Bicyclic RGD-peptides with high affinity and selectivity for αvβ3, αvβ5, and α5β1 integrins
D. Bernhagen[a], L. De Laporte[b], P. Timmerman[a,c]
[a] Pepscan Therapeutics, Zuidersluisweg 2, 8243 RC Lelystad, Netherlands; [b] DWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany; [c] Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, Sciencepark 904 XH Amsterdam, Netherlands
The ‘bicyclic peptide’ platform recently attracted considerable interest as a powerful platform for novel therapeutic drugs because of their high binding affinities and selectivities in combination with an appreciable proteolytic stability. So far, only few cellular proteins have been evaluated for their binding to bicyclic peptides. The integrins represent an interesting target class for novel therapeutic agents because of their significant role in key pathological processes. We therefore synthesized libraries of hundreds of different bicyclic peptides, one loop comprising the well-known “RGD”-motif and providing basic integrin-affinity, and second loop consisting of a random sequence (XXX) that allows for controlling the binding selectivity. In order to screen for affinity and selectivity to each integrin (αvβ3, αvβ5, and α5β1) we developed a robust and cost-effective screening assay using a knottin-RGD peptide as a reference binder.[4,5] After selection of the best binders for each integrin, we designed 2nd and 3rd generation diversity libraries and thus gradually improved the affinity and selectivity for each integrin. The best IC50 values that we obtained in this way were, for example, 30 nM for αvβ3 (GRGDS: 5 µM, knottin-RGD: 250 nM), and 90 nM for α5β1 (GRGDS: >30 µM, knottin-RGD: 100 nM). We also labeled the best bicyclic integrin-binders with a fluorescent dye and studied their integrin-binding on live cells. The most potent bicyclic integrin-binders showing highest were also immobilized on 2D hydrogels in order to investigate their effect on cell adhesion, cell proliferation and viability as compared to the conventional RGD-peptides.
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