Vascular Endothelial Growth Factor Peptide Ligands Explored by Competition Assay and Isothermal Titration Calorimetry

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• 作者：Marie Reille-Seroussi ; Jean-Fran莽ois Gaucher ; Claudia Desole ; Nathalie Gagey-Eilstein ; Franck Brachet ; Isabelle Broutin ; Michel Vidal ; Sylvain Broussy
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：August 25, 2015
• 年：2015
• 卷：54
• 期：33
• 页码：5147-5156
• 全文大小：429K
• ISSN：1520-4995

文摘

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 螖螖G掳 and 螖螖H掳 of binding, which correlated with free peptide CD spectra changes. We also propose a biochemical model to extrapolate K_i from IC₅₀ values measured in the displacement assay. These calculated K_i correlate well with the K_d values determined by extensive direct and reverse ITC measurements.