Conformational mAb as a tool for integrin ligand discovery

Ben H. Njus, Alexandre Chigaev, Anna Waller, Danuta Wlodek, Liliana Ostopovici-Halip, Oleg Ursu, Wei Wang, Tudor I. Oprea, Cristian G. Bologa, Larry A. Sklar

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

41-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-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 EC50 for antibody binding was equal to previously reported Kd 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. EC50 values for HUTS-21 binding showed good correlation with Kis 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.

Original languageEnglish (US)
Pages (from-to)507-515
Number of pages9
JournalAssay and Drug Development Technologies
Volume7
Issue number5
DOIs
StatePublished - Oct 1 2009
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

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