title: Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: ligand affinity screening and stereoselective ligand-macromolecule interaction.
abstract: In this study we report the development of new chromatographic tools for binding studies based on the gamma isoform ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARγ) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ subtype plays important roles in the functions of adipocytes, muscles, and macrophages with a direct impact on type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In order to set up a suitable immobilization chemistry, the LBD of PPARγ receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPARγ-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPARγ-OT capillaries following different immobilization conditions. The capillaries were used in frontal affinity chromatography coupled to mass spectrometry (FAC-MS) experiments to determine the relative binding affinities of a series of chiral fibrates. The relative affinity orders obtained for these derivatives were consistent with the EC(50) values reported in literature. The optimized PPARγ-OT capillary was validated by determining the K(d) values of two selected compounds. Known the role of stereoselectivity in the binding of chiral fibrates, for the first time a detailed study was carried out by analysing two enantioselective couples on the LBD-PPARγ capillary by FAC and a characteristic two-stairs frontal profile was derived as the result of the two saturation events. All the obtained data indicate that the immobilized form of PPARγ-LBD retained the ability to specifically bind ligands.
authors: Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, 27100 Pavia, Italy.
authors: E
authors: E
authors: Calleri
authors: G
authors: G
authors: Fracchiolla
authors: R
authors: R
authors: Montanari
authors: G
authors: G
authors: Pochetti
authors: A
authors: A
authors: Lavecchia
authors: F
authors: F
authors: Loiodice
authors: A
authors: A
authors: Laghezza
authors: L
authors: L
authors: Piemontese
authors: G
authors: G
authors: Massolini
authors: C
authors: C
authors: Temporini
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D055808:Drug Discovery / Q000379:methods; D056945:Hep G2 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D047495:PPAR gamma / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D055767:Immobilized Proteins; D008024:Ligands; D046911:Macromolecular Substances; D047495:PPAR gamma