Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics. |
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Authors: | Robbert J Kok Astrid J Schraa Erwin J Bos Henk E Moorlag Sigridur A Asgeirsdóttir Maaike Everts Dirk K F Meijer Grietje Molema |
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Institution: | Groningen University Institute for Drug Exploration, Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. r.j.kok@farm.rug.nl |
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Abstract: | Tumor blood vessels can be selectively targeted by RGD-peptides that bind to alpha(v)beta(3) integrin on angiogenic endothelial cells. By inhibiting the binding of these integrins to its natural ligands, RGD-peptides can serve as antiangiogenic therapeutics. We have prepared multivalent derivatives of the cyclic RGD-peptide c(RGDfK) by covalent attachment of the peptide to side chain amino groups of a protein. These RGDpep-protein conjugates inhibited alpha(v)beta(3)-mediated endothelial cell adhesion in vitro, while conjugates prepared with a control RAD-peptide showed no activity. Radiobinding and displacement studies with endothelial cells demonstrated an increased affinity of the RGDpep-protein conjugates compared to the free peptide, with IC(50) values ranging from 23 to 0.6 nM, depending on the amount of coupled RGDpep per protein. Compared to the parental RGD-peptide and the related RGD-peptide ligand c(RGDfV), the RGDpep-protein conjugates showed a considerable increase in affinity (IC(50) parent RGDpep: 818 nM; IC(50) c(RGDfV): 158 nM). We conclude that the conjugation of RGD-peptides to a protein, resulting in products that can bind multivalently, is a powerful approach to increase the affinity of peptide ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins. |
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