噻唑衍生物WSF-SN-10激活AMPK的机制研究

目的:在噻唑衍生物中筛选新型AMPK激活剂并探究其激活AMPK的分子机制,以期找到副作用少的II型糖尿病治疗药物。方法:用14种噻唑衍生物处理293T细胞,用Western Blot筛选能显著提高AMPK磷酸化水平的化合物;用HPLC和FACS分别检测该化合物对细胞内AMP/ATP比值和Ca~(2+)浓度的影响,探究其激活AMPK的分子机制。结果:WSF-SN-10(2-(2-(6,6-二甲基双环[3,1,1]庚-2-亚基)肼基)-4-(4-氰基苯基)噻唑)为14种噻唑衍生物中活性最强的AMPK激活剂;20μM下WSF-SN-10激活AMPK的活性最强;用20μM WSF-SN-10处理293T细胞后,细胞内的AMP/ATP比值和LKB1的磷酸化水平分别上升至空白对照的1.94和3.04倍,同时Ca~(2+)浓度无明显变化,说明WSF-SN-10通过增加细胞内的AMP/ATP比值来激活AMPK。结论:噻唑衍生物WSF-SN-10能抑制细胞内的ATP合成来间接激活AMPK,是治疗II型糖尿病和肥胖症的潜在药物。

The Mechanism of AMPK Activation by WSF-SN-10, a Thiazole Derivative

ABSTRACT Objective: AMP-activated protein kinase (AMPK) is a prime therapeutic candidate for obesity-related metabolic disorders. It is activated (phosphorylated at Thr172) mainly by increasing the ratio of AMP/ATP and increasing intracellular Ca²⁺ concentration. Biguanides and Thiazolidinediones are both AMPK activators used in the treatment of type II diabetes. However, they are associated with various undesirable side effects. In this study, we search among thiazole derivatives for the strongest AMPK activator and investigate its molecular mechanism of AMPK activation, to identify potent antidiabetic drugs with few side effects. Methods: 293T cells were treated with 14 thiazole derivatives and Western Blot was used to find out the strongest AMPK activator. HPLC and FACS were used respectively to measure intracellular ratio of AMP/ATP and intracellular Ca²⁺ concentration, to investigate its molecular mechanism of AMPK activation. Results: WSF-SN-10 was the strongest AMPK activator among the 14 thiazole derivatives; WSF-SN-10 increased the ratio of AMP/ATP and the level of LKB1 phosphorylation, but did not increase Ca²⁺ concentration in 293T cells. Therefore, WSF-SN-10 activates AMPK by increasing intracellular ratio of AMP/ATP. Conclusion: WSF-SN-10 is a novel AMPK activator which activates AMPK by inhibiting intracellular ATP synthesis, making it a potential drug candidate for the treatment of Type II diabetes and obesity.