Conformational mAb as a tool for integrin ligand discovery. Academic Article uri icon

start page

  • 507

end page

  • 515

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.

date/time value

  • 2009

Digital Object Identifier (DOI)

  • 10.1089/adt.2009.0203

PubMed Identifier

  • 19754304

volume

  • 7

number

  • 5

keywords

  • Antibodies, Monoclonal
  • Antigens, CD29
  • Binding, Competitive
  • Epitopes
  • Flow Cytometry
  • Fluorescein-5-isothiocyanate
  • Fluorescent Dyes
  • Humans
  • Integrins
  • Kinetics
  • Ligands
  • Protein Conformation
  • U937 Cells