Bicyclic RGD-peptides with Exquisite Selectivity for the Integrin αvβ3 Receptor using a 'Random Design' Approach.

We describe the identification of bicyclic RGD-peptides with high affinity and selectivity for integrin αvβ3 via high-throughput screening of partly randomized libraries. Peptide libraries (~700 different compounds) comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop and a randomized sequence XXX (X being one of 18 canoni-cal L-amino acids) in the second loop, both enclosed by either an L- or D-Cys residue, were converted to bicyclic peptides via reaction with 1,3,5-tris(bromomethyl)benzene (T3). Screening of 1st generation libraries yielded lead bicyclic inhibitors dis-playing submicromolar affinities for integrin αvβ3 (e.g. CT3HEQcT3RGDcT3, IC50: 195 nM). Next generation (2nd and 3rd) librar-ies were obtained by partially varying the structure of the strongest lead inhibitors and screening for improved affinities and selectivities. In this way, we identified the highly selective bicyclic αvβ3-binders CT3HPQcT3RGDcT3 (IC50=30 nM), CT3HPQCT3RGDcT3 (IC50=31 nM), and CT3HSQCT3RGDcT3 (IC50=42 nM) with affinities comparable to that of a knottin-RGD-type peptide (32 amino acids, IC50=38 nM) and outstanding selectivities over integrins αvβ5 (IC50 >10,000 nM) and α5β1 (IC50 >10,000 nM). Affinity measurements using Surface Plasmon-enhanced Fluorescence Spectroscopy (SPFS) yielded Kd values of 0.4 and 0.6 nM for Cy5-labeled bicycle CT3HPQcT3RGDcT3 and RGD 'knottin' peptide, respectively. In vitro stain-ing of HT29 cells with Cy5-labeled bicycles using confocal microscopy revealed strong binding to integrins in their natural environment, which highlights the high potential of these peptides as markers of integrin-expression.