A Potent Integrin Antagonist from a Small Library of Cyclic RGD Pentapeptide Mimics Including Benzyl‐Substituted Azabicycloalkane Amino Acids

Abstract

A small library of cyclic RGD pentapeptide mimics, including benzyl-substituted azabicycloalkane amino acids, was synthesized with the aim of developing active and selective integrin antagonists. In vitro binding assays established one particular compound with affinity for both the alpha(v)beta(3) and the alpha(v)beta(5) integrins. The synthesis in solution and the in vitro screening of these RGD derivatives, as well as the determination of the conformational properties of the integrin ligands by spectroscopic and computational methods are described.

STD and trNOESY NMR Study of Receptor–Ligand Interactions in Living Cancer Cells

“…As already reported, [7] the conformational preferences of these compounds in the free state are modulated by the configurations of the bicyclic lactam, and so are their affinities for a v b 3 integrin. These activities were estimated by competition experiments with [ 125 I]echistatin for binding to purified a v b 3 receptors.…”

“…The binding epitope, as determined from the STD spectra, agrees with the docking-derived binding model [7] for ligand 1 shown in Figure 3 A. In the docked orientation, ligand 1 interacts with the receptor site through ionic interactions (ligand Arg-guanidinium group with integrin a v -Asp218 and ligand Asp-carboxylate with the bivalent ion in the integrin b 3 subunit), a p-p interaction (between the ligand benzylic group and the aromatic ring of b 3 -Tyr122) and a complex hydrogen bond network that includes the Asp-NH and the C=O of b 3 -Arg216.…”

“…[7] We show that the interactions between these small ligands and membrane-bound proteins can be observed by NMR directly in H 2 O buffer suspensions of living cells. The data allowed us to identify (by STD) the portions of the ligands in closest contact with the protein and to define (by trNOESY) the preferred conformations of the bound ligands.…”

“…These activities were estimated by competition experiments with [ 125 I]echistatin for binding to purified a v b 3 receptors. [7] The resulting IC 50 values for 1 and 2 were 6.4 and 154 nm, respectively. Adhesion assay tests were also performed for 1 with ECV304 bladder cancer cells, a cell line derived from solid tumours that highly expresses a v b 3 integrin.…”

“…The data allowed us to identify (by STD) the portions of the ligands in closest contact with the protein and to define (by trNOESY) the preferred conformations of the bound ligands. These NMR techniques [8] focus on the NMR signals of the ligands, exploiting NOE effects between protein and ligands in the proteinligand complexes.The ligands, the cyclic RGD pentapeptide mimics 1 and 2 (Scheme 1), previously described by workers from our laboratory, [7] are each characterized by a benzylic group connected to carbon 3 of the lactam unit, but differ in the configurations of the C-3 and C-7 lactam stereocentres. As already reported, [7] the conformational preferences of these compounds in the free state are modulated by the configurations of the bicyclic lactam, and so are their affinities for a v b 3 integrin.…”