靶向PRMT5和BRD4蛋白的药物发现及机制研究

发布时间：2018-01-15 17:01

本文关键词：靶向PRMT5和BRD4蛋白的药物发现及机制研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

更多相关文章： 表观遗传 PRMT5 BRD4 抗肿瘤活性

【摘要】：表观遗传学指的是基因表达发生可遗传变化,但是在染色体不改变DNA序列的前提下。即由环境因素引起遗传物质稳定的,可逆的,可遗传的变化。DNA甲基化,组蛋白修饰,染色质重塑和非编码RNA调控,这四种方式是表观遗传在基因表达调节过程中作用的主要方式。过去几十年里,大量研究报道证实,表观遗传发生在多种生物反应过程中,参与调控肿瘤、糖尿病、免疫和代谢性疾病等人类疾病的发生和病理进程。阐明表观遗传与疾病的相关机制,有助于为研制药物和治疗疾病方案设计提供依据和方向。蛋白精氨酸甲基转移酶是催化组蛋白或非组蛋白甲基化修饰的一种酶,并且其甲基化修饰是真核细胞常见的转录后修饰。已证实,在几种人类恶性肿瘤中,蛋白质精氨酸甲基转移酶5(PRMT5)表达均出现上调,可见其在肿瘤形成发展过程中发挥重要的作用。此外,PRMT5已经被确证为套细胞淋巴瘤(MCL)的治疗靶标。在本研究中,我们基于计算辅助药物设计虚拟筛选的手段,以靶标的三维结构、药效团和分子对接等信息为基础对化合物库进行高通量虚拟筛选,结合药物化学合成修饰的方法,发现了有效地且有选择性地PRMT5小分子抑制剂。通过讨论结构-活性关系(SAR),确证了其骨架的有效性,为合成优化先导化合物并增强其选择性提供线索和依据。最终筛选出具有0.33 μM的半数抑制浓度(IC5Q)和广泛选择性的苗头化合物17。在细胞水平实验中,化合物17表现出对白血病和淋巴瘤细胞增殖抑制的效果,同时,选择性的表现出最有效抑制人白血病MV4-11细胞的增殖作用。此外,化合物17通过以一定时间和浓度依赖性抑制PRMT5底物SmD3对称二甲基化修饰的表达水平。表明化合物17的生物抗增殖作用是抑制PRMT5底物SmD3表达的直接结果。这些靶标实验证实化合物17的确为靶向PRMT5蛋白的新型小分子探针。Bromodomain是能够特异识别乙酰化组蛋白的结构域。BRD4作为BET蛋白家族的一部分,是识别组蛋白乙酰化的识别因子。BRD4在维持体内组蛋白乙酰化水平上发挥着关键的作用,也可以转录调控CDK6,BCL2,C-MYC等下游靶基因,最终起到稳定的抗癌作用。已经确证BRD4作为治疗多种血液病及实体瘤的一种靶点,包括AML,ALL,MLL,MM以及肺癌和中线癌等。JQ1,IBET151以及IBET762等阳性化合物的发现,推动了靶向BET家族小分子抑制剂的研究发展。在本研究中,将对体外筛选出的苗头化合物进行细胞表征研究。在细胞增殖实验中,化合物DCBD008对THP-1细胞增殖表现出明显的抑制活性,IC50=8.098 μM,结果与阳性化合物JQ1,IBET表征结果一致。并且证实DCBD008通过诱导THP-1细胞周期阻滞在G0/G1期,导致细胞无法进入正常分裂期。并通过调控C-MYC等重要基因的转录表达,进而影响细胞周期以及凋亡发挥抗癌作用。结果显示,给药后,THP-1细胞中CDK6,BCL2和C-MYC基因的表达量均呈现出浓度依赖性的下降,表明化合物DCBD008在THP-1细胞内是通过靶向抑制BRD4来发挥抗癌作用的。该课题为靶向BRD4蛋白的药物发现及机制研究提供了新的小分子骨架。
[Abstract]:Epigenetics refers to heritable changes in gene expression, but in the premise of chromosomes do not alter the sequence of DNA. From the environmental factors caused by genetic material stable, reversible, but genetic changes in.DNA methylation, histone modification, chromatin remodeling and non encoding RNA control, the four way is to watch the concept of expression mainly in the regulation process of the role of genetic gene. In the past few decades, a large number of studies confirmed that epigenetic inheritance occurs in a variety of biological processes, involved in the regulation of tumor, diabetes, occurrence and pathological process of immune and metabolic diseases such as human disease. Clarify the mechanism of epigenetic form and illness. Helps to provide basis and direction for the development of drug design and treatment of the disease. The protein arginine methyltransferase is an enzyme that catalyzes histone and non histone methylation, and its methylation modification Is the common transcription of eukaryotic cells. After modification has been confirmed in several human malignancies, protein arginine methyltransferase 5 (PRMT5) expression were up-regulated in tumor formation, which play an important role in the process of development. In addition, PRMT5 has been confirmed as a cell lymphoma (MCL). Treatment targets. In this study, we design computer aided drug virtual screening method based on the three-dimensional structure, target, pharmacophore model and molecular docking information for high-throughput virtual screening of compound libraries, combined with the method of chemical synthesis drug modified, found effective and selective small molecule inhibitor of PRMT5. Through the discussion of structure-activity relationship (SAR), confirmed the effectiveness of the framework, for the synthesis optimization of lead compounds and enhance the selectivity to provide clues and basis. Finally screened with 0.33 M half inhibitory concentration (IC5Q) and wide selection of signs of compound 17. at the cellular level in the experiment, 17 compounds showed inhibition of leukemia and lymphoma cell proliferation effect, at the same time, exhibit selective proliferation of the most effective inhibition of human leukemia MV4-11 cells. In addition, 17 compounds by inhibiting expression levels of PRMT5 substrate SmD3 methylation in symmetric two a certain period of time and concentration dependent. Show that the anti proliferative effect of biological compound 17 is a direct result of the inhibition of PRMT5 SmD3 expression. These target substrate experiments confirmed that compound 17 is indeed targeting new type PRMT5 protein small molecule probe.Bromodomain is able to identify specific histone acetylation domains of.BRD4 as part of the BET protein family that is to play a key role recognition factor.BRD4 recognition of histone acetylation in vivo to maintain the level of histone acetylation and transcriptional regulation can also be BCL2, control CDK6, C-MYC downstream target genes and eventually play anticancer effect and stability. Have identified BRD4 as a target for the treatment of a variety of hematologic and solid tumors including AML, ALL, MLL, MM and lung cancer and cancer line.JQ1, IBET151 and IBET762 positive compounds is promoted study on the targeted development of small molecule inhibitors of the BET family. In this study, the signs of compounds were screened in vitro cell characterization. In cell proliferation experiment, compound DCBD008 on the proliferation of THP-1 cells showed obvious inhibitory activity of IC50=8.098, M, and the positive compounds JQ1, IBET and characterization results. DCBD008 confirmed by inducing THP-1 cell cycle arrest in G0/G1 phase, leading to cell division can not enter the normal period. And the expression of key genes regulating transcription through C-MYC, thereby affecting the cell cycle and apoptosis play a role in cancer . the results showed that after administration of CDK6, THP-1 cells, expression of BCL2 and C-MYC genes showed a concentration dependent decrease, showed that the compound DCBD008 is through targeted inhibition of BRD4 play a role in cancer in THP-1 cells. The research target provides a new research framework of small molecule drugs to BRD4 protein the discovery and mechanism.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

【相似文献】