Discovery of sub-nanomolar RGD-based alphavbeta3/alphavbeta5 integrin binders embedding 4-aminoproline residues

The embodiment of 4-aminoproline residues (Amp) into the arginine-glycine-aspartate (RGD) sequence led to the discovery of a novel class of high-affinity RV3/RV5 integrin binders [IC50h(RV3) 0.03–5.12 nM; IC50h(RV5) 0.88–154 nM]. A total of eight cyclopeptides of type cyclo-[-Arg-Gly-Asp-Amp-], 5-12, were assembled by a standard solid-phase peptide synthesis protocol that involved the C2-carboxyl and C4- amino functionalities of the proline scaffolds, leaving the NR-nuclear site untouched. Functionalization of this vacant proline site with either alkyl or acyl substituents proved feasible, with significant benefit to the integrin binding capabilities of the ligands. Notably, six out of eight cyclopeptide inhibitors, 5-7 and 9-11, showed moderate yet significant selectivity toward the RV3 receptor. The three-dimensional structure in water was determined by NMR techniques and molecular dynamics calculations. Docking studies to the X-ray crystal structure of the extracellular segment of integrin RV3 complexed with reference compound 1 were also performed on selected analogues to highlight the structural features required for potent ligand binding affinity.