Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding

Peptides involving the ROD motive (arginine glycine aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for alpha(v)beta(3) integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG(n)) with different chain lengths. Therefore, the dependence of multivalent ROD systems with and without EG(n) linkers were investigated on their binding properties to cultured alpha(v)beta(3) integrin-expressing U87MG cells. Methods: We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with I-125-labeled echistatin. Results: While c(RGDyK) function is a relative weak competitor against [I-125]echistatin (Ki, 329 +/- 18 nM) for alpha(v)beta(3) integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K 64 23 nM). This effect was even more pronounced with the ROD trimers (K1, 40 +/- 7 nM) and tetramers (K1, 26 +/- 9 nM). The introduction of EG(n) spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [1251]echistatin. The EG(6) group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer). Conclusion: The binding experiments performed with the three forms of multivalent ROD ligands indicate the weakening of competitive potency against [I-125]echistatin with the introduction of EG(n) spacers. This effect may be related to the decrease of the effective ROD molarity, which becomes most prominent within the tetravalent series. (C) 2010 Elsevier Inc. All rights reserved.