title: Vascular Endothelial Growth Factor Peptide Ligands Explored by Competition Assay and Isothermal Titration Calorimetry.
abstract: The v114* cyclic peptide has been identified as a tight vascular endothelial growth factor (VEGF) ligand. Here we report on the use of isothermal titration calorimetry (ITC), 96-well plate competition assay, and circular dichroism (CD) to explore the binding determinants of a new set of related peptides. Anti-VEGF antibodies are currently used in the clinic for regulating angiogenesis in cancer and age-related macular degeneration treatment. In this context, our aim is to develop smaller molecular entities with high affinity for the growth factor by a structure activity relationship approach. The cyclic disulfide peptide v114* was modified in several ways, including truncation, substitution, and variation of the size and nature of the cycle. The results indicated that truncation or substitution of the four N-terminal amino acids did not cause severe loss in affinity, allowing potential peptide labeling. Increase of the cycle size or substitution of the disulfide bridge with a thioether linkage drastically decreased the affinity, due to an enthalpy penalty. The leucine C-terminal residue positively contributed to affinity. Cysteine N-terminal acetylation induced favorable ΔΔG° and ΔΔH° of binding, which correlated with free peptide CD spectra changes. We also propose a biochemical model to extrapolate Ki from IC50 values measured in the displacement assay. These calculated Ki correlate well with the Kd values determined by extensive direct and reverse ITC measurements.
authors.forename: Marie
authors.initials: M
authors.lastname: Reille-Seroussi
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Gaucher
authors.forename: Claudia
authors.initials: C
authors.lastname: Desole
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gagey-Eilstein
authors.forename: Franck
authors.initials: F
authors.lastname: Brachet
authors.forename: Isabelle
authors.initials: I
authors.lastname: Broutin
authors.affiliation: UF Pharmacocinétique et Pharmacochimie, hôpital Cochin, AP-HP, 27 rue du Faubourg Saint Jacques, Paris 75014, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Vidal
authors.forename: Sylvain
authors.initials: S
authors.lastname: Broussy
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D002151:Calorimetry; D003164:Competitive Bidding; D015195:Drug Design; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008024:Ligands; D010456:Peptides, Cyclic; D042461:Vascular Endothelial Growth Factor A