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

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 chromatog. 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 biochromatog. systems were used in both ranking and Kd expts. toward new ureidofibrate-like dual agonists for subtype selectivity ratio detn. In order to validate the system, the Kd values detd. by frontal anal. chromatog. were compared to the affinity consts. obtained by ITC expts. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes.