【摘要】：鞘磷脂合成酶(SMS)是脂代謝通路中鞘磷脂生物合成最后一步關(guān)鍵酶。SMS以卵磷脂和神經(jīng)酰胺為底物,催化反應生成鞘磷脂和甘油二酯。神經(jīng)酰胺和二酰基甘油是細胞內(nèi)重要的第二信使,它們在細胞內(nèi)濃度的改變與細胞信號傳導以及細胞凋亡密切相關(guān)。最近大量的研究表明,鞘磷脂合成酶與動脈粥樣硬化癥密切相關(guān),抑制鞘磷脂合成酶可以有效緩解動脈粥樣硬化癥。因此鞘磷脂合成酶可以作為一個重要的、潛在的抗動脈粥樣硬化藥物的靶點。目前尚無選擇性的鞘磷脂合成酶抑制劑,發(fā)現(xiàn)第一代SMS酶抑制劑具有前沿性和創(chuàng)新性,這對于深入研究鞘磷脂合成酶在動脈粥樣硬化的作用機制和開發(fā)新一類抗動脈粥樣硬化藥物有著重要的價值。 為了尋找第一代SMS小分子抑制劑,我們以人類SMS1為研究對象,以同源模建方法模擬hSMSl三維結(jié)構(gòu),采用選擇高度三維空間拓撲結(jié)構(gòu)相似性蛋白的策略,選取了Escherichia coli GlpG (PDB編號：2IC8)作為模板蛋白,運用Discovery Studio2.0的Modeller模塊進行同源模建。對于胞外重要的Loop2,則選取了同源性高的蛋白1BW0作模板模建。分子動力學優(yōu)化后的結(jié)構(gòu)能夠很好的解釋天然底物卵磷脂和鞘磷脂與hSMSl的作用機制。隨即用驗證后的hSMSI三維結(jié)構(gòu)對SPECS庫進行虛擬篩選,使用Gold初篩,然后Glide精細篩選,對200余個命中物根據(jù)結(jié)構(gòu)多樣性手動挑選出了93個分值高的化合物。 在本實驗室建立起來的SMS酶活性篩選平臺上,我們檢驗了93個化合物的SMS酶抑制活性,成功的得到了兩個先導化合物34和72。根據(jù)化合物34的結(jié)構(gòu),進行結(jié)構(gòu)相似性搜索,成功的找到化合物104、105和107,對化合物104和105的合成證實了這個骨架是一類有效的SMS酶抑制劑結(jié)構(gòu)。經(jīng)對化合物72的合成和結(jié)構(gòu)鑒定,證實SPECS庫提供的是未知成分的混合物,已經(jīng)成功分離出了兩個活性成分。從化合物72結(jié)構(gòu)中還找到了一個SMS激動劑。 本課題還從鞘磷脂天然底物結(jié)構(gòu)出發(fā),進行結(jié)構(gòu)改造,合成了17個含苯環(huán)結(jié)構(gòu)的底物類似物,從中發(fā)現(xiàn)了4個活性化合物,并考察了其初步的構(gòu)效關(guān)系。 我們還對文獻中關(guān)于MS-209是潛在SMS抑制劑的報道進行了驗證。經(jīng)合成和活性測試,證實MS-209并不是SMS的抑制劑。
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R341

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