非激动剂PPARγ配体的筛选方法建立和应用  

Establishment and application of screening methods for non-agonist PPARγ ligand

作　　者：环奕 彭军 王悦 贾春明 王克 王克华 冯志强 申竹芳 

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室,北京100050

出　　处：《药学学报》2014年第12期1658-1664,共7页Acta Pharmaceutica Sinica

基　　金：国家新药创制重大专项基金(2012ZX09301002-004);中央级公益性科研院所基本科研业务费专项基金(2013CHX18)

摘　　要：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。In-vitro assay methods were established to evaluate transactivation and binding activity of compounds on peroxisome proliferator-activated receptor γ （PPARγ）. Firstly, plasmids were constructed for transactivation assay of PPARγ response element （PPRE） triggered reporter gene expression, and for cell-based binding activity assay of the chimeric receptor, which was fused with PPARγ ligand binding domain （LBD） and yeast transcriptional activator Ga14. Secondly, by using PPARγ competitive binding assay based on time resolved-fluorescence resonance energy transfer （TR-FRET）, affinities of compounds and drugs to PPARγ were evaluated. In application of these above methods, the PPARγ activating potency and characteristics of different compounds were evaluated, and a novel benzeneselfonamide derivative, ZLJ01, was found to have comparable binding activity and affinity with the well-known PPARγ, agonist, but lack of PPRE mediated transactivation activity. In preliminary study on in-vitro hypoglycemic activity, ZLJ01 was found to promote insulin-stimulated glucose uptake by liver ceils. Therefore, we believe that combining transactivation and binding activity as wellas affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator.

关 键 词：过氧化物酶体增殖物激活受体Γ 激动剂 配体 转录激活 结合活性 亲和力 

分 类 号：R943[医药卫生—药剂学]