Insights into molecular bases of cadherin : small ligands interaction by STD : NMR

Cadherins are calcium-dependent adhesive membrane proteins involved in intercellular junctions (“adherent junctions”) and overexpressed in several solid tumors.1 Classical cadherins are characterized by an N–terminal extracellular domain, constituted by five EC1-EC5 domains rigidified at the interface by calcium ions, by a single pass transmembrane region and by a conserved C–terminal cytoplasmic tail. They mediate cell–cell adhesion by forming homophilic dimers between the N–terminal extracellular domains of two cadherins localized on adjacent cells. Cadherins are known to play a major role in physiological and pathological processes such as tissue morphogenesis and stability, as well as in the immune system regulation.2 Cadherins’ dysregulation has been shown to contribute to different aspects of tumor progression.3 Thus, cadherins are becoming valuable diagnostic and prognostic indicators, as well as potential therapeutic targets. Despite a growing interest in the field, the rational design of small ligands targeting cadherins protein –protein interactions is still in its infancy. So far, only a N (neuronal subtype) – cadherins antagonist cyclic peptide, ADH-1, has entered clinical trials as a systemic anticancer agent.4 Our group designed and synthesized a small library of peptidomimetics based on the tetrapeptide sequence Asp1 – Trp2 – Val3 – Ile4 (DWVI) of the N–terminal “adhesive arm” (identified by the crystallographic structures) of cadherins. These compounds were obtained by replacing the central dipeptide Trp2 – Val3 unit of the DWVI adhesive motif with several scaffolds developed in our laboratories. Critical aspects concerning the initial recognition process of cadherins and complete adhesion mechanisms as well as structural and in silico studies of small-molecule – cadherin interactions need to be fully investigated. So, we began structural investigation by NMR of the interaction between cadherins’ constructs and some peptidomimetics. NMR techniques, tr – NOESY and STD,5 are being applied in order to detect and study the binding epitopes of these compounds with the EC1 – EC2 construct of the epithelial subtype E – cadherins. NMR and X-ray experiments are being used to obtain insights into peptidomimetics – cadherin interactions at a molecular level, providing information on the ligand epitope as well as on the receptor - bound conformation.