Relative binding affinities of integrin antagonists by equilibrium dialysis and liquid chromatography-mass spectrometry

The integrin α_vβ₆ is a potential target for treatment of idiopathic pulmonary fibrosis (IPF). Equilibrium dialysis (ED) was investigated for its ability to report ligand binding in an α_vβ₆ inhibitor screening assay. As a preliminary experiment, an established peptidomimetic inhibitor of the integrin was dialyzed against α_vβ₆, and the fraction bound (f_b) and percentage saturation determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Quantitation of the inhibitor in the two chambers of the ED cartridge revealed an uneven distribution in the presence of α_vβ₆, corresponding to near saturation binding to the protein (93 ± 3%), while the control (without integrin) showed an equal partitioning of the inhibitor on either side of the dialysis membrane. A competitive ED assay with a 12 component mixture of antagonists was conducted, and the results compared with an established cell adhesion assay for quantifying α_vβ₆ inhibition of individual antagonists. Compounds clustered into three groupings: those with pIC₅₀ values between ca. 5.0 and 5.5, which possessed ED f_b values indistinguishable from the controls, those with pIC₅₀s of 6.5 ± 0.2, which exhibited detectable integrin binding (f_b 13-25%) in the ED assay, and a single compound of pIC₅₀ 7.2 possessing an f_b value of 38%. A good correlation between ED-derived f_b and pIC₅₀ was observed despite the two assays utilizing quite different outputs. These results demonstrate that ED with LC-MS detection shows promise as a rapid α_vβ₆ integrin antagonist screening assay for mixtures of putative ligands.