title: A vascular endothelial growth factor high affinity receptor 1-specific peptide with antiangiogenic activity identified using a phage display peptide library.
abstract: Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 microm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel was completely inhibited in the presence of 10 microm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and beta-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-beta-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure.
authors.affiliation: Supratek Pharma Inc., 215 Bouchard Street, Dorval, Quebec H9S 1A9, Canada.
authors.forename: Mayada
authors.initials: M
authors.lastname: El-Mousawi
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Tchistiakova
authors.forename: Ludmila
authors.initials: L
authors.lastname: Yurchenko
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Pietrzynski
authors.forename: Maria
authors.initials: M
authors.lastname: Moreno
authors.forename: Danica
authors.initials: D
authors.lastname: Stanimirovic
authors.forename: Darakhshan
authors.initials: D
authors.lastname: Ahmad
authors.forename: Valery
authors.initials: V
authors.lastname: Alakhov
mesh_terms: D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D017104:Bacteriophage M13; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006801:Humans; D019151:Peptide Library; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D014471:Umbilical Veins / Q000166:cytology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D016326:Extracellular Matrix Proteins; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1