title: Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.
abstract: Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.
authors.affiliation: HTS Core Facility, Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center , New York, New York 10065, USA.
authors.forename: Christophe
authors.initials: C
authors.lastname: Antczak
authors.forename: Alun
authors.initials: A
authors.lastname: Bermingham
authors.forename: Paul
authors.initials: P
authors.lastname: Calder
authors.forename: Dmitry
authors.initials: D
authors.lastname: Malkov
authors.forename: Keming
authors.initials: K
authors.lastname: Song
authors.forename: John
authors.initials: J
authors.lastname: Fetter
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
mesh_terms: D000970:Antineoplastic Agents / Q000032:analysis* / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D010865:Pilot Projects; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D034741:RNA, Small Interfering; D066246:ErbB Receptors