Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding.
Peptides involving the RGD 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 α(v)β(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 RGD systems with and without EG(n) linkers were investigated on their binding properties to cultured α(v)β(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 (125)I-labeled echistatin.


RESULTS
While c(RGDyK) function is a relative weak competitor against [(125)I]echistatin (K(i), 329 ± 18 nM) for α(v)β(3) integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K(i), 64 ± 23 nM). This effect was even more pronounced with the RGD trimers (K(i), 40 ± 7 nM) and tetramers (K(i), 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 [(125)I]echistatin. The EG(6) group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer).


CONCLUSIONS
The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [(125)I]echistatin with the introduction of EG(n) spacers. This effect may be related to the decrease of the effective RGD molarity, which becomes most prominent within the tetravalent series.
Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Holger
H
Kubas
Martin
M
Schäfer
Ulrike
U
Bauder-Wüst
Matthias
M
Eder
Dörte
D
Oltmanns
Uwe
U
Haberkorn
Walter
W
Mier
Michael
M
Eisenhut
D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008024:Ligands; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding
D039302:Integrin alphaVbeta3; D008024:Ligands; D010456:Peptides, Cyclic; C507169:cyclic arginine-glycine-aspartic acid peptide; D011092:Polyethylene Glycols