设计合成组蛋白去乙酰化酶的小分子抑制剂

发布时间：2018-05-18 15:30

本文选题：组蛋白去乙酰化酶 + 组蛋白去乙酰化酶抑制剂　；参考：《大连理工大学》2014年硕士论文

【摘要】：在肿瘤的发生和发展过程中,表观遗传学修饰起着非常重要的作用。组蛋白修饰是其中的主要方式之一,主要包括乙酰化、甲基化、磷酸化、泛素化等。组蛋白修饰主要由两种拮抗酶共同调控：组蛋白乙酰基转移酶(Histone Acetyl Transferases, HATs)、组蛋白去乙酰化酶(Histone Deacetylases, HDACs)、HDACs将组蛋白N-端赖氨酸残基侧链上的ε-氨基去乙酰化,因此,电荷量增多会促进组蛋白与DNA链之间的相互作用,从而影响染色质结构及基因的表达。HDACs的高表达会导致平衡失调从而引起肿瘤的发生,寻找高效抑制HDACs为靶点的抑制剂成为研发新型抗癌药物的热点。组蛋白去乙酰化酶抑制剂(Histone Deacetylases Inhibitors, HDACIs)的结构分为三部分：金属结合区(Zinc Binding Group, ZBG)、连接区(Linker Group)和表面识别区(Surface Recognition Group)。通过对三部分的结构进行设计,寻找高活性与高选择性的抑制剂。本论文根据上市药物SAHA、FK228及报道的抑制剂结构特点,设计了两个系列化合物。第一系列化合物基于2-氨基-7溴庚酸为骨架,引入不同的疏水结构作为表面识别区；5个亚甲基作为连接区；硫代乙酰基作为金属结合区。第二系列化合物主要以对苯硝基作为金属结合区。基于以上结构特点,设计出300余种抑制剂小分子,通过Autodock4.2分子对接软件初步筛选出13种化合物,并采用多肽液相合成法成功合成了13种化合物10A1-10A6、11B1、12C1-12C2、13-1至16-1。通过了HPLC、ESI-MS、1H NMR与13C NMR的结构验证,与目标产物结构一致。目标化合物均显示很好的酶抑制活性。其中第一系列的12C1(IC50=0.04μm)与12C2(IC50=0.092μm)活性最好,在表面识别区引入2-氨基-4-苯基噻唑可以提高抑制剂活性。对HDAC1与HDAC6的抑制活性,与对HDAC的抑制活性呈现相同趋势。在目标化合物对肿瘤细胞的抗增殖活性实验中,对HeLa(宫颈癌细胞系)的选择性要高于MCF-7(乳腺癌细胞系)与SMMC-7721(肝癌细胞系)。化合物12C1(IC50=40.3μM)对HeLa的抑制作用优于TSA (IC50=48.5μM)。12C1、12C2较对照TSA的酶抑制活性低10倍,但在细胞活性实验中,12C1活性优于TSA1.21倍。测试化合物中的硫代乙酰基类化合物与对照TSA分别进行抗增殖活性水平与酶抑制活性水平比较,硫代乙酰基类化合物在抗增殖活性水平上比对照TSA有更高的提升。说明硫代乙酰基在细胞内水解为巯基,促进与Zn2+发生作用,达到与TSA相同的抑制水平。由于脂溶性,渗透性的原因造成其它化合物的抑制活性不高。通过对接实验中的结合能分析,化合物中可旋转的单键数目过多,与复合物之间的静电能作用太大,都不利于与酶的相互作用。HDAC4-HDACIs复合物之间的对接能量与HDAC2-HDACIs复合物具有相同的分布趋势。在目标化合物与HDAC2的对接实验中,12C1与HDAC2形成的氢键数目多于其它,且只有12C1表面识别区的2-氨基-4-苯基噻唑中的N原子参与氢键的形成。在目标化合物与HDAC4的对接实验中,TSA中异羟肟酸基形成的氢键多于硫代乙酰基,与体外酶抑制活性一致。巯基与酶的相互作用高于硫代乙酰基,因此,硫代乙酰基在细胞内水解为巯基后会提高与酶的相互作用,这与体外抗增殖活性实验较酶抑制活性有较大提高相符。
[Abstract]:Epigenetic modification plays a very important role in the development and development of tumor. Histone modification is one of the main ways, including acetylation, methylation, phosphorylation, and ubiquitination. Histone modification is mainly regulated by two kinds of antagonists: histone acetyltransferase (Histone Acetyl Transferases, H) ATs), the histone deacetylation enzyme (Histone Deacetylases, HDACs), HDACs deacetylation of the epsilon amino group on the side chain of the protein N- terminal lysine residue, therefore, the increase of the charge will promote the interaction between the histone and the DNA chain, thus affecting the chromatin structure and the gene expression of the.HDACs, resulting in the imbalance of the balance resulting from the expression of.HDACs. The occurrence of tumor and the search for the inhibitors that target the HDACs as the target are the hot spots in the development of new anticancer drugs. The structure of Histone Deacetylases Inhibitors (HDACIs) is divided into three parts: the metal binding zone (Zinc Binding Group, ZBG), the connection area (Linker Group) and the surface recognition area (Surface) Tion Group). Through the design of the three part of the structure, we look for inhibitors with high activity and high selectivity.
In this paper, two series of compounds were designed based on the structure characteristics of SAHA, FK228 and reported inhibitors. The first series of compounds were based on 2- amino -7 bromoheptanic acid as the skeleton, and different hydrophobic structures were introduced as surface recognition areas; 5 methylene was used as the connection area; thioacetyl was used as the metal binding zone. Second series of compounds were used. Based on the above structural characteristics, more than 300 small molecules of inhibitor are designed based on the above structural characteristics. 13 compounds are screened by Autodock4.2 molecular docking software, and 13 compounds, 10A1-10A6,11B1,12C1-12C2,13-1 to 16-1., are successfully synthesized through the synthesis of HPLC, ESI-MS, 1H NMR and 13C. The structural verification of the NMR is consistent with the structure of the target product.
The target compounds all showed good enzyme inhibitory activity. The first series of 12C1 (IC50=0.04 m) and 12C2 (IC50=0.092 mu m) activity was the best. The introduction of 2- amino -4- phenyl thiazole in the surface recognition area could improve the activity of the inhibitor. The inhibitory activity of HDAC1 and HDAC6 showed the same trend as the inhibitory activity to HDAC.
In the experiment of anti proliferation activity of target compounds to tumor cells, the selectivity of HeLa (cervical cancer cell line) was higher than that of MCF-7 (breast cancer cell line) and SMMC-7721 (liver cancer cell line). Compound 12C1 (IC50=40.3 mu M) inhibited HeLa better than TSA (IC50=48.5 micron M).12C1,12C2 10 times lower than that of control TSA enzyme inhibitory activity, but in cell In the activity test, the activity of 12C1 was better than that of TSA1.21. The antiproliferative activity level of the thioacetyl group in the test compound compared with the control TSA was compared with that of the enzyme inhibitory activity. The thioacetyl group had a higher increase in the anti proliferative activity level than that of the control TSA. It can promote the function of Zn2+ and achieve the same inhibition level as TSA. Due to fat solubility and permeability, the inhibitory activity of other compounds is not high.
Through the binding energy analysis in the docking experiments, the number of rotatable single bonds in the compound is too much, and the electrostatic energy between the compounds is too large, and the interaction between the.HDAC4-HDACIs complex and the HDAC2-HDACIs complex has the same distribution trend. In the docking experiment between the target compound and the HDAC2, The number of hydrogen bonds formed by 12C1 and HDAC2 is more than that of the others, and only the N atoms of the 2- amino -4- phenyl thiazole in the 12C1 surface recognition region are involved in the formation of hydrogen bonds. In the docking experiment between the target compound and HDAC4, the hydrogen bonds formed by the hydroxamic acid group in TSA are more than the thioacetyl group, which is consistent with the inhibitory activity of the enzyme in vitro. The interaction of the sulfhydryl group with the enzyme is high. Therefore, the thioacetyl group can increase the interaction with the enzyme after the hydrolysis of thioacetyl in the cell, which is more consistent with the inhibition activity of anti proliferative activity in vitro than in the enzyme inhibition activity.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91;O629

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