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 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.

Please refer to other AIDs (995, 1454, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732) for detailed assay protocols.

This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Molecules pending validation are given a score of 10. Inactive analogues from these series are given a score of 0. The present results summarize results for select compounds chosen for validation in secondary assays. ML102 (CID 2303746) was declared a probe from this project.

Grant Number: MH082406-01