Conformational mAb as a Tool for Integrin Ligand Discovery

Department of Chemistry, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Assay and Drug Development Technologies (Impact Factor: 1.53). 09/2009; 7(5):507-15. DOI: 10.1089/adt.2009.0203
Source: PubMed


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.

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    • "This conformation results in the rapid formation of a large number of aggregates in cell suspension (Chigaev et al., 2008), with high tether capture frequency and long tether duration in the rolling assay (Table 2). The exposure of the hybrid domain (LIBS) epitope can be also used to determine VLA-4 ligand binding affinity for unlabeled ligands (Chigaev et al., 2009; Njus et al., 2009). translated into the well documented inability of LFA-1 to support tethering and rolling under natural conditions (Lawrence and Springer, 1991; von Andrian et al., 1991). "
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    • "Previously, we described and characterized in detail a model ligand an LDV-FITC containing small molecule ([14,42-44], and references therein) for the detection of VLA-4 conformational regulation. This VLA-4 specific fluorescent probe was based on a highly specific α4β1-integrin inhibitor BIO1211, which contains the Leu-Asp-Val (LDV) ligand binding motif from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin [17,45]. "
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