A High-Content Assay to Screen for Modulators of EGFR Function

Methods Mol Biol. 2016:1360:97-106. doi: 10.1007/978-1-4939-3073-9_8.

Abstract

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.

Keywords: Cancer; Drug discovery; EGFR; HTS; High-content assay; INCA2000; INCA3000; Inhibitor; Iressa; Kinase; Microscopy; RNAi; siRNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosensing Techniques*
  • Cell Line, Tumor
  • Cytoplasmic Granules / ultrastructure
  • Drug Discovery / methods
  • Enzyme Activation / drug effects
  • ErbB Receptors / antagonists & inhibitors
  • ErbB Receptors / drug effects*
  • ErbB Receptors / genetics
  • GRB2 Adaptor Protein / metabolism
  • Genes, erbB-1
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Image Processing, Computer-Assisted
  • Indicators and Reagents
  • Microscopy, Fluorescence / methods
  • Neoplasm Proteins / antagonists & inhibitors
  • Phosphorylation
  • Protein Binding
  • Protein Conformation
  • Protein Kinase Inhibitors / isolation & purification
  • Protein Kinase Inhibitors / pharmacology
  • Protein Processing, Post-Translational
  • Protein Transport
  • RNA Interference
  • RNA, Small Interfering / pharmacology
  • Receptor Protein-Tyrosine Kinases / antagonists & inhibitors
  • Receptor Protein-Tyrosine Kinases / drug effects
  • Small Molecule Libraries
  • src Homology Domains

Substances

  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Indicators and Reagents
  • Neoplasm Proteins
  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • Small Molecule Libraries
  • Green Fluorescent Proteins
  • EGFR protein, human
  • ErbB Receptors
  • Receptor Protein-Tyrosine Kinases