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人源一磷酸腺苷激活蛋白激酶其小分子激活剂的发现
发表日期: 2008-06-11
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    体内AMPK活化后在糖代谢、脂代谢中产生的有益效应,使之成为治疗II型糖尿病及肥胖症的一个新的靶点。寻找直接激活AMPK的小分子药物,将是未来治疗II型糖尿病的一条最可行的途径。
    利用建立的AMPK激活剂分子水平高通量筛选模型,通过对国家新药筛选中心化合物库中的3600个化合物进行随机筛选,得到了结构新颖的AMPK小分子激活剂PT1。基于建立的激活剂PT1与人源AMPKα1催化亚基结构的对接模型,通过点突变实验结果表明激活剂PT1可能通过与人源AMPKα1催化亚基上的Glu96残基,Lys156残基,Asp291残基相互作用从而调控了AMPK的活性。PT1能剂量依赖性地增加L6肌细胞内的AMPK及其下游底物ACC的磷酸化水平;PT1处理Hela细胞的结果表明其刺激AMPK和ACC的活性是不依赖于AMPK的上游激酶LKB1的。而且PT1处理人肝癌细胞HepG2后能剂量依赖性地减少细胞内甘油三酯的含量。这些结果表明:得到的AMPK小分子激活剂对于细胞内的糖代谢和脂代谢上的调节作用是有效的,初步证明通过直接激活AMPK来改善细胞内的糖脂代谢这一途径是可行的,进一步动物水平的药效药理学研究仍在进行中。
    本研究由李佳课题组和南发俊课题组共同完成,已发表在J. Biol. Chem.杂志,该化合物的发现对发现新的AMPK激活剂、作为工具阐述AMPK自抑制及构象变化都有积极的意义。
 
Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells
Pang T, Zhang ZS, Gu M, Qiu BY, Yu LF, Cao PR, Shao W, Su MB, Li JY, Nan FJ*, Li J*.
2008 Mar 5 [Epub ahead of print]
AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. Previous report has shown that mammalian AMPK a1 catalytic subunit including autoinhibitory domain, was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in a subunits, we screened a chemical library with inactive human a1394 (a1, residues 1-394), and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK a1394, a1335, a2398, and even heterotrimer a1ss11. Based on PT1-docked AMPK a1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition.
Further studies using L6 myotubes, showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase (ACC), were dose-dependently and time-dependently increased by PT1 without an increase in cellular AMP:ATP ratio. Moreover, in Hela cells deficient with LKB1, PT1 enhanced AMPK hosphorylation, and which can be inhibited by the CaMKKs inhibitor STO-609, and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.
 

(供稿部门:国家新药筛选中心,供稿人:李佳课题组、南发俊课题组)
 
 
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