title: Substrate-specific conformational regulation of the receptor tyrosine kinase VEGFR2 catalytic domain.
abstract: The contributions of the phosphoacceptor and the catalytic domain context to protein kinase biology and inhibitor potency are routinely overlooked, which can lead to mischaracterization of inhibitor and receptor functions. The receptor tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR2) is studied as a model system using a series of phosphoacceptor substrates (k(cat)/K(m) 684-116,000 M(-1) s(-1)) to assess effects on catalysis and inhibitor binding. ATP-competitive inhibitor potency toward the VEGFR2 catalytic domain (VEGFR2-CD) varies with different phosphoacceptor substrates, which is unexpected because the phosphoacceptors do not affect K(m,ATP) values. Indazole-based inhibitors are up to 60-fold more potent with two substrates (gastrin, minigastrin) relative to the others. Thus there is a component of uncompetitive inhibition because a specific phosphoacceptor enhances potency but is not strictly required. This substrate-specific inhibitory potency enhancement correlates with phosphoacceptor active site saturation and is not observed with other related kinases. The effect is confined to a specific catalytic domain conformation because autophosphorylation eliminates the potency enhancement as does the addition of the juxtamembrane domain (20 amino acids). Indazole inhibitor structure-activity analysis reveals that the magnitude of potency enhancement correlates with the size of the substituent that binds in a regulatory region of the active site. VEGFR drugs profiled with VEGFR2-CD using minigastrin have potency well-correlated with inhibition of full-length, cellular VEGFR2 autophosphorylation, an indication that the minigastrin-induced conformation is biologically relevant. These findings raise the possibility that inhibitors directed toward a common target can have different biological effects based on the kinase-substrate complexes present in different cellular contexts.
authors: Oncology Research Unit, Pfizer Worldwide Research and Development, 10777 Science Center Drive, San Diego, CA 92121, USA.
authors: James
authors: J
authors: Solowiej
authors: Jeffrey H
authors: JH
authors: Chen
authors: Helen Y
authors: HY
authors: Zou
authors: Stephan K
authors: SK
authors: Grant
authors: Brion W
authors: BW
authors: Murray
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002364:Caseins / Q000378:metabolism; D020134:Catalytic Domain; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology; D005755:Gastrins / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002364:Caseins; D005755:Gastrins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D042461:Vascular Endothelial Growth Factor A; C008530:minigastrin; C027606:L-glutamic acid-L-tyrosine copolymer; D000255:Adenosine Triphosphate; D040301:Vascular Endothelial Growth Factor Receptor-2