【作者】 李中华；

【导师】 王鹏；

【作者基本信息】 南开大学 ， 化学生物学， 2013， 博士

【摘要】 酶蛋白几乎参与机体所有的生命活动,其功能对于机体的正常运行至关重要,许多疾病的发生都与酶功能的失调有关。因此,以酶为靶点的药物开发一直都是研究的热点。其中,酶抑制剂类药物占据临床用药的三分之一,成为新药来源的主要途径。糖昔酶是一类将寡糖等糖基复合物的糖苷键水解的酶,主要参与食物的消化、多糖和糖脂复合物的降解。糖苷酶抑制剂可以抑制糖苷酶的活性,阻断碳水化合物的分解,对一些糖代谢紊乱疾病如溶酶体贮积症、糖尿病等有重要药用价值。同时,以糖苷酶抑制剂为工具,可以更好的了解糖苷酶的水解机制,分析糖基化的生物学作用,利于揭示阿尔茨海默病、肿瘤等重大疾病的发生机制。因此,围绕酶抑制剂药物的开发,本课题开展了糖苷酶抑制剂和组蛋白去乙酰化酶抑制剂的设计、筛选和机制分析,为相关疾病靶向药物的开发提供了依据和新思路。戈谢病是一种由于酸性p-葡萄糖脑苷酶(acid β-glucocerebrosidase, GCase)基因突变引起的发病率最高的溶酶体贮积病。该酶基因的突变将导致GCase的提前降解,最终不能定位于溶酶体中发挥活性,从而使底物葡萄糖脑苷脂(Glucosylcera-mide, GC)大量积累。基于酶的竞争性抑制剂机制,人体提出了分子伴侣设想：竞争性抑制剂与突变酶的活性中心结合,诱导酶形成正确折叠,并辅助酶定位于溶酶体,在高浓度底物的溶酶体中,竞争性抑制剂被底物竞争性解离,酶分子得以释放,从而发挥生物功能。依据该酶的催化机制,本课题组前期设计、合成系列葡萄糖咪唑类化合物,本课题中通过生物学活性测试,发现化合物具有强抑制活性和高选择性,且细胞膜透过性好,细胞毒性低。以发病率最高的两种成淋巴细胞(N370S, L444P)为模型,我们发现该类化合物均具有较好的伴侣活性,尤其是化合物11(3,3-二甲基-N-苯基-4-酰胺-1-丁基取代的葡萄糖咪唑),2.5μM的用药浓度下,可使N370S型GCase酶活提高2.1倍。利用计算机模拟、脉冲酶解稳定性试验、Western blot和细胞免疫荧光试验等方法分析,发现化合物11可以结合并稳定突变GCase酶的结构,提高其蛋白水平,促进其溶酶体定位,最终提高细胞中GCase的活性。N-乙酰葡萄糖胺(0-linked N-acetylglucosamine,O-GlcNAc)修饰是一种重要的蛋白翻译后修饰,参与生物体中的多种细胞活动,O-GlcNAcase (OGA)是体内唯一水解该修饰的糖苷酶。有人发现阿尔茨海默病人的大脑中Tau蛋白的O-GlcNAc修饰严重不足,因此,我们设想通过OGA抑制剂来提高患者体内的O-GlcNAc修饰水平。利用前期设计、合成的系列化合物,通过酶学筛选,获得了一个高效选择性的OGA抑制剂(Ki=5.9μM),与阳性化合物PUGNAc相比它能更多地提高细胞内的O-GlcNAc水平。同时,利用体外分段表达,我们获得了一个OGA变体(细胞凋亡过程中的OGA剪切体,nOGA)的专一性抑制剂。所筛选的这些OGA抑制剂将有助于进一步揭示细胞内O-GlcNAc修饰的机制,阐明与O-GlcNAc修饰相关疾病的病因,为相关药物的研发提供了基础。组蛋白去乙酰化酶(Histone deacetylases, HDACs)是一类调控染色体形成和基因表达的关键酶。当组蛋白处于低乙酰化状态时,核小体结构紧密,使各种促进细胞分化和凋亡的基因受到抑制,引起肿瘤的发生。HDACs抑制剂(HDACi)通过抑制HDAC去乙酰化活性,阻滞肿瘤细胞生长,诱导其分化和凋亡。因此,以HDACs为靶点,设计合成抗肿瘤药物具有广阔的前景。依据前期设计、合成的系列HDACi,通过体外酶活和细胞毒性筛选,我们获得了化合物Z-13,它对HDAC酶活性和肿瘤细胞增殖的抑制都要比阳性药SAHA (suberoylanilide hydroxamic acid)高一个数量级。通过对它抗肿瘤机制的研究,发现它主要通过阻遏细胞周期和诱导细胞凋亡,来抑制肿瘤细胞增殖,延缓体内乳腺癌肿瘤的生长。同时,我们做了SAHA和taxol联合用药对乳腺癌细胞抑制的影响,发现,SAHA能增强taxol对乳腺癌细胞的抑制,尤其是增强了taxol对其不敏感的乳腺癌细胞增殖的抑制。在对体内建立的移植瘤模型上,也表现出了SAHA能增强taxol对肿瘤抑制的作用。所以二者的联合用药为临床上的进一步研究提供了理论基础。更多还原

【Abstract】 Enzymes involve in almost all of our life process, so their activities play a vital role in maintaining the normal operation of the body. Many diseases are caused by disorder of the activity of some enzymes, so it has been widely recognized in recently years that enzymes are promising targets for drug development. So far, enzyme inhibitor drugs supply a third of drugs clinical applied, and as a mainly source of new drugs.Glycosidases catalyze the hydrolysis of the glycosidic linkage to release smaller sugars. They widely involve in digesting of food, polysaccharides and glycoconjugates metabolism, glycosidase inhibitors can inhibit the activity of glycosidase, and block the decomposition of carbohydrates. they have important therapeutic value against some glycometabolism disorder diseases, such as lysosomal storage diseases, diabetes, etc. Meanwhile, The study of the glycosidase inhibitors contribute to better understanding of the the hydrolytic mechanism and biological functions of glycosylation, revealing the pathogenesises of Alzheimer’s disease, cancer, and other major diseases. Surrounding the development of enzyme inhibitor drugs, we carried out the topic:design, screening and mechanism analysis of glycosidase inhibitors and HDAC inhibitors. These results provide a basis for the development of the targeted drugs.Gaucher disease, the highest incidence of lysosomal storage diseases, is caused by the mutations in acid β-glucocerebrosidase (GCase). The mutation of GCase leads to the earlier degradation of GCase in endoplasmic reticulum, so GCase cannot be located in Iysosome, eventually, cause the accumulation of glucosylceramide (GlcCer). Based on the mechanism of competitive enzymes inhibitors, people put forward the hypothesis of pharmacological chaperone therapy (PCT), which refers to the use of competitive inhibitors to specifically bind and potentially stabilize catalytically competent GCase variants at neutral pH in the ER, assisting with the folding of these mutant proteins and helping these proteins pass the quality control checks for trafficking to the lysosome. Due to the high substrate concentrations and low pH in the lysosome, the pharmacological chaperones (PCs) would be displaced from the active site of the enzyme, and the stored GlcCer would be degraded. According to the catalytic mechanism of GCase, we synthesized a series of glucoimidazoles. By biological activity screening, we found all of the compounds had strong inhibitory activity, high selectivity, good cell membrane permeability, and low cytotoxicity. A cell-based assay using patient-derived lymphoblasts (N370S or L444P mutation) demonstrated that the administration of these compounds can significantly increase GCase activity. Interestingly, the moderate inhibitor, compound11, which bears a3,3-dimethyl-N-phenyl-4-amide-l-butyl substituent, had the greatest effect on activity, yielding a2.1-fold increase in N370S lymphoblasts at2.5μM and a1.2-fold increase in L444P at0.5μM following a three-day incubation. Next, computer docking studies and a protease protection assay were used to elucidate the ligand-enzyme interactions responsible for the chaperone activity of11. Western blot and immuno-fluorescence assays verified the trafficking of the restored GCase to the lysosome.O-linked N-acetylglucosamine (O-GlcNAc) modification, which involes in a variety of cellular processes, is an essential posttranslational modification in metazoans. O-GlcNAcase (OGA) is the only glycosidase which is responsible for the removal of O-GlcNAc. The study found that O-GlcNAcylation of tau in the brains of alzheimer’s patients is severe insufficient. So, We wanted to improve the level of O-GlcNAcby OGA inhibitors. We designed and synthesized series of compounds. By enzyme activity screening, we got a powerful selective OGA inhibitor (Kj=5.9μM) which effectively induced more cellular hyper-O-GlcNAcylation than PUGNAc. Using piecewise expression of OGA in vitro, we obtained a specific nOGA (N r terminal fragment of OGA during apoptosis) inhibitor (Kj=48μM). These OGA inhibitors contributed to revealing the mechanism of O-GlcNAc modifications, and pathogenesises of diseases for the abnormalities of O-GlcNAc modification, in order to provide a basis for the development of the relevant drugs.HDACs (Histone deacetylases) are a kind of key enzymes which play fundamental roles in the regulation of gene expression and maintenance of chromatin structures. When the histones are in low acetylation state, which can condense chromatin, promote cell growth, differentiation and inhibit the expression of apoptosis related genes, finally cause the occurrence of tumor. Histone deacetylase inhibitors (HDACi) can reverse the hypoacetylation status of histones, thereby inducing the tumor cell differentiation and apoptosis. It has been widely recognized in recently years that HDACs are promising targets for cancer therapy. We synthesized a novel calss of HDAC inhibitors. After in vitro enzyme activity and cell toxicity screening, we discovered a compound, Z-13, which showed about one order of magnitude more potent than SAHA (suberoylanilide hydroxamic acid) in both enzymatic and cellular assays. Based on the research of antitumor mechanism of Z-13, we found that it inhibited the proliferation of tumor cell in vitro and in vivo mainly by inducing cell apoptosis and cell cycle arrest. We also want to know whether SAHA can enhance the growth inhibitory effect induced by taxol against breast cancer cell. By experiment, we found that SAHA can enhance taxol-induced cell death against human breast cancer cells, especially in taxol-resistant and multi-resistant breast cancer cells. Based on BALB/c nude mice breast cancer xenograft model, the synergetic effect was also observed in the in vivo xenograft tumor model. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.更多还原