Insights into the Binding of Cyclic RGD Peptidomimetics to α₅β₁ Integrin by using Live-Cell NMR And Computational...

The interaction of a small library of cyclic DKP-RGD peptidomimetics with α₅β₁ integrin has been investigated by means of an integrated experimental and computational approach. Bioaffinity NMR techniques, including saturation transfer difference (STD) and transferred NOESY, were applied to the ligands in a suspension of intact MDA-MB-231 breast cancer cells, in which integrin α₅β₁ is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the α₅β₁ binding site, and were integrated with competitive binding assays to the purified α₅β₁ integrin. Ligand binding epitopes involve protons of both the RGD moiety and the DKP scaffold, although the stereochemistry and the functionalization of the DKP scaffold as well as the macrocycle conformation determine a great variability in the interaction. The ligand showing the highest number of STD signals is also the most potent α₅β₁ ligand of the series, displaying a nanomolar IC₅₀ value.