Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: Ligand affinity screening and stereoselective ligand-macromolecule interaction
Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: Ligand affinity screening and stereoselective ligand-macromolecule interaction
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DOI：
10.1016/j.chroma.2011.10.037
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发表时间：
2012-04-06
影响因子：
4.1
通讯作者：
Temporini, C.
中科院分区：
文献类型：
--
作者：
Calleri, E.;Fracchiolla, G.;Temporini, C.
在这项研究中，我们报告的开发新的色谱工具的结合研究的基础上的γ亚型配体结合域（LBD）的过氧化物酶体增殖物激活受体（PPAR γ）属于核受体超家族的配体激活的转录因子。PPAR γ亚型在脂肪细胞、肌肉和巨噬细胞的功能中起重要作用，对2型糖尿病、血脂异常、动脉粥样硬化和心血管疾病有直接影响。为了建立合适的固定化化学，首先将PPAR γ受体的LBD共价固定到氨丙基二氧化硅颗粒的表面上以产生用于区域洗脱实验的PPAR γ-二氧化硅柱，然后在不同的固定化条件下将其固定到开放管状（OT）毛细管的表面上以产生PPAR γ-OT毛细管。该毛细管用于前端亲和色谱-质谱联用（FAC-MS）实验，以确定一系列手性贝特类化合物的相对结合亲和力。这些衍生物获得的相对亲和力顺序与文献中报道的EC 50值一致。通过测定两种化合物的Kd值，对优化的PPARgamma-OT毛细管柱进行了验证。已知的手性贝特类化合物的结合中的立体选择性的作用，第一次进行了详细的研究，通过分析两个对映体选择性对LBD-PPAR γ毛细管FAC和一个特征的两个台阶的前沿轮廓，来自两个饱和事件的结果。所有获得的数据表明，PPAR γ-LBD的固定化形式保留了特异性结合配体的能力。(C)2011 Elsevier B. V.保留所有权利。
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 gamma) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPAR gamma 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 gamma receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPAR gamma-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPAR gamma-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 gamma-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 gamma 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 gamma-LBD retained the ability to specifically bind ligands. (C) 2011 Elsevier B.V. All rights reserved.