A High-Content Assay to Screen for Modulators of EGFR Function.
Cell-based assays have the potential and advantage to identify cell-permeable modulators of kinase function, and hence provide an alternative to the conventional enzymatic activity-driven discovery approaches that rely on purified recombinant kinase catalytic domains. Here, we describe a domain-based high-content biosensor approach to study endogenous EGFR activity whereby EGF-induced receptor activation, subsequent trafficking, and internalization are imaged and quantified using time-dependent granule formation in cells. This method can readily be used to search for EGFR modulators in both chemical and RNAi screening; with potential applicability to other receptor tyrosine kinases.
HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA.|Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA, 02139, USA.
Christophe
C
Antczak
HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA. hakim.djaballah@ip-korea.org.|Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea. hakim.djaballah@ip-korea.org.
Hakim
H
Djaballah
D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D055808:Drug Discovery / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000235:genetics; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D018773:Genes, erbB-1; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence / Q000379:methods; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects; D054852:Small Molecule Libraries; D018909:src Homology Domains
D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D054852:Small Molecule Libraries; D049452:Green Fluorescent Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases
Cancer; Drug discovery; EGFR; HTS; High-content assay; INCA2000; INCA3000; Inhibitor; Iressa; Kinase; Microscopy; RNAi; siRNA