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 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.

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. Vasopressin 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 vasopressin). 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 vasopressin (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, type-1 vasopressin receptors, homogenous, cell-based kinase assay, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC

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.