The R V β 3 integrin is implicated in human tumor metastasis and in angiogenesis. The design of lowmolecular-mass R V β 3 antagonists by "spatial screening" led to the highly active peptides c(RGDFV) and c(RGDFV).Here the influence of the amino acids in positions 4 and 5 flanking the RGD-sequence on the inhibition of vitronectin and fibrinogen binding to the isolated R V β 3 and R IIb β 3 receptors was investigated. The influence of the side chain and the backbone conformation on activity and selectivity was studied. The compounds were divided into conformational classes. For each class at least one representative peptide was subjected to detailed structure determination in solution. The peptides of classes 1, 2, and 3 show a βII′/γ-turn arrangement with the D-amino acid in the i + 1 position of the βII′-turn. By contrast, the peptides of class 4 reveal a modified βII′/γ-turn pattern with glycine in the i + 1 position of the βII′-turn and the D-amino acid in the i + 1 position of the γ-turn. Class 1 is divided into two subclasses: besides the βII′/γ-turn arrangement a γ/γ-turn motif is found for two members of this class. Structure-activity relationship (SAR) investigations show that the amino acid in position 4 and the proton of the amide bond between residues 3 and 4 are essential for high biological activities toward R V β 3 . By contrast, the amino acid in position 5 has no influence on the activity. A bent conformation of the RGD-sequence, as observed for the peptides of classes 1 and 2, fits the R V β 3 better than the R IIb β 3 receptor and so increases the selectivity of these peptides.

Abstract

We have synthesized by solution methods and fully characterized the Nα-blocked heptapeptide methylamide mBrBz-[L-Iva-L-(αMe)Val]2-L-(αMe)Phe-L-(αMe)Val-L-Iva-NHMe, fully based on conformationally constrained C α-methylated α-amino acids. An X-ray diffraction investigation of the N α-benzyloxycarbonylated analogue showed that in the crystal state both independent molecules (A and B) in the asymmetric unit of the peptide adopt a fully developed, regular, right-handed 310-helical structure, although molecule A would be slightly distorted at the C-terminal residue. Solution conformational analysis on the mBrBz-blocked peptide was carried out in CDCl3 by means of NMR spectroscopy. For structure determination we performed restrained molecular dynamics simulations in CDCl3 based on a search of the conformational space derived from a simulated annealing strategy. For this peptide the NMR observables can be described by a single backbone conformation, more specifically a rigid 310-helix spanning the amino acid sequence from residue 1 to residue 6. The C-terminal methylamido NH group seems to be involved simultaneously in two H-bonds (with the preceding i − 3 and i − 4 carbonyl groups). Although in this peptide model there are no distinct NOE distances for discriminating 310- versus α-helix conformation, the sum of all NMR-derived restraints clearly results in a 310-helical structure. Convergence from different starting structures (including an α-helix) into a 310-helix was observed.