Insights into the Binding of Cyclic RGD Peptidomimetics to ?5?1 Integrin by using Live-Cell NMR And Computational Studies

The interaction of a small library of cyclic DKP-RGD peptidomimetics with ?5?1 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 ?5?1 is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the ?5?1 binding site, and were integrated with competitive binding assays to the purified ?5?1 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 ?5?1 ligand of the series, displaying a nanomolar IC50 value.