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), 96well 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 K i from IC 50 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.

ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2

“…The latter limitation may however be overcome by application of the reverse ITC experiment, in which the macromolecule is used as a titrant instead of the small-mass ligand [31]. It should also be noted that there are very few studies in which both modes of calorimetric titrations were applied in parallel [32, 33] or accompanied by alternative experimental techniques [34, 35]. …”

A Cyclic Peptide Epitope of an Under-Explored VEGF-B Loop 1 Demonstrated In Vivo Anti-Angiogenic and Anti-Tumor Activities

“…Meanwhile, many other interaction structures of different growth factors with VEGFRs have been reported, but only with D2 of VEGFR1, such as placental growth factor (PlGF) dimer with D2 of VEGFR1 (PDB code 1RV6) ( Christinger et al, 2004 ) and VEGF-B dimer with D2 of VEGFR1 (PDB: 2XAC) ( Iyer et al, 2010 ). All these structure data promoted the rational design of peptide inhibitors targeting VEGF or VEGFRs for the purpose of blocking VEGF/VEGFR interaction to inhibit angiogenesis ( Basile et al, 2011 ; Garcia-Aranda et al, 2013 ; Assareh et al, 2019 ), including our works ( Goncalves et al, 2007a ; Gautier et al, 2010 ; Wang et al, 2014 ; Reille-Seroussi et al, 2015 ; Wang et al, 2017 ; Wang et al, 2019 ).…”

A Structural Overview of Vascular Endothelial Growth Factors Pharmacological Ligands: From Macromolecules to Designed Peptidomimetics

“…Although the folding process results in a thermodynamically unfavorable entropy variation [119], the peptide v107 was considered a suitable starting point for further optimization because of its high affinity for VEGF-A, its structuration upon binding, and because it is binding with the growth factor was mainly mediated by specific hydrophobic interactions with its side chains. Additional studies on this v107 peptide include a study in the gas phase of D-amino acids variants [120], the modification with an electrophile chemical function for covalent labeling of VEGF-A [121], the radiolabeling for imaging [122,123], the extension with peptide fragments to improve its affinity as a capture agent [122], the incorporation of non-natural beta-amino acids for increased proteolytic stability (structure of peptide HH4, PDB 6D3O, to be published) [119,124], the shortening of the sequence to 15 amino acids [125], and the stabilization of the α-helix C-terminal end by cyclization for increased affinity (PDB 6Z13, 6ZCD, 6Z3F, 6ZBR, to be published). A bacterial display screening independently identified peptides that were very similar to v107, with comparable affinities for VEGF-A and the same core sequence WE/DWE/D [126].…”