Identification potent kinase inhibitors against EGFR and its signaling pathways using a label-free cell based assay Free

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The epidermal growth factor receptor (EGFR; erbB1) is a member of a tyrosine kinase family that includes erbB2 (Her2/neu), erbB3, and erbB4. Disregulation of EGFR function as result of mutation, change in expression levels of receptor or its ligand, has been implicated in malignant transformation of several cancers of various origin including lung, breast, colorectal, squamous cell cancer of the head and neck, and prostate cancer. We report the identification of potent small molecule inhibitors of EGFR using a novel, quantitative, real-time, and label-free cellular assay. This cellular assay allows for the identification of both competitive and allosteric type of EGFR inhibitors. Using EGF-mediated changes in morphology as a read-out, a focused kinase library of about 1254 compounds was initially screened. From this screen, a benzothiazole based compound that inhibits EGFR kinase activity was identified. Initial characterization of the compound showed an in vitro IC50 activity of 46 nM. The compound also showed inhibition of EGF-dependent survival of LoVo cell at IC50 of 197 nM.In addition, a compound was also identified that inhibited EGF dependent activity but not EGFR tyrosine kinase activity. The activities of these compounds on proliferation of a panel of normal and cancer cell lines including EGFR and erbB2 overexpressing lines, migration, and different signaling pathways activated by EGFR were also examined. As a result of the initial screen, an additional 10,000 compounds of a focused kinase library was screened. Several more compounds that either inhibited EGFR directly or inhibited the EGF dependent signaling were additionally identified. The unbiased nature of this screen can lead to classes of compounds that have novel mechanism of inhibition and utility against resistant forms of the kinase.