title: Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains. Demonstration of differential sensitivity to kinase inhibitors.
abstract: Epidermal growth factor receptor (EGFR), ErbB-2, and ErbB-4 are members of the type 1 receptor tyrosine kinase family. Overexpression of these receptors, especially ErbB-2 and EGFR, has been implicated in multiple forms of cancer. Inhibitors of EGFR tyrosine kinase activity are being evaluated clinically for cancer therapy. The potency and selectivity of these inhibitors may affect the efficacy and toxicity of therapy. Here we describe the expression, purification, and biochemical comparison of EGFR, ErbB-2, and ErbB-4 intracellular domains. Despite their high degree of sequence homology, the three enzymes have significantly different catalytic properties and substrate kinetics. For example, the catalytic activity of ErbB-2 is less stable than that of EGFR. ErbB-2 uses ATP-Mg as a substrate inefficiently compared with EGFR and ErbB-4. The three enzymes have very similar substrate preferences for three optimized peptide substrates, but differences in substrate synergies were observed. We have used the biochemical and kinetic parameters determined from these studies to develop an assay system that accurately measures inhibitor potency and selectivity between the type 1 receptor family. We report that the selectivity profile of molecules in the 4-anilinoquinazoline series can be modified through specific aniline substitutions. Moreover, these compounds have activity in whole cells that reflect the potency and selectivity of target inhibition determined with this assay system.
authors.affiliation: Department of Systems Research, GlaxoSmithKline Inc., Research Triangle Park, North Carolina 27709, USA.
authors.forename: Perry S
authors.initials: PS
authors.lastname: Brignola
authors.forename: Karen
authors.initials: K
authors.lastname: Lackey
authors.forename: Sue H
authors.initials: SH
authors.lastname: Kadwell
authors.forename: Christine
authors.initials: C
authors.lastname: Hoffman
authors.forename: Earnest
authors.initials: E
authors.lastname: Horne
authors.forename: H Luke
authors.initials: HL
authors.lastname: Carter
authors.forename: J Darren
authors.initials: JD
authors.lastname: Stuart
authors.forename: Kevin
authors.initials: K
authors.lastname: Blackburn
authors.forename: Mary B
authors.initials: MB
authors.lastname: Moyer
authors.forename: Krystal J
authors.initials: KJ
authors.lastname: Alligood
authors.forename: Wilson B
authors.initials: WB
authors.lastname: Knight
authors.forename: Edgar R
authors.initials: ER
authors.lastname: Wood
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D066247:Receptor, ErbB-4
chemical_list: D000255:Adenosine Triphosphate; D002413:Cations, Divalent; D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D066247:Receptor, ErbB-4; C587689:ERBB4 protein, human