title: Conformational mAb as a tool for integrin ligand discovery.
abstract: alpha(4)beta(1)-Integrin (very late antigen-4 (VLA-4)) mediates cell adhesion to cell surface ligands (VCAM-1). Binding of VLA-4 to VCAM-1 initiates rolling and firm adhesion of leukocytes to vascular endothelium followed by the extravasation into the tissue. VLA-4-dependent adhesion plays a key role in controlling leukocyte adhesive events. Small molecules that bind to the integrin ligand-binding site and block its interaction with natural ligands represent promising candidates for treatment of several diseases. Following a flow cytometric screen for small molecule discovery, we took advantage of a conformationally sensitive anti-beta(1)-integrin antibody (HUTS-21) and a small LDV-containing ligand (LDV-FITC) with known affinity to study binding affinities of several known and recently discovered integrin ligands. We found that binding of the LDV-containing small molecule induced exposure of HUTS-21 epitope and that the EC(50) for antibody binding was equal to previously reported K(d) for fluorescent LDV (LDV-FITC). Thus, binding of HUTS-21 can be used to report ligand-binding site occupancy. We studied binding of two known integrin ligands (YLDV and TR14035), as well as of two novel compounds. EC(50) values for HUTS-21 binding showed good correlation with K(i)s determined in the competition assay with LDV-FITC for all ligands. A docking model suggests a common mode of binding for the small molecule VLA-4 ligands. This novel approach described here can be used to determine ligand-binding affinities for unlabeled integrin ligands, and can be adapted to a high-throughput screening format for identification of unknown integrin ligands.
authors.affiliation: Department of Chemistry, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
authors.forename: Ben H
authors.initials: BH
authors.lastname: Njus
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chigaev
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.forename: Danuta
authors.initials: D
authors.lastname: Wlodek
authors.forename: Liliana
authors.initials: L
authors.lastname: Ostopovici-Halip
authors.forename: Oleg
authors.initials: O
authors.lastname: Ursu
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Tudor I
authors.initials: TI
authors.lastname: Oprea
authors.forename: Cristian G
authors.initials: CG
authors.lastname: Bologa
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001667:Binding, Competitive; D000939:Epitopes; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D019012:Integrin beta1 / Q000187:drug effects; D016023:Integrins / Q000187:drug effects*; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D020298:U937 Cells
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D005456:Fluorescent Dyes; D019012:Integrin beta1; D016023:Integrins; D008024:Ligands; D016650:Fluorescein-5-isothiocyanate