药学学报, 2014, 49(12): 1658-1664
引用本文:
环奕, 彭军, 王悦, 贾春明, 王克, 王克华, 冯志强, 申竹芳. 非激动剂PPARγ配体的筛选方法建立和应用[J]. 药学学报, 2014, 49(12): 1658-1664.
HUAN Yi, PENG Jun, WANG Yue, JIA Chun-ming, WANG Ke, WANG Ke-hua, FENG Zhi-qiang, SHEN Zhu-fang. Establishment and application of screening methods for non-agonist PPARγ ligand[J]. Acta Pharmaceutica Sinica, 2014, 49(12): 1658-1664.

非激动剂PPARγ配体的筛选方法建立和应用
环奕, 彭军, 王悦, 贾春明, 王克, 王克华, 冯志强, 申竹芳
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
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 receptor γ, 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 (PPARγ response 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.
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     
Establishment and application of screening methods for non-agonist PPARγ ligand
HUAN Yi, PENG Jun, WANG Yue, JIA Chun-ming, WANG Ke, WANG Ke-hua, FENG Zhi-qiang, SHEN Zhu-fang
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
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 Gal4. 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 cells. Therefore, we believe that combining transactivation and binding activity as well as affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator.
Key words:    peroxisome proliferator-activated receptor γ    agonist    ligand    transactivation    binding activity    affinity    
收稿日期: 2014-06-04
基金项目: 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).
通讯作者: 申竹芳
Email: shenzhf@imm.ac.cn
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参考文献:
[1] Camp HS, Li O, Wise SC, et al. Differential activation of peroxisome proliferator-activated receptor-γ by troglitazone and rosiglitazone [J]. Diabetes, 2000, 49: 539-547.
[2] Leesnitzer LM, Parks DJ, Bledsoe RK, et al. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662 [J]. Biochemistry, 2002, 41: 6640-6650.
[3] Guan YF, Hao CM, Cha DR, et al. Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption [J]. Nat Med, 2005, 11: 861-866.
[4] Home PD, Pocock SJ, Beck-Nielsen H, et al. Rosiglitazone evaluated for cardiovascular outcomes in oral agent combination therapy for type 2 diabetes (RECORD): a multicentre, randomised, open-label trial [J]. Lancet, 2009, 373: 2125- 2135.
[5] Shockley KR, Lazarenko OP, Czernik PJ, et al. PPARγ 2 nuclear receptor controls multiple regulatory pathways of osteoblast differentiation from marrow mesenchymal stem cells [J]. J Cell Biochem, 2009, 106: 232-246.
[6] Wei W, Wang XQ, Yang M, et al. PGC1β mediates PPARγ activation of osteoclastogenesis and rosiglitazone-induced bone loss [J]. Cell Metab, 2010, 11: 503-516.
[7] Higgins LS, Mantzoros CS. The development of INT131 as a selective PPARγ modulator: approach to a safer insulin sensitizer [J]. PPAR Res, 2008, doi: 10.1155/2008/936906.
[8] Fujimura T, Sakuma H, Konishi S, et al. FK614, a novel peroxisome proliferator-activated receptor γ modulator, induces differential transactivation through a unique ligand-specific interaction with transcriptional coactivators [J]. J Pharmacol Sci, 2005, 99: 342-352.
[9] Yue LD, Ye F, Du Y, et al. Four screening methods of PPARγ agonists [J]. Chem Life (生命的化学), 2004, 24: 243-244.
[10] Choi JH, Banks AS, Estall JL, et al. Obesity-linked phos­phorylation of PPARγ by cdk5 is a direct target of the anti-diabetic PPARγ ligands [J]. Nature, 2010, 466: 451-456.
[11] Acton JJ, 3rd, Black RM, Jones AB, et al. Benzoyl 2-methyl indoles as selective PPARγ modulators [J]. Bioorg Med Chem Lett, 2005, 15: 357-362.
[12] Choi JH, Banks AS, Kamenecka TM, et al. Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation [J]. Nature, 2011, 477: 477-481.
[13] Amato AA, Rajagopalan S, Lin JZ, et al. GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain [J]. J Biol Chem, 2012, 287: 28169-28179.
[14] Huan Y, Li LY, Liu Q, et al. A cell-based fluorescent glucose transporter assay for SGLT2 inhibitor discovery [J]. Acta Pharm Sin B, 2013, 3: 97-101.