AID	Name	Type	Comment
995	qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay	confirmatory
1454	qHTS Assay for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay; Stimulation with EGF	confirmatory
1742	Quantitative High-Throughput Screen for Inhibitors of the ERK Signaling Pathway using a Homogeneous Screening Assay: Summary	summary

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.

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 Kinase TK assay kit (Cisbio, Bedford, MA) was used to develop the MEK assay (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). MEK protein was 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 Kinase 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 MEK 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

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.

Grant Number: MH082406-01