Simple, mammalian cell-based assay for identification of inhibitors of the Erk MAP kinase pathway

Abstract

The Erk MAP kinase pathway contributes to tumor development and thus represents an important therapeutic target. Several inhibitors of the Erk pathway are presently being evaluated in clinical trials for cancer, but show limited efficiency thus warranting discovery of more potent inhibitors. We have developed a novel mammalian cell-based assay that should facilitate the identification of such compounds by screening molecular libraries. In rat chondrosarcoma (RCS) cells, treatment with fibroblast growth factor 2 (FGF2) leads to sustained activation of the Erk pathway, resulting in growth arrest with more than an 80% cell count difference between control and FGF2-treated cells after 72 h of treatment. The extent of both Erk activation and the growth arrest can be precisely modulated by the FGF2 dose. We also demonstrate that FGF2-mediated activation of the Erk pathway is robust and has only a limited sensitivity to the available MEK inhibitors. The assay is rapid, sensitive and easily adapted to high throughput screening. A major advantage of this system is exclusion of toxic compounds as false-positive hits, given the nature of the RCS response to inhibition of the Erk pathway, i.e. growth.

NF449 Is a Novel Inhibitor of Fibroblast Growth Factor Receptor 3 (FGFR3) Signaling Active in Chondrocytes and Multiple Myeloma Cells

“…Here, we took advantage of RCS chondrocytes, which represent the most extensively studied cell model for FGFR3-related skeletal dysplasia (8 -12). We used the well characterized growth-inhibitory response of RCS chondrocytes to FGFR3 activation as a reporter for compound library screening aimed at identification of novel inhibitors of FGFR3 signaling (7). A molecular library consisting of 1120 molecules was screened, leading to identification of several potential antagonists of FGFR3 signaling.…”

“…1A). This activity of NF449 is remarkable, considering the robust nature of the RCS growth arrest phenotype (7).…”

“…The RCS cell growth arrest experiments utilizing crystal violet staining to quantify cell growth are described in detail elsewhere (7). For the RCS growth arrest experiments here, ϳ1 ϫ 10 4 cells were seeded in 24-well tissue culture plates (Costar, Cambridge, MA), treated as indicated for 72 h, and counted.…”

“…RCS chondrocytes respond to FGFR3 activation (via exogenous FGF2 addition) with potent growth arrest, loss of the cartilage-like extracellular matrix, and marked alteration of their cellular shape (9,11,12). We have taken advantage of this response and adapted the FGF2/FGFR3-mediated growth arrest for rapid screening of chemical compounds for their activity against FGFR3 signaling (7).…”

“…The aim of this study was to identify novel inhibitors of FGFR3 signaling in cellular environments relevant to FGFR3-related disorders. We recently established a chondrocyte cell-based reporter assay suitable for identification of inhibitors of FGFR3 signaling via molecular library screening (7). Here, we used this assay to identify and characterize the compound named NF449 as a novel antagonist of FGFR3 signaling active in both chondrocytes and multiple myeloma cells.…”