Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

Abstract

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, highthroughput screen using a novel ERF1 Redistribution ® assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution ® assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC 50 = < 5 µM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution ® screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant. 1 The pathway is deregulated in various cancer types, and pathway-associated proteins are important targets in cancer drug discovery. Overexpression or mutations in receptor tyrosine kinases (RTKs) that result in excessive pathway activation are common in various types of carcinomas, and approximately 50% of several cancers, especially melanoma and colorectal and pancreatic cancers, exhibit aberrant MAPK pathway signaling.2-4 The MAPK pathway is activated by various growth factors that bind to RTKs, resulting in the activation of the RAS proteins. These proteins are GTPases that, when active, form a complex with RAF protein kinases. 5 This complex translocates to the plasma membrane where the RAF proteins subsequently activate MEK proteins. 5The downstream MEK1 and 2 proteins are dual-specificity protein kinases and constitute a focal point in the pathway because the kinases ERK1 and 2 (also named p44 and p42 MAPKs) are the only identified MEK substrates. Inactive ERK proteins are sequestered in the cytoplasm by the MEK kinases. Activated MEK1/2 release ERK1/2 from their anchorage in the cytoplasm by phosphorylating ERK1/2 on tyrosine and threonine residues.1,6 Activated ERK proteins phosphorylate multiple downstream effectors and also move into the nucleus to regulate gene expression by phosphorylating certain transcription factors.7,8 As a result of stimulating these transcriptional regulators, key cell cycle regulatory proteins are expressed, which enable the cell to progress through the G1 phase of the cell cycle. 9 In the nucleus, ERK1/2 is inactivated by the MAPK phosphatases (MKPs). 10-12The Ets domain transcriptional repressor ERF1 is a ubiquitously ex...