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建立体外活性评价方法考察化合物对过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)转录激活活性和结合活性。首先通过质粒构建,分别建立PPARγ反应原件(PPARγresponse element,PPRE)介导报告基因表达的转录激活筛选方法以及PPARγ配体结合区(ligand binding domain,LBD)与酵母转录激活因子Gal4组成的嵌合受体在细胞水平的结合活性筛选方法。并采用基于时间分辨-荧光共振能量转移(time resolved-fluorescence resonance energy transfer,TR-FRET)技术的PPARγ竞争性结合检测方法,考察化合物或药物对PPARγ的亲和力。采用以上3种不同的体外活性评价方法,评价不同PPARγ配体的效能和作用特点,并发现了一个新的苯磺酰胺衍生化合物,ZLJ01,具有类似于已知PPARγ激动剂的结合活性及亲和力,但无PPRE介导的转录激活活性。初步体外降糖活性检测发现,ZLJ01可促进肝细胞胰岛素依赖的葡萄糖摄取。综上所述,结合转录激活活性和亲和力的评价方法,可用于筛选非激动剂PPARγ配体,以发现新型PPARγ调节剂。
The in vitro activity assay was established to investigate the transcriptional activation and the binding activity of the compound to peroxisome proliferator-activated receptorγ (PPARγ). Firstly, the transcriptional activation screening method of PPARγresponse element (PPRE) -mediated reporter gene expression and the chimeric receptor consisting of PPARγ ligand binding domain (LBD) and yeast transcriptional activator Gal4 Binding activity of cell at the cellular level by screening methods. The PPARγ competitive binding assay based on the time resolved-fluorescence resonance energy transfer (TR-FRET) technique was used to investigate the affinity of the compounds or drugs for PPARγ. Using the above three different in vitro activity evaluation methods, the potency and action characteristics of different PPARγ ligands were evaluated and a new benzenesulfonamide-derived compound, ZLJ01, having similar binding activity and affinity as known PPARγ agonists, But no PPRE-mediated transcriptional activation. Preliminary in vitro hypoglycemic activity test found that, ZLJ01 can promote insulin-dependent glucose uptake in liver cells. In summary, combined with the evaluation of transcriptional activation activity and affinity, it can be used to screen non-agonist PPARγ ligands to find novel PPARγ modulators.