Design and evaluation of benzophenone-containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin α(v)β₃-ligand bimolecular interaction

Integrins are cell-surface adhesion molecules involved in mediating cell-extracellular matrix interactions. High-resolution structural data are not available for these heterodimeric receptors. In order to generate tools for photoaffinity scanning of the RGD-binding site of human integrin α(v)β₃, new conformationally constrained ligands were designed. The ligands were based on five different cyclic peptidic or peptidomimetic scaffolds with high affinity for α(v)β₃. A single photoreactive group (a benzophenone moiety) was introduced at different positions relative to the RGD triad. In addition, an ¹²⁵I or a biotin group was introduced as a reporting tag. Twenty-four cyclic ligands were prepared and their binding affinity for α(v)β₃ was determined. In most cases, the modifications resulted in a 5- to 500-fold decrease in affinity relative to the unmodified scaffold. Analogs representing three of the five families were screened for their cross-linking efficiency. Ligands with submicromolar affinities cross- linked efficiently and specifically to the integrin receptor, whereas ligands with weaker affinities gave specific cross-linking, but with lower efficiency. Almost all of the screened ligands cross-linked predominantly to the β₃ subunit.