x lysis buffer.
(7) Incubate at 37C for 10 min.
(8) Dispense 1.5uL of mixture of activation buffer and reaction buffer with AlphaScreen beads (streptavidin donor beads and protein-A acceptor beads, 10 ng/well for each).
(9) Incubate at room temperature for 5 hr.
(10) Detect the assay plate in an EnVision plate reader (PerkinElmer) using the AlphaScreen detection mode.

Keywords: ERK, MEK, epidermal growth factor, homogenous, cell-based kinase assay, MLSMR, MLSCN, NIH Roadmap, qHTS and 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". 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
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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 signficant, 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 0.041 uM (0.0411345μM**)% Activity at given concentration.Float%
15Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
16Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
17Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
18Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
19Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
20Activity at 5.141 uM (5.14086μM**)% Activity at given concentration.Float%
21Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
22Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
23Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
24Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: X01MH082406-01

Data Table (Concise)
Classification
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