Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay
文献类型：期刊论文
| 作者 | Xu, Yazhou2; Wang, Yunjie2; Xu, Yuan2; Li, Jia3 ; Liao, Hong2; Zhang, Luyong2; Pang, Tao1,2,4
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| 刊名 | JOURNAL OF BIOMOLECULAR SCREENING
 |
| 出版日期 | 2015-08 |
| 卷号 | 20期号:7页码:906-912 |
| 关键词 | AMPK phosphatase PP2A high-throughput screening TR-FRET AMPK activator |
| ISSN号 | 1087-0571 |
| DOI | 10.1177/1087057115585471 |
| 文献子类 | Article |
| 英文摘要 | AMP-activated protein kinase (AMPK), a conserved heterotrimeric kinase, serves as an energy sensor maintaining energy balance at both cellular and whole-body levels and plays multiple beneficial roles in carbohydrate and lipid metabolism, which makes AMPK an attractive target for diabetes and other metabolic disorders. To date, establishment of the physiologically relevant biochemical assay for AMPK has not been reported. Here we developed a phosphorylated AMPK protection assay based on a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, using the protein phosphatase 2A (PP2A) to dephosphorylate AMPK. The partially dephosphorylated AMPK by PP2A had lower activity than phosphorylated AMPK. This specific TR-FRET assay for AMPK was optimized in the 384-well format and produced similar EC50 values for AMPK activators AMP and A769662 and a similar IC50 value for AMPK inhibitor compound C, as previously reported. Under the optimized conditions, the assay Z factor calculated over 160 data points has an optimal value greater than 0.5, which is suitable for high-throughput screening. In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions. |
| WOS关键词 | ACTIVATED PROTEIN-KINASE ; FATTY-ACID OXIDATION ; METABOLIC SYNDROME |
| 资助项目 | National Natural Science Foundation of China[21402241] ; National Natural Science Foundation of China[81320108029] ; Program for Jiangsu Province Innovative Research Team[00000000] ; Natural Science Foundation of Jiangsu Province[BK20130653] ; Open Project Program of the State Key Laboratory of Drug Research[SIMM1403KF-02] ; Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University[SKLNMKF201407] ; Fundamental Research Funds for the Central Universities[JKZD2013006] ; Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry[00000000] |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Chemistry |
| 语种 | 英语 |
| WOS记录号 | WOS:000358266100009 |
| 出版者 | SAGE PUBLICATIONS INC |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/276458]  |
| 专题 | 国家新药筛选中心 中科院受体结构与功能重点实验室 新药研究国家重点实验室 药物安全性评价中心 |
| 通讯作者 | Pang, Tao |
| 作者单位 | 1.Georgetown Univ, Med Ctr, Dept Physiol & Pharmacol, Washington, DC 20007 USA 2.China Pharmaceut Univ, Jiangsu Key Lab Drug Screening, State Key Lab Nat Med, Nanjing 210009, Peoples R China; 3.Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Natl Ctr Drug Screening, Shanghai 200031, Peoples R China; 4.China Pharmaceut Univ, Jiangsu Key Lab Drug Discovery Metab Dis, Nanjing 210009, Peoples R China; |
| 推荐引用方式 GB/T 7714 | Xu, Yazhou,Wang, Yunjie,Xu, Yuan,et al. Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay[J]. JOURNAL OF BIOMOLECULAR SCREENING,2015,20(7):906-912. |
| APA | Xu, Yazhou.,Wang, Yunjie.,Xu, Yuan.,Li, Jia.,Liao, Hong.,...&Pang, Tao.(2015).Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay.JOURNAL OF BIOMOLECULAR SCREENING,20(7),906-912. |
| MLA | Xu, Yazhou,et al."Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay".JOURNAL OF BIOMOLECULAR SCREENING 20.7(2015):906-912. |
入库方式： OAI收割
来源：上海药物研究所
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