title: Relative binding affinities of integrin antagonists by equilibrium dialysis and liquid chromatography-mass spectrometry.
abstract: The integrin αvβ6 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 αvβ6 inhibitor screening assay. As a preliminary experiment, an established peptidomimetic inhibitor of the integrin was dialyzed against αvβ6, 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 αvβ6, 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 αvβ6 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 αvβ6 integrin antagonist screening assay for mixtures of putative ligands.
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: William J
authors.initials: WJ
authors.lastname: Tipping
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: Nkazimulo
authors.initials: N
authors.lastname: Tshuma
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: James
authors.initials: J
authors.lastname: Adams
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: Harvey T
authors.initials: HT
authors.lastname: Haywood
authors.affiliation: Fibrosis Discovery Performance Unit, GlaxoSmithKline, Medicines Research Centre , Gunnels Wood Road, Stevenage SG1 2NY, U.K.
authors.forename: James E
authors.initials: JE
authors.lastname: Rowedder
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: M Jonathan
authors.initials: MJ
authors.lastname: Fray
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: Thomas
authors.initials: T
authors.lastname: McInally
authors.affiliation: Fibrosis Discovery Performance Unit, GlaxoSmithKline, Medicines Research Centre , Gunnels Wood Road, Stevenage SG1 2NY, U.K.
authors.forename: Simon J F
authors.initials: SJ
authors.lastname: Macdonald
authors.affiliation: School of Chemistry, University of Nottingham , University Park, Nottingham NG7 2RD, U.K.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
keywords: Idiopathic pulmonary fibrosis; equilibrium dialysis; integrin; liquid chromatography−mass spectrometry; αvβ6