RGD-based peptides targheting αIIbβ3 and αvβ3 integrin receptors: an NMR point of view

Integrins are transmembrane receptors that link the cytoskeleton to the extracellular matrix (ECM),1 mediating cell–cell and cell–matrix adhesion and providing the traction for cell mobility and invasion.1 Integrin αvβ3 and αvβ5 expression is correlated with disease progression in various tumor types (melanoma, prostate, glioblastoma) while the platelet integrin αIIbβ3 plays a key role in adhesion of platelet to protein-coated surfaces and in platelet/platelet aggregation (thrombus formation). These integrins show a similar drug-receptor site and bind to the Arg-Gly-Asp (RGD) motif as their primary recognition sequence in their protein ligands and the conformation of the RGD sequence is critical for the specificity of this recognition. Since the RGD motif occurs in many extracellular matrix ligands, the recognition specificity is expected to be modified by other residues and to depend on the conformation of the RGD sequence. Detailed comparison to different ligands of the same integrin binding site could shed light on the essential elements that determine their interaction, specificity and affinity, and allow the rational design of new antagonists. Our work focuses on the application of NMR techniques2 (STD and tr-NOESY) for understanding, at the molecular and atomic levels, the diverse mechanisms by which peptidomimetics are recognised by the binding sites of integrins αvβ3 and αIIbβ3. The data allowed us to identify (by STD) the portions of the ligands that are in closest contact with the protein and to define (by trNOESY) the preferred conformation of the bound ligands. The properties of integrins embedded into cell membranes may differ from those of purified receptors, therefore, we show that the interaction between a small library of RGD-peptidomimetics3 and membrane-bound proteins can be observed by NMR directly in a H2O-buffer-suspension of living cells, without the need of isolating the protein receptor. We performed the NMR experiments on a suspension of ECV304, bladder cancer cells in which the integrin αvβ3 is highly expressed, or in the presence of whole human platelets (where integrin IIb3 is the most abundant platelet cell surface glycoprotein). The NMR data are interpreted with the aid of docking calculations affording an improved understanding of integrin-ligand interactions.