Frontal affinity chromatography with MS detection of the ligand binding domain of PPAR纬 receptor: Ligand affinity screening and stereoselective ligand-macromolecule interaction

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• 作者：E. Calleri^a ; G. Fracchiolla^b ; R. Montanari^c ; G. Pochetti^c ; A. Lavecchia^d ; F. Loiodice^b ; A. Laghezza^b ; L. Piemontese^b ; G. Massolini^a ; C. Temporini^a ; ^{caterina.temporini@unipv.it}
• 关键词：HPAC ; FAC&ndash ; MS ; Immobilized PPAR纬 receptor ; Binding assay ; Ligand affinity screening ; Enantioselective binding
• 刊名：Journal of Chromatography A
• 出版年：2012
• 期刊代码：47_00219673
• 类别：ch
• 出版时间：6 April, 2012
• 卷：1232
• 期：Complete
• 页码：84-92
• 文件大小：647 K

摘要

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 (PPAR纬) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPAR纬 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 PPAR纬 receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPAR纬-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPAR纬-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 EC₅₀ values reported in literature. The optimized PPAR纬-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-PPAR纬 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 PPAR纬-LBD retained the ability to specifically bind ligands.