Cyclic RGD peptidomimetics: insights into the binding to integrin-rich cancer cells by NMR and computational studies

The integrin family of adhesion molecules regulates diverse cell functions crucial to tumor angiogenesis, progression and metastasis. In particular, recent studies provide evidence of the importance of Arg-Gly-Asp (RGD) binding integrins in angiogenesis and cancer, and suggest the development of dual and multi-antagonists to efficiently target these processes [1]. Among tumor associated integrins from the RGD-binding subfamily, α5β1 along with αVβ3 and other αv integrins are highly expressed on a wide range of tumors and surrounding vasculature, where they are correlated with disease progression. The most used strategy to inhibit integrin function consists in blocking natural ligand binding by small-molecule integrin antagonists designed around the RGD motif. In particular, since linear peptides have obvious drawbacks, a large number of cyclic peptidic and peptidomimetic ligands have been developed that target with high affinity and specificity tumor associated integrin heterodimers and show potential as both anti-tumor and anti-angiogenic agents [2]. In this context, we have recently reported a small library of cyclic peptidomimetics containing a bifunctional diketopiperazine scaffold and the tripeptide sequence RGD as potent αVβ3 integrin ligands [3]. The molecular basis of this activity was rationalized in terms of preferred ligand conformations featuring an extended arrangement of the RGD sequence, which are highly preorganized for the interaction with integrin αVβ3, as demonstrated by docking studies and NMR experiments with αVβ3-rich bladder cancer cells [4]. Prompted by these results and by the perspective of targeting two key players in angiogenesis and cancer in the RGD-binding integrin subfamily, we have investigated the interaction of our cyclic RGD peptidomimetics with α5β1 integrin by exploiting the same integrated computational and experimental approach applied in the study of αVβ3 integrin. Bioaffinity NMR techniques, including Saturation Transfer Difference and transferred NOE, were applied to ligands in a suspension of MDA-MB-231 breast cancer cells in which integrin α5β1 is highly expressed. The NMR data were interpreted with the aid of docking calculations of the RGD ligands in the crystal structure of the α5β1 binding site, thus affording an improved understanding of ligand–integrin interactions.