Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells Journal Article
| Authors: | Antczak, C.; Bermingham, A.; Calder, P.; Malkov, D.; Song, K.; Fetter, J.; Djaballah, H. |
| Article 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. © 2012, Mary Ann Liebert, Inc. |
| Keywords: | controlled study; unclassified drug; human cell; sunitinib; antineoplastic agents; protein function; imatinib; epidermal growth factor receptor; green fluorescent protein; rna, small interfering; rna interference; receptor, epidermal growth factor; high throughput screening; drug screening; dose-response relationship, drug; drug screening assays, antitumor; cell line, tumor; dasatinib; cell assay; temsirolimus; pilot projects; screening; gefitinib; vatalanib; gene silencing; wortmannin; apigenin; midostaurin; mepacrine; calyculin a; 12 (2 cyanoethyl) 6,7,12,13 tetrahydro 13 methyl 5 oxoindolo[2,3 a]pyrrolo[3,4 c]carbazole; alpha [amino(4 aminophenylthio)methylene] 2 (trifluoromethyl)phenylacetonitrile; biosensing techniques; lycorine; protein sh2; biosensor; 4 (3 bromoanilino) 6,7 dimethoxyquinazoline; erbstatin; 2,4 thiazolidinedione; 4 (4 fluorophenyl) 2 (4 nitrophenyl) 5 (4 pyridyl)imidazole; adaphostin; lavendustin a; pd 16916 |
| Journal Title: | Assay and Drug Development Technologies |
| Volume: | 10 |
| Issue: | 1 |
| ISSN: | 1540-658X |
| Publisher: | Mary Ann Liebert, Inc |
| Date Published: | 2012-02-01 |
| Start Page: | 24 |
| End Page: | 36 |
| Language: | English |
| DOI: | 10.1089/adt.2011.423 |
| PROVIDER: | scopus |
| PMCID: | PMC3277729 |
| PUBMED: | 22280060 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 2 April 2012" - "CODEN: ADDTA" - "Source: Scopus" |
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