新型调脂分子WS070117激活靶蛋白AMPK作用方式探究

The new compound, WS070117 (abbreviated as 117), derived from natural products with intellectual property rights of China, is the national class 1.1 lipid-regulating candidate drug, which is developed by the applicant team independently. 117 has been approved to preclinical study and named as one of the twenty most outstanding chemical candidate drugs in the "eleventh five years" major project candidate drug acceptance, In the previous study funded by national natural science foundation, AMP-activated protein kinase (AMPK) was proved to be the lipid-regulating target of 117 in vivo. .In this study, we are going to fine describe the path and pattern of 117 activating AMPK , not only to provide theoretical support for the compound's clinical study, but also to supply experimental evidence for the expansion of the basic theory of lipid-regulating molecular pharmacology. . . LC-MS/MS hyphenated technique is employed to determine the molecular form of 117 activating AMPK in vivo. Then, the data about the transmembrance transport, activation of intracellular molecules and the drug target by 117 will be collected by using methods, such as Surface-Enhanced Raman Scattering, Fluorescence Resonance Energy Transfer, Radionuclide Labeling etc. On the basis of molecular docking results and molecules interaction key parameters acquired by Isothermal Titration Calorimetry, Cold Spray Drying Mass Spectrometry and Point Mutation etc, this study is planning to bulid the molecular model of compound activating drug target, further to make clear the action mode of 117 activating AMPK.

源于天然产物、具有我国知识产权的新结构化合物WS070117（简称117）是申请者团队自主研制的国家1.1类调血脂候选新药，在国家"十一五"重大专项候选药物项目验收中被评为全国最优秀20个化学候选药物之一进入临床前研究。申请者在前期国家自然基金项目资助下确认AMP激活蛋白激酶（AMPK）是117调血脂作用的体内药靶。.本研究拟从分子层面精细描述117激活药靶行为路径和效应模式，不仅为该化合物进入临床研究提供理论支持，而且为分子药理学基础理论领域的拓展提供实验依据。.采用液质联用技术确定117体内激活AMPK的分子活性形式；以表面增强拉曼散射、荧光能量共振转移、核素标记等技术手段捕捉117细胞跨膜转运、胞内分子活化、激活药靶蛋白等行为信息；依据分子对接结果，以等温滴定量热、冷喷雾干燥质谱、点突变等技术获取分子间相互作用关键参数，构建化合物激活靶蛋白分子模型，阐明117激活AMPK的作用方式。

源于天然产物、具有我国知识产权的新结构化合物WS070117（又名IMM-H007）是申请者团队自主研制的国家1.1类调血脂候选新药，申请者在前期国家自然基金项目资助下确认AMP激活蛋白激酶（AMPK）是WS070117调血脂作用的体内药靶。本研究拟从分子层面精细描述WS070117激活药靶行为路径和效应模式，不仅为该化合物进入临床研究提供理论支持，而且为分子药理学基础理论领域的拓展提供实验依据。.研究结果总结如下：采用同位素标记酶学活性检测技术、免疫印迹技术和间接量热技术发现，M1可呈时间和剂量依赖性激活AMPK，同时抑制脂肪酸氧化和合成限速酶ACC的活性，抑制胞内甘油三酯的累积，促进C57BL/6J小鼠全身脂肪酸氧化。采用siRNA干扰技术、免疫印迹技术和高效液相技术发现，M1激活AMPK/ACC信号通路不依赖于上游激酶LKB1和CaMKKβ以及细胞能量状态（AMP/ATP）的改变，提示M1可能是AMPK直接激动剂。采用同位素标记酶学活性检测和免疫印迹技术发现，在分子水平M1不能直接激活AMPK。于是通过HPLC-MS/MS分析M1胞内代谢产物，发现一种新型小分子化合物MP，已申请国家新物质发明专利。采用腺苷激酶小分子抑制剂与M1共处理，MP生成明显受阻，此时M1激活AMPK，抑制甘油三酯累积的作用消失，说明MP是M1发挥作用的主要活性分子。同时我们在体内外研究中发现MP自身也可以激活AMPK/ACC信号通路，抑制胞内甘油三酯累积，促进C57BL/6J小鼠全身脂肪酸氧化。进一步研究发现MP在分子水平可以别构激活AMPK且抑制磷酸酶PP2C对其α亚基上Thr172去磷酸化作用，与AMP竞争性调控AMPK活性。于是通过构建五种AMPKγ亚基突变体稳转细胞系发现MP确实结合在AMPKγ亚基上，其中R69、R151和R298三个位点突变可以明显抑制MP增加AMPK磷酸化表达。.根据以上实验结果申请者在Oncotarget, Journal of Proteome Research，Journal of Lipid Research，Plos One，Vascular Pharmacology等SCI源国际知名期刊发表文章6篇。申请国家新物质发明专利2项，举办2次国际性会议，参加国际性学术会议并做大会报告3次，培养博士和硕士研究生共计15人。