Rational Design, Structure, and Biological Evaluation of Cyclic Peptides Mimicking the Vascular Endothelial Growth Factor

Abstract

Angiogenesis is the development of a novel vascular network from a pre-existing structure. Blocking angiogenesis is an attractive strategy to inhibit tumor growth and metastasis formation. Based on structural and mutagenesis data, we have developed novel cyclic peptides that mimic, simultaneously, two regions of the VEGF crucial for the interaction with the VEGF receptors. The peptides, displaying the best affinity for VEGF receptor 1 on a competition assay, inhibited endothelial cell transduction pathway, migration, and capillary-like tubes formation. The specificity of these peptides for VEGF receptors was demonstrated by microscopy using a fluorescent peptide derivative. The resolution of the structure of some cyclic peptides by NMR and molecular modeling has allowed the identification of various factors accounting for their inhibitory activity. Taken together, these results validate the selection of these two regions as targets to develop molecules able to disturb the development of cancer and angiogenesis-associated diseases.

Helical peptides from VEGF and Vammin hotspots for modulating the VEGF–VEGFR interaction

“…For the design of VEGF [13][14][15][16][17][18][19][20][21][22][23][24][25] analogues, key amino acids Phe17, Tyr21 and Tyr25 (corresponding to residues 5, 9 and 13 in the designed peptides) were maintained while the other residues were replaced. Alignment of the N-terminal regions of VEGF-A and Vammin permitted the identification of the fragment 1-13 of Vammin as the sequence corresponding to the VEGF [13][14][15][16][17][18][19][20][21][22][23][24][25] fragment.…”

“…For the design of VEGF [13][14][15][16][17][18][19][20][21][22][23][24][25] analogues, key amino acids Phe17, Tyr21 and Tyr25 (corresponding to residues 5, 9 and 13 in the designed peptides) were maintained while the other residues were replaced. Alignment of the N-terminal regions of VEGF-A and Vammin permitted the identification of the fragment 1-13 of Vammin as the sequence corresponding to the VEGF [13][14][15][16][17][18][19][20][21][22][23][24][25] fragment. In this case, residues Phe5, His9, and Ala13, located at the equivalent positions identified as key in VEGF [13][14][15][16][17][18][19][20][21][22][23][24][25] , were kept intact and only the rest of the residues were modified.…”

“…VEGF and its receptors represent one of the best validated signaling pathways in angiogenesis, 22 however very little attention has been paid to the rational design of compounds to modulate the protein-protein interactions involved in the molecular recognition between VEGF and VEGFRs. [23][24][25][26][27][28][29] In this respect, disruption of the above-mentioned protein-protein interaction could be a valid target for the search for inhibitors of angiogenesis. Mutagenesis data indicated that the VEGF binding sites for VEGFR-1 and VEGFR-2 receptors are very similar.…”

“…30,31 these peptides indicated a tendency to be structured around the central β-turn of the VEGF 81-91 β-hairpin and some of them showed significant affinity for VEGFR-1, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF-VEGFR-1 interaction. Concerning the less explored N-terminal fragment of VEGF [17][18][19][20][21][22][23][24][25] , D'Andrea and co-workers have reported a 15-mer analogue of VEGF [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] (Ac-KLTWMELYQLAYKGI-NH 2 ) that exhibits stable helical structure in solution, and pro-angiogenic properties both in vitro and in vivo. 39,40 More recently, the same authors described the preparation of a new peptide analogue (Ac-KLTWQELYQLKYKGI-NH 2 ) that shows anti-angiogenic properties.…”

“…The crystal structure of Vammin, reported by Morita and co-workers, revealed high structural similarities with VEGF-A, 44 and these authors centered their structural studies particularly on the Vammin fragment 69-80, which corresponds to VEGF [81][82][83][84][85][86][87][88][89][90][91] . Based on all the above precedents, we describe herein our efforts towards the de novo design of helical peptides able to mimic VEGF [13][14][15][16][17][18][19][20][21][22][23][24][25] and Vammin 1-13 fragments, selected as the minimum sequence to maintain the native helix conformation. Our main goal is to interfere with the VEGF-VEGFR interaction and try to identify key elements driving the molecular recognition between these two proteins.…”