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

ABSTRACT: The integrin ?v?6 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 ?v?6 inhibitor screening assay. As a preliminary experiment, an established peptidomimetic inhibitor of the integrin was dialyzed against ?v?6, 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 ?v?6, 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 ?v?6 inhibition of individual antagonists. Compounds clustered into three groupings: those with pIC 50 values between ca. 5.0 and 5.5, which possessed ED f b values indistinguishable from the controls, those with pIC 50s of 6.5 ± 0.2, which exhibited detectable integrin binding (f b 13-25%) in the ED assay, and a single compound of pIC 50 7.2 possessing an f b value of 38%. A good correlation between ED-derived f b and pIC 50 was observed despite the two assays utilizing quite different outputs. These results demonstrate that ED with LC-MS detection shows promise as a rapid ?v?6 integrin antagonist screening assay for mixtures of putative ligands.

SUBMITTER: Tipping WJ 

PROVIDER: S-EPMC4329580 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature