2008 Articles

Gene expression-based screening for inhibitors of PDGFR signaling

Antipova, Alena A.; Stockwell, Brent R.; Golub, Todd R.

Here we describe a proof-of-concept experiment designed to explore the possibility of using gene expression-based high-throughput screening (GE-HTS) to find inhibitors of a signaling cascade, using platelet derived growth factor receptor (PDGFR) signaling as the example. The previously unrecognized ability of aurintricarboxylic acid to inhibit PDGFR signaling, discovered through a screen of 1,739 compounds, demonstrates the feasibility and generalizability of GE-HTS for the discovery of small molecule modulators of any signaling pathway of interest.
High throughput screening of small-molecule libraries is a well-established and highly productive tool for the identification of chemical compounds targeting a specific protein function of interest. Traditionally, the high-throughput screening for modulators of molecular pathways involves cell-free biochemical assays, or in some cases, highly specialized cell-based phenotypic assays. However, in many cases the optimal target for therapeutic intervention is not known, or the development of a suitable phenotypic read-out is not technically feasible. For example, it is becoming increasingly of interest to modulate the activity of particular signal transduction pathways, but the components of such pathways are in many cases only partially known. It would therefore be of interest to develop a screening approach that could identify inhibitors of such pathways without first defining the biochemical target of candidate small molecules. Here we demonstrate that it is possible to use mRNA expression levels as a read-out to infer activity of a signal transduction pathway, thus establishing a general approach to screening for modulators of signal transduction pathways.
Endogenous mRNA expression has been previously successfully used as a surrogate of cellular states in high-throughput screening for compounds inducing differentiation of acute myeloid leukemia cells, and for identifying inhibitors of androgen receptor-mediated transcriptional activation in prostate cancer. It is not obvious, however, that gene expression signatures could be used to identify inhibitors of signal transduction pathways that are regulated at the level of post-translational modification (phosphorylation), as opposed to transcriptional regulation.
To test the feasibility of using gene expression-based high-throughput screening (GE-HTS) to identify inhibitors of a signaling pathway, we chose platelet derived growth factor receptor (PDGFR) signaling for a proof-of-concept study, with particular emphasis on downstream activation of the extracellular regulated kinase (ERK) pathway (also known as the p42/p44 mitogen activated protein (MAP) kinase pathway) as a target pathway for the screen. The ERK pathway plays a major role in the control of cell growth, cell differentiation and cell survival. The protein kinase cascade Raf>mitogen/extracellular signal-regulated kinase (MEK)>ERK, also referred to as the MAP kinase module, is activated in mammalian cells through receptor tyrosine kinases, G-protein coupled receptors and integrins. Activated ERKs translocate to the nucleus where they phosphorylate transcription factors. The ERK pathway is often upregulated in human tumors, and as such is an attractive target for anticancer therapy. Furthermore, because the pathway has been extensively studied, many experimental tools are available with which to interrogate the pathway. We demonstrate here that indeed small molecule inhibitors of the PDGFR/ERK pathway can be discovered using the GE-HTS approach.