title: Indolinones and anilinophthalazines differentially target VEGF-A- and basic fibroblast growth factor-mediated responses in primary human endothelial cells.
abstract: OBJECTIVE
  The potent pro-angiogenic growth factors VEGF-A and basic fibroblast growth factor (bFGF) exert their effects by binding VEGF receptor 2 and FGF receptor tyrosine kinases, respectively. Indolinones (e.g. SU5416 and Sutent) and anilinophthalazines (e.g. PTK787) are potent small molecule inhibitors of VEGFR2 and other tyrosine kinases, but their effects on VEGF-A- and bFGF-stimulated endothelial responses are unclear. Here we assess the ability of these compounds to inhibit pro-angiogenic responses through perturbation of receptor activity and endothelial function(s).
  
  
  METHODS
  We used in silico modelling, in vitro tyrosine kinase assays, biochemistry and microscopy to evaluate the effects of small molecules on receptor tyrosine kinase activation and intracellular signalling. Primary human endothelial cells were used to assess intracellular signalling, cell migration, proliferation and tubulogenesis.
  
  
  RESULTS
  We predicted that the anilinophthalazine PTK787 binds the tyrosine kinase activation loop whereas indolinones are predicted to bind within the hinge region of the split kinase domain. Sutent is a potent inhibitor of both VEGFR2 and FGFR1 tyrosine kinase activity in vitro. The compounds inhibit both ligand-dependent and -independent VEGFR2 trafficking events, are not selective for endothelial cell responses and inhibit both VEGF-A- and bFGF-mediated migration, wound healing and tubulogenesis at low concentrations. CONCLUSIONS AND IMPLICATIONS; We propose that these compounds have novel properties including inhibition of bFGF-mediated endothelial responses and perturbation of VEGFR2 trafficking. Differential inhibitor binding to receptor tyrosine kinases translates into more potent inhibition of bFGF- and VEGF-A-mediated intracellular signalling, cell migration and tubulogenesis. Indolinones and anilinophthalazines thus belong to a class of multi-kinase inhibitors that show clinical efficacy in disease therapy.
authors.affiliation: Endothelial Cell Biology Unit, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Latham
authors.forename: A F
authors.initials: AF
authors.lastname: Bruns
authors.forename: J
authors.initials: J
authors.lastname: Kankanala
authors.forename: A P
authors.initials: AP
authors.lastname: Johnson
authors.forename: C W G
authors.initials: CW
authors.lastname: Fishwick
authors.forename: S
authors.initials: S
authors.lastname: Homer-Vanniasinkam
authors.forename: S
authors.initials: S
authors.lastname: Ponnambalam
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D003198:Computer Simulation; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008954:Models, Biological; D008958:Models, Molecular; D010793:Phthalazines / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
chemical_list: D007211:Indoles; D010793:Phthalazines; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D000255:Adenosine Triphosphate; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1; D040301:Vascular Endothelial Growth Factor Receptor-2