多胺类组蛋白甲基转移酶DOT1L抑制剂筛选及诱导混合谱系白血病细胞凋亡的研究
发布时间：2018-10-05 15:09
【摘要】：混合谱系白血病(Mixed Lineage Leukemia,MLL),因11q23处MLL基因易位重排形成融合蛋白而得名,是一类致死率极高、预后差的高危恶性急性白血病。DOT1L蛋白是目前发现的唯一H3K79甲基化转移酶,也是MLL白血病的重要靶点,开发DOT1L小分子抑制剂成为治疗MLL白血病的一条新思路。多胺类化合物也因其多靶点性成为抗肿瘤药物的研究热点,目前对于DOT1L抑制剂的设计也主要为多胺类衍生物。本课题首先建立组蛋白甲基转移酶DOT1L抑制剂的虚拟筛选方法,筛选出与DOT1L受体对接评分较高的化合物;设计合成路线合成、分离纯化目标化合物,并进行结构确认;最后考察目标化合物对混合谱系白血病细胞的体外活性研究。研究结果如下:(1)根据实验室前期研究基础结合相关文献资料,设计一系列多胺类衍生物,与正处于一期临床实验的阳性化合物EPZ-5676共同构成配体化合物库,从蛋白质晶体数据库PDB中选取受体模型4HRA。用Sybyl-X2.0软件将受体与配体进行分子对接试验,筛选出了分数最高的化合物DUOA003作为目标化合物;(2)设计合成路线并完成目标化合物DUOA003的合成工作,通过LC-MS、1HNMR,13CNMR确定目标化合物的结构为:N,N'-(propane-1,3-diyl)bis(2,5-dihydroxybenzamide);(3)以混合谱系白血病细胞MV4-11和巨噬细胞作为研究对象,采用CCK-8法测定两株细胞在目标化合物和阳性药物阿糖胞苷不同浓度(120、100、50、25、10、1、0.1μM)作用24h后的增殖抑制率。结果发现DUOA003和阳性药物阿糖胞苷对MV4-11细胞均有显著的抑制作用,IC50分别为25和28μM,对巨噬细胞的IC50分别为71μM和11μM;选择对正常细胞安全浓度范围考察不同作用时间24h、48h、72h对细胞抑制作用的影响,结果反映出DUOA003对MV4-11细胞的抑制率展现出时间和剂量的依赖性;(4)采用Annexin V-FITC/PI法对经高、中、低浓度的药物诱导24h和48h的MV4-11细胞进行染色,在荧光显微镜下观察诱导48h后细胞状态,并用流式细胞仪检测目标化合物诱导MV4-11细胞的凋亡率。结果发现DUOA003能够显著地诱导MV4-11细胞产生凋亡。诱导48h后,与阴性对照组(17.3±5%)相比,DUOA003的低浓度组(53.9±8%)、中浓度组(80.9±9%)、高浓度组(88.5±6%)、以及阿糖胞苷高浓度组(82.6±9%)均呈现显著性差异(P0.001),DUOA003诱导的细胞48h后凋亡多处于晚期,阿糖胞苷诱导的细胞凋亡则主要出现在早期;(5)用Caspase-3活性测定试剂盒,检测经高、中、低浓度的目标化合物诱导细胞凋亡24h后Caspase-3的活性变化。Caspase-3活性检测显示化合物DUOA003在诱导细胞凋亡24后,与阴性对照组Caspase-3活性测定OD值(0.146±0.002)比较,DUOA003的低浓度组(0.227±0.003)、中浓度组(0.367±0.004)、高浓度组(0.554±0.005)以及阿糖胞苷高浓度组(0.477±0.006)均呈现显著性差异(P0.001)。DUOA003诱导混合谱系白血病细胞MV4-11凋亡过程中存在Caspase-3介导的细胞凋亡。
[Abstract]:Mixed lineage leukemia (Mixed Lineage Leukemia,MLL), named after MLL gene translocation and fusion protein in 11q23, is the only H3K79 methyltransferase found in high risk acute leukemia patients with high mortality and poor prognosis. It is also an important target of MLL leukemia. The development of small molecular inhibitors of DOT1L has become a new idea in the treatment of MLL leukemia. Polyamines have become the research focus of antitumor drugs because of their multi-target, and the current design of DOT1L inhibitors is mainly polyamine derivatives. Firstly, a virtual screening method of histone methyltransferase DOT1L inhibitor was established to screen the compounds with high docking score with DOT1L receptor, then the synthesis route was designed, the target compounds were isolated and purified, and the structure of the target compounds was confirmed. Finally, the in vitro activity of the target compounds to mixed lineage leukemia cells was investigated. The results are as follows: (1) A series of polyamines derivatives were designed according to the basis of pre-laboratory research and related literature, and the ligands library was constructed together with the positive compound EPZ-5676, which is in the primary clinical trial. The receptor model 4HRAwas selected from protein crystal database PDB. Molecular docking test between receptor and ligand was carried out by Sybyl-X2.0 software and the highest fraction compound DUOA003 was selected as the target compound. (2) the synthetic route was designed and the synthesis of target compound DUOA003 was completed. The structure of the target compound was determined by LC-MS,1HNMR,13CNMR to be propane-1,3-diyl) bis (2-dihydroxybenzamide); (3. The mixed lineage leukemic cells MV4-11 and macrophages were used as the research objects. CCK-8 assay was used to determine the proliferation inhibition rate of the two cell lines at different concentrations of target compound and cytarabine at different concentrations (120 ~ 100g / 100) for 24 hours after exposure to 0.1 渭 M of cytosine arabinoside, the target compound and the positive drug cytarabine. The results showed that DUOA003 and cytarabine had significant inhibitory effects on MV4-11 cells, IC50 were 25 渭 M and 28 渭 M, and IC50 on macrophages were 71 渭 M and 11 渭 M, respectively. The effect of cell inhibition, The results showed that the inhibition rate of DUOA003 on MV4-11 cells was time-and dose-dependent. (4) Annexin V-FITC/PI method was used to stain the MV4-11 cells induced by high, medium and low concentrations of drugs for 24 and 48 hours, and the cells were observed under fluorescence microscope after 48 hours of induction. The apoptosis rate of MV4-11 cells induced by target compounds was detected by flow cytometry. The results showed that DUOA003 could induce apoptosis of MV4-11 cells significantly. 48 h after induction, there were significant differences between the low concentration group (53.9 卤8%), the middle concentration group (80.9 卤9%), the high concentration group (88.5 卤6%) and the high concentration group of cytarabine (82.6 卤9%) compared with the negative control group (17.3 卤5%). The apoptosis induced by cytarabine mainly occurred in the early stage. (5) the Caspase-3 activity assay kit was used to detect the cell apoptosis. The changes of Caspase-3 activity after 24 hours of apoptosis induced by low concentration of target compound. The activity of Caspase-3 showed that the compound DUOA003 induced apoptosis 24 hours later. Compared with the negative control group (0.146 卤0.002), the low concentration group (0.227 卤0.003), the middle concentration group (0.367 卤0.004), the high concentration group (0.554 卤0.005) and the high concentration group (0.477 卤0.006) showed significant difference in the process of MV4-11 apoptosis induced by DUOA003. There is Caspase-3 mediated apoptosis.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
[Abstract]:Mixed lineage leukemia (Mixed Lineage Leukemia,MLL), named after MLL gene translocation and fusion protein in 11q23, is the only H3K79 methyltransferase found in high risk acute leukemia patients with high mortality and poor prognosis. It is also an important target of MLL leukemia. The development of small molecular inhibitors of DOT1L has become a new idea in the treatment of MLL leukemia. Polyamines have become the research focus of antitumor drugs because of their multi-target, and the current design of DOT1L inhibitors is mainly polyamine derivatives. Firstly, a virtual screening method of histone methyltransferase DOT1L inhibitor was established to screen the compounds with high docking score with DOT1L receptor, then the synthesis route was designed, the target compounds were isolated and purified, and the structure of the target compounds was confirmed. Finally, the in vitro activity of the target compounds to mixed lineage leukemia cells was investigated. The results are as follows: (1) A series of polyamines derivatives were designed according to the basis of pre-laboratory research and related literature, and the ligands library was constructed together with the positive compound EPZ-5676, which is in the primary clinical trial. The receptor model 4HRAwas selected from protein crystal database PDB. Molecular docking test between receptor and ligand was carried out by Sybyl-X2.0 software and the highest fraction compound DUOA003 was selected as the target compound. (2) the synthetic route was designed and the synthesis of target compound DUOA003 was completed. The structure of the target compound was determined by LC-MS,1HNMR,13CNMR to be propane-1,3-diyl) bis (2-dihydroxybenzamide); (3. The mixed lineage leukemic cells MV4-11 and macrophages were used as the research objects. CCK-8 assay was used to determine the proliferation inhibition rate of the two cell lines at different concentrations of target compound and cytarabine at different concentrations (120 ~ 100g / 100) for 24 hours after exposure to 0.1 渭 M of cytosine arabinoside, the target compound and the positive drug cytarabine. The results showed that DUOA003 and cytarabine had significant inhibitory effects on MV4-11 cells, IC50 were 25 渭 M and 28 渭 M, and IC50 on macrophages were 71 渭 M and 11 渭 M, respectively. The effect of cell inhibition, The results showed that the inhibition rate of DUOA003 on MV4-11 cells was time-and dose-dependent. (4) Annexin V-FITC/PI method was used to stain the MV4-11 cells induced by high, medium and low concentrations of drugs for 24 and 48 hours, and the cells were observed under fluorescence microscope after 48 hours of induction. The apoptosis rate of MV4-11 cells induced by target compounds was detected by flow cytometry. The results showed that DUOA003 could induce apoptosis of MV4-11 cells significantly. 48 h after induction, there were significant differences between the low concentration group (53.9 卤8%), the middle concentration group (80.9 卤9%), the high concentration group (88.5 卤6%) and the high concentration group of cytarabine (82.6 卤9%) compared with the negative control group (17.3 卤5%). The apoptosis induced by cytarabine mainly occurred in the early stage. (5) the Caspase-3 activity assay kit was used to detect the cell apoptosis. The changes of Caspase-3 activity after 24 hours of apoptosis induced by low concentration of target compound. The activity of Caspase-3 showed that the compound DUOA003 induced apoptosis 24 hours later. Compared with the negative control group (0.146 卤0.002), the low concentration group (0.227 卤0.003), the middle concentration group (0.367 卤0.004), the high concentration group (0.554 卤0.005) and the high concentration group (0.477 卤0.006) showed significant difference in the process of MV4-11 apoptosis induced by DUOA003. There is Caspase-3 mediated apoptosis.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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