Design and evaluation of benzophenone-containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin alpha(v)beta(3)-ligand bimolecular interaction

Integrins are cell-surface adhesion molecules involved in mediating cell-ex tracellular matrix interactions. High-resolution structural data are not av ailable for these heterodimeric receptors. In order to generate tools for p hotoaffinity scanning of the RGD-binding site of human integrin alpha(v)bet a(3), new conformationally constrained ligands were designed. The ligands w ere based on five different cyclic peptidic or peptidomimetic scaffolds wit h high affinity for alpha(v)beta(3). A single photoreactive group (a benzop henone moiety) was introduced at different positions relative to the RGD tr iad. In addition, an I-125 Or a biotin group was introduced as a reporting tag. Twenty-four cyclic ligands were prepared and their binding affinity fo r alpha(v)beta(3), was determined. In most cases, the modifications resulte d in a 5- to 500-fold decrease in affinity relative to the unmodified scaff old. Analogs representing three of the five families were screened for thei r cross-linking efficiency. Ligands with submicromolar affinities cross-lin ked efficiently and specifically to the integrin receptor, whereas ligands with weaker affinities gave specific cross-linking, but with lower efficien cy. Almost all of the screened ligands cross-linked predominantly to the be ta(3) Subunit.