title: Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with mass spectrometry detection.
abstract: The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of frontal affinity chromatography coupled to mass spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments toward new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes.
authors: Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, 27100 Pavia, Italy.
authors: C
authors: C
authors: Temporini
authors: G
authors: G
authors: Pochetti
authors: G
authors: G
authors: Fracchiolla
authors: L
authors: L
authors: Piemontese
authors: R
authors: R
authors: Montanari
authors: R
authors: R
authors: Moaddel
authors: A
authors: A
authors: Laghezza
authors: F
authors: F
authors: Altieri
authors: L
authors: L
authors: Cervoni
authors: D
authors: D
authors: Ubiali
authors: E
authors: E
authors: Prada
authors: F
authors: F
authors: Loiodice
authors: G
authors: G
authors: Massolini
authors: E
authors: E
authors: Calleri
mesh_terms: D002151:Calorimetry; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D055808:Drug Discovery / Q000379:methods; D055767:Immobilized Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D047493:PPAR alpha / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D047495:PPAR gamma / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results
chemical_list: D055767:Immobilized Proteins; D008024:Ligands; D047493:PPAR alpha; D047495:PPAR gamma