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鞘磷脂合成酶蛋白结构设计、小分子抑制剂发现及活性评价

发布时间:2019-03-31 17:40
【摘要】:鞘磷脂合成酶(SMS)是脂代谢通路中鞘磷脂生物合成最后一步关键酶。SMS以卵磷脂和神经酰胺为底物,催化反应生成鞘磷脂和甘油二酯。神经酰胺和二酰基甘油是细胞内重要的第二信使,它们在细胞内浓度的改变与细胞信号传导以及细胞凋亡密切相关。最近大量的研究表明,鞘磷脂合成酶与动脉粥样硬化症密切相关,抑制鞘磷脂合成酶可以有效缓解动脉粥样硬化症。因此鞘磷脂合成酶可以作为一个重要的、潜在的抗动脉粥样硬化药物的靶点。目前尚无选择性的鞘磷脂合成酶抑制剂,发现第一代SMS酶抑制剂具有前沿性和创新性,这对于深入研究鞘磷脂合成酶在动脉粥样硬化的作用机制和开发新一类抗动脉粥样硬化药物有着重要的价值。 为了寻找第一代SMS小分子抑制剂,我们以人类SMS1为研究对象,以同源模建方法模拟hSMSl三维结构,采用选择高度三维空间拓扑结构相似性蛋白的策略,选取了Escherichia coli GlpG (PDB编号:2IC8)作为模板蛋白,运用Discovery Studio2.0的Modeller模块进行同源模建。对于胞外重要的Loop2,则选取了同源性高的蛋白1BW0作模板模建。分子动力学优化后的结构能够很好的解释天然底物卵磷脂和鞘磷脂与hSMSl的作用机制。随即用验证后的hSMSI三维结构对SPECS库进行虚拟筛选,使用Gold初筛,然后Glide精细筛选,对200余个命中物根据结构多样性手动挑选出了93个分值高的化合物。 在本实验室建立起来的SMS酶活性筛选平台上,我们检验了93个化合物的SMS酶抑制活性,成功的得到了两个先导化合物34和72。根据化合物34的结构,进行结构相似性搜索,成功的找到化合物104、105和107,对化合物104和105的合成证实了这个骨架是一类有效的SMS酶抑制剂结构。经对化合物72的合成和结构鉴定,证实SPECS库提供的是未知成分的混合物,已经成功分离出了两个活性成分。从化合物72结构中还找到了一个SMS激动剂。 本课题还从鞘磷脂天然底物结构出发,进行结构改造,合成了17个含苯环结构的底物类似物,从中发现了4个活性化合物,并考察了其初步的构效关系。 我们还对文献中关于MS-209是潜在SMS抑制剂的报道进行了验证。经合成和活性测试,证实MS-209并不是SMS的抑制剂。
[Abstract]:Sphingomyelin synthetase (SMS) is a key enzyme in the biosynthesis of sphingomyelin in lipid metabolism pathway, which is catalyzed by lecithin and ceramide to produce sphingomyelin and glycerodiester. Ceramide and diacylglycerol are important second messengers in cells. Their intracellular concentrations are closely related to cell signal transduction and apoptosis. A large number of recent studies have shown that sphingomyelin synthetase is closely related to atherosclerosis. Inhibition of sphingolipin synthetase can effectively alleviate atherosclerosis. Therefore, sphingolipin synthetase can be used as an important, potential target for anti-atherosclerosis drugs. At present, there is no selective inhibitor of sphingomyelin synthetase. It is found that the first generation of SMS enzyme inhibitors are forward and innovative. It is of great value to study the mechanism of sphingomyelin synthetase in atherosclerosis and to develop a new kind of antiatherosclerotic drugs. In order to search for the first generation of SMS inhibitors, we took human SMS1 as the research object, simulated the three-dimensional structure of hSMSl by homologous modeling method, and adopted the strategy of selecting highly three-dimensional topological similarity proteins. Escherichia coli GlpG (PDB number: 2IC8 was selected as template protein and Modeller module of Discovery Studio2.0 was used for homologous modeling. For the extracellular important Loop2, the highly homologous protein 1BW0 was selected as the template model. The structure optimized by molecular dynamics can explain the mechanism of the interaction of lecithin and sphingomyelin with hSMSl. Then the virtual screening of SPECS library was carried out with the verified three-dimensional structure of hSMSI. The SPECS library was screened by Gold and then fine-screened by Glide. 93 compounds with high score were selected manually from more than 200 hit objects according to their structural diversity. On the screening platform of SMS enzyme activity established in our laboratory, we tested the SMS enzyme inhibitory activity of 93 compounds, and obtained two lead compounds 34 and 72. According to the structure of compound 34, the structure similarity search was carried out, and compounds 104105 and 107 were successfully found. The synthesis of compounds 104 and 105 confirmed that the framework was an effective structure of SMS enzyme inhibitors. The synthesis and structural identification of compound 72 showed that the SPECS library provided a mixture of unknown components and two active components were successfully isolated. A SMS agonist was also found from the structure of compound 72. Based on the natural substrate structure of sphingomyelin, 17 substrate analogues containing benzene ring structure were synthesized. Four active compounds were found, and their preliminary structure-activity relationship was investigated. We also confirmed that MS-209 is a potential SMS inhibitor in the literature. The synthesis and activity test showed that MS-209 was not an inhibitor of SMS.
【学位授予单位】:复旦大学
【学位级别】:博士
【学位授予年份】:2009
【分类号】:R341

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