Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells
Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells
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DOI：
10.1074/jbc.m710114200
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发表时间：
2008-06-06
影响因子：
4.8
通讯作者：
Li, Jia
中科院分区：
文献类型：
--
作者：
Pang, Tao;Zhang, Zhen-Shan;Li, Jia
AMP活化蛋白激酶（AMPK）作为一种能量传感器，被认为是治疗II型糖尿病和肥胖的一个有前途的药物靶点。先前的报道表明哺乳动物AMPK α 1催化亚基包括自抑制结构域是无活性的。为了验证小分子可以通过拮抗α亚基中的自抑制来激活AMPK的假设，我们筛选了具有失活的人α 1的化学文库（394）。（α 1，残基1 - 394），并发现了一种新的小分子激活剂，PT1，其剂量依赖性地激活AMPK α 1（394），α 1（335），α 2（398），甚至异源三聚体α 1 β 1 γ 1。基于PT1-docked AMPK α 1亚基结构模型和不同的突变，我们发现PT1可能与自身抑制结构域附近的Glu-96和Lys-156残基相互作用，直接解除自身抑制。使用L6肌管的进一步研究表明，AMPK及其下游底物乙酰辅酶A羧化酶的磷酸化通过PT1呈剂量依赖性和时间依赖性增加，而不增加细胞AMP：ATP比率。此外，在LKB 1缺陷的HeLa细胞中，PT1增强AMPK磷酸化，这可以被钙/钙调素依赖性蛋白激酶抑制剂STO-609和AMPK抑制剂化合物C抑制。PT1还通过激活HepG2细胞中的AMPK以剂量依赖性方式降低肝脂质含量，并且该作用被化合物C减弱。总之，这些数据表明，这种小分子激活剂可以直接激活AMPK通过拮抗在体外和细胞中的自抑制。该化合物突出了发现新型AMPK激活剂的努力，并且可以成为阐明AMPK构象变化和自抑制调节机制的有用工具。
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. A previous report has shown that mammalian AMPK alpha 1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha 1(394) (alpha 1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha 1(394), alpha 1(335), alpha 2(398), and even heterotrimer alpha 1 beta 1 gamma 1. Based on PT1-docked AMPK alpha 1 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, were dose-dependently and time-dependently increased by PT1 without an increase in cellular AMP: ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases 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.