Method for integrin ligand discovery

A method for determining whether or not a compound of unknown integrin activity is an allosteric inhibitor of an integrin molecule selected from the group consisting of Very Late Antigen 4 (VLA-4, CD49d/CD29) and Leukocyte Function-Associated Antigen (LFA-1, CD18/CD11a) comprising exposing said integrin molecule to a known competitive ligand of said integrin molecule in the presence of an integrin-epitope binding antibody comprising a reporter moiety, measuring the binding of said antibody to the integrin molecule in the presence of the competitive ligand;

• thereafter exposing said bound integrin molecule in the presence of known competitive ligand and bound antibody to a compound of unknown activity and measuring the binding of the antibody to the integrin molecule, wherein the decrease in binding of antibody to integrin is evidence that the compound of unknown activity is an allosteric inhibitor of integrin, wherein said binding antibody for said VLA-4 integrin molecule is monoclonal antibody clone HUTS-21, said binding antibody for said LFA-1 integrin molecule is monoclonal antibody clone MEM-148, said competitive ligand of said VLA-4 integrin molecule is a compound according to the chemical structure:
• where R″ is a peptide of SEQ ID NO:1 (compound LDV) or SEQ ID NO:2 (compound YLDV), N-(2,6-dichlorobenzoyl)-(L)-4-(2′,6′-bis-methoxyphenyl)phenylalanine (TR14035), 3-(adamantane-1-carbonylamino)-3-(4-ethoxyphenyl) propanoic acid (compound D) or 3-(adamantane-1-carbonylamino)-3-(4-propoxyphenyl) propanoic acid (compound E) and said competitive ligand of said LFA-1 integrin molecule is (2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-succinamic acid) (compound 3 of FIG. 9) or (2-[2,6-Dichloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(3,5-dihydroxy-benzoylamino)-propionic acid) (XVA143), wherein said integrin molecule is expressed on a cell.