Bookmark and Share
BioAssay: AID 1731

Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: EGFR Kinase Inhibition

The Ras/extracellular-signal-regulated kinase (ERK) mitogen activated protein (MAP) kinase signaling pathway (ERK1/2 cascade) plays is 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. ..more
_
   
 Tested Compounds
 Tested Compounds
All(26)
 
 
Active(8)
 
 
Inactive(17)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Active(8)
 
 
Inactive(19)
 
 
Inconclusive(1)
 
 
AID: 1731
Data Source: NCGC (ERKS584)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-05-07

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 8
Depositor Specified Assays
AIDNameTypeComment
995qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assayconfirmatory
1454qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay; Stimulation with EGFconfirmatory
1742Quantitative High-Throughput Screen for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: Summarysummary
Description:
qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay

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 is 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.

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-throughput 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 validate pathway hits from the cell-based assay, enzyme assays consisting of EGFR tyrosine kinase, Raf and MEK were used as a means of deciphering the enzymes of target for the prioritized group of compounds derived from the HEK-293 cell based assay. The compounds were tested against significant members of the ERK signaling pathway EGFR and EGFR mutants (EGFR L858R, EGFR T790M, and EGFR L858R T790M). In order to establish the specificity of the prioritized compounds for EGFR and EGFR mutants, the compounds were also assayed against other important ERK signaling pathway enzymes, c-RAF and MEK-1.
Protocol
NCGC Assay Protocol Summary:

The HTRF kinase assay (components supplied as kit by Cisbio) was chosen for the EGFR and EGFR mutant enzyme assays that applied the time resolved fluorescence resonance energy transfer (TR-FRET) principle. A peptide substrate is labeled with a biotin that can bind to XL665 labeled streptavidin and the anti-phosphoresidue antibody is labeled with Eu+. Upon phosphorylation of the substrate, the antibody binds to phosphorylated substrate that enables TR-FRET detection in homogenous assay format.

An HTRF Kinease TK assay kit (Cisbio, Bedford, MA) was used to develop the EGFR and EGFR mutant assays (Harbert et al., 2008. Harbert C, Marshall J, Soh S, Steger K. Development of a HTRF Kinase Assay for Determination of Syk Activity. Current Chemical Genomics. 1: 20-26. 2008). EGFR and EGFR mutants were obtained from Invitrogen. The assay buffer was composed of 50 mM HEPES (pH 7.0), 5 mM MgCl2, 5mM DTT, 0.1 % NaN3, 0.1 % BSA and 0.1 mM orthovanadate.

The HTRF assays were preformed according to the HTRF Kinease TK kit. Optimization for each enzyme was preformed in 384 well format (data not included). All reagents were dispensed into 1536 well plates.

(1) Enzyme, 2 nl EGFR at 3 nM final
(2) Compound, 22 nl, Library Compounds in 0.128 to 10 mM titration series or control
(3) Reagent, 1.5 ul 10 uM ATP and 0.25 uM substrate final
(4) Incubation for 30 min, Ambient conditions
(5) Reagent, 3 ul antibody and XL-665
(6) Incubation for 30 min, Ambient conditions
(7) Detector#Ex 320, Em 615/665, EnVision plate reader
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
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 0.0000137549 uM (1.37549e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0000412648 uM (4.12648e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0001237945 uM (0.000123794μM**)% Activity at given concentration.Float%
17Activity at 0.0003713835 uM (0.000371384μM**)% Activity at given concentration.Float%
18Activity at 0.00111 uM (0.00111415μM**)% Activity at given concentration.Float%
19Activity at 0.00334 uM (0.00334245μM**)% Activity at given concentration.Float%
20Activity at 0.010 uM (0.0100274μM**)% Activity at given concentration.Float%
21Activity at 0.030 uM (0.0300821μM**)% Activity at given concentration.Float%
22Activity at 0.090 uM (0.0902462μM**)% Activity at given concentration.Float%
23Activity at 0.271 uM (0.270739μM**)% Activity at given concentration.Float%
24Activity at 0.812 uM (0.812216μM**)% Activity at given concentration.Float%
25Activity at 2.437 uM (2.43665μM**)% Activity at given concentration.Float%
26Activity at 7.310 uM (7.30994μM**)% Activity at given concentration.Float%
27Activity at 21.93 uM (21.9298μM**)% Activity at given concentration.Float%
28Activity at 65.79 uM (65.7895μM**)% Activity at given concentration.Float%
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH082406-01

Data Table (Concise)
Classification
PageFrom: