基于HDAC和NO双靶点抗癌先导化合物的设计、合成与活性研究

发布时间：2018-06-20 04:40

本文选题：表观遗传学 + 组蛋白去乙酰化酶抑制剂　；参考：《山东大学》2015年博士论文

【摘要】：本研究论文是以表观遗传学为基础,利用药物分子杂交策略,以发现多靶点抗肿瘤药物为目标,将时下抗肿瘤研究最热门的领域组蛋白去乙酰化酶抑制剂(HDACI)与能够产生“明星分子”一氧化氮(NO)的药物小分子片段一氧化氮供体(NO donor)结合在一起,设计合成了25个分子结构新颖的NO-HDACI,并对其进行了系统的体内外生物活性评价并对其结果进行了详细的讨论。全文共分四个部分进行分章论述。一、研究背景组蛋白去乙酰化酶(HDACs)是一类可以水解掉组蛋白或非组蛋白末端赖氨酸残基上乙酰基的蛋白酶家族,可以使染色质形成一个转录抑制的状态。HDACs活性的异常与基因的异常表达及包括癌症在内的多种人类疾病的发生发展有着密切的联系。抑制HDACs可以恢复正常基因的表达,从而可以阻滞癌细胞的细胞周期,促进细胞分化及细胞凋亡。因此,阻断HDACs非正常去乙酰化作用的组蛋白去乙酰化酶抑制剂(HDACI)作为潜在的有效的抗肿瘤药物便应运而生。HDACI主要包括四类：异羟肟酸类,短链脂肪酸类,环状四肽类以及苯甲酰胺类。异羟肟酸类是研究最广最深入的一类,目前该类已有三个药物(SAHA, PXD-101和LBH-589)被FDA批准上市,分别用来治疗皮肤T细胞淋巴瘤、外周T细胞淋巴瘤及多发性骨髓瘤。一氧化氮(NO)作为有机体内重要的信使小分子,参与体内诸多生理过程,比如血管调节,神经传递,炎症以及免疫反应等过程。此外,研究表明,NO还可以抑制肿瘤细胞的增殖,转移,血管生成以及促进肿瘤细胞的凋亡。在体内,NO的产生在一氧化氮合成酶(iNOS)参与下生成以外,一氧化氮供体(NO donor)也是产生高浓度NO的重要方式。1,2,5-恶二唑-2-氧化物(呋咱氮氧化物)是2008年通过高通量筛选而得到的一个重要的具有开发潜力的NO供体,它可以在体外生成高浓度的NO以及在体内具有抑制肿瘤生长的作用。因此,多年来,很多新药特别是抗肿瘤药物的研发过程中都会看到它的身影。近年来,越来越多的研究发现,由NO引起的巯基亚硝酸化或蛋白质酪氨酸硝化等共价修饰显著影响着细胞功能。HDAC的很多家族成员可以直接或间接的以NO为靶标,而且已经有报道证明了部分HDAC的功能也依赖于NO的调节。更重要的是,曾有研究证明了NO和HDACI在治疗心肌肥厚及伤口愈合方面存在着协同作用。这就为两者在某些疾病的联合应用上提供了充分的证据。分子杂交策略是新药研发过程中发现多靶点药物的重要手段之一,本论文的研究主要是以设计多靶点药物为原则,以分子杂交策略手段将NO供体融入进HDACI中来,合理设计、定向合成具有全新结构的NO-HDACI,以期待可以得到体内外抗肿瘤活性更好,靶向更明确,选择性更高的先导化合物,为进一步开发具有我国自主知识产权的创新性抗肿瘤药物奠定基础。二、苯磺酰呋咱氮氧化物一氧化氮供体型组蛋白去乙酰化酶抑制剂的设计,合成及抗肿瘤活性研究苯磺酰呋咱氮氧化物是一个经典的1,2,5-恶二唑-2-氧化物的一氧化氮供体,在各种组织及器官中不需要酶的作用即可释放出大量的NO。由于其对酸碱相对稳定,该片段一直被用来与多种药物分子进行偶联且大部分显示出了比原药更强的药效。鉴于此,本课题首选该片段作为NO供体参与一氧化氮供体型HDACI的设计及合成。根据苯磺酰呋咱氮氧化物的结构特点以及HDACI的结构特征,共设计合成出了15个全新的化合物。所有的目标化合物先后经过了对Hela细胞提取物(主要包含HDAC1和HDAC2)和8株肿瘤细胞增殖抑制活性的评价,结果显示大部分化合物都具有明显的抑制活性,特别是目标化合物5c与阳性药SAHA相比,抑制效果尤为突出,其敏感的肿瘤细胞株为HEL细胞。而且经对NO释放含量的测定发现,目标化合物5c可以释放大量的NO,对5c经过NO清除剂Hemoglobin (Hb)的预处理后,发现其对HEL细胞增殖的抑制活性与Hb的浓度增加而呈梯度递减,说明其对肿瘤细胞增殖的抑制是由释放NO和抑制HDACs酶两方面的共同作用。进一步的机制研究表明相对于阳性药SAHA,目标化合物5c对HEL细胞能引起比较强的细胞凋亡,而且主要作用在细胞周期的G1期。裸鼠体内实验结果发现,5c相较于阳性药SAHA在同等剂量,相同给药方式及给药时间的情况下,具有明显优于SAHA的抑制肿瘤生长的作用,而且几乎不存在体内毒性。此外,目标化合物5c在分子水平上显示出对HDACs的亚型HDAC6明显的选择性,该发现可以作为进一步设计探索HDAC6选择性抑制剂的重要依据。三、苯并氧化呋咱氮氧化物一氧化氮供体型组蛋白去乙酰化酶抑制剂的设计,合成及抗肿瘤活性研究苯并氧化呋咱是1,2,5-恶二唑-2-氧化物的另一个应用广泛的NO供体片段,因为可以释放大量的NO而应用在心血管疾病及抗肿瘤药物的研发上。因此,本论文的另一部分研究内容就是将该片段作为NO供体融入目标化合物的设计,共合成出10个全新的NO-HDACI。生物活性评价结果表明,大部分化合物都表现出明显的抑制酶活的作用,而且根据初步的构效关系可以看出其连接臂长度与苯并氧化呋咱的位置对其抑制酶活性强弱具有重要影响。通过MTT实验可以看出,目标化合物10d和15d在抑制HCT116肿瘤细胞株的增殖上明显的优于阳性药SAHA。此外,作为有NO供体存在的HDACI,在NO释放量的评价上,目标产物10d和15d在细胞内能产生较大量的NO,而且随着NO清除剂Hemoglobin浓度的增加其抑制肿瘤细胞增殖的作用减弱,充分说明了我们设计合成的目标化合物在体外同时具有抑制HDACs酶活的作用和释放NO的作用,这对多靶点抗肿瘤药物设计合成具有积极的指导意义。四、全文总结与展望本课题是以发现多靶点抗肿瘤药物为前提,综合运用药物化学、化学生物学、计算机化学等前沿学科的交叉,利用分子杂交策略将NO供体融入组蛋白去乙酰化酶抑制剂(HDACI)的研设计中,定向合成了两个系列共25个具有全新骨架结构的NO-HDACI。对所有目标化合物进行了分子水平的抑酶活性筛选,从中选取活性较好的化合物逐层进行细胞水平、动物水平的抗肿瘤活性测试,利用得到的构效关系信息对表现优异的化合物进行进一步的结构改造、修饰和优化。结果表明：当把NO供体融进HDACI分子中时,其抗肿瘤效果会明显提高,就目前得到的化合物5c来讲,无论是在体内还是体外的抗肿瘤活性上都明显优于上市药物SAHA,同时在分子水平还表现出比较显著的选择性。化合物5c的临床前药理活性还是进一步的评价中,同时,对其进一步的结构优化及活性筛选也在进行中。该论文的呈现,无论是为多靶点药物的设计发现还是为选择性HDACI的研究都奠定了良好的基础。
[Abstract]:This study is based on epigenetics, using the drug molecular hybridization strategy to find multi target antitumor drugs as the target, the most popular domain of antitumor research histone deacetylase inhibitor (HDACI) and a small molecule fragment of nitric oxide (NO do) that can produce "star molecule" nitrogen oxide (NO). Nor) combined together, 25 novel NO-HDACI molecular structures were designed and synthesized, and the biological activity of the system was evaluated and the results were discussed in detail. The full text was divided into four chapters. First, the research background histone deacetylase (HDACs) is a class of proteins that can hydrolyze histone or non egg group. The family of acetylated proteinases on the white terminal lysine residue can make the chromatin form a transcriptional inhibition state of the.HDACs activity, which is closely related to the abnormal expression of genes and the development of a variety of human diseases, including cancer. Inhibition of HDACs can restore the expression of normal genes, thus blocking cancer fines. The cell cycle of the cell promotes cell differentiation and apoptosis. Therefore, the histone deacetylase inhibitor (HDACI), which blocks the abnormal deacetylation of HDACs as a potential effective antitumor drug, arises at the historic moment of.HDACI mainly including four types: hydroxamic acid, short chain fatty acids, ring four peptides, and benzamides. The hydroxamic acid is the most widely studied class. At present, three drugs (SAHA, PXD-101 and LBH-589) have been approved by FDA to treat skin T cell lymphoma, peripheral T cell lymphoma and multiple myeloma. Nitric oxide (NO), as an important messenger in the body, is involved in many physiological processes in the body. Such as vascular regulation, neurotransmission, inflammation and immune response. In addition, NO can also inhibit the proliferation, metastasis, angiogenesis, and apoptosis of tumor cells. In vivo, the production of NO is produced by nitric oxide synthase (iNOS), and the nitric oxide donor (NO donor) is also a high concentration of NO. The important way.1,2,5- oxide two azole -2- oxide (furazono NOx) is an important development potential NO donor by high throughput screening in 2008. It can produce high concentration of NO in vitro and inhibit tumor growth in vivo. Therefore, many new drugs, especially antitumor drugs, have been developed for many years. In recent years, more and more studies have found that covalent modification of mercapto nitrification or protein tyrosine nitrification caused by NO significantly affects a number of family members of the cell function.HDAC directly or indirectly with NO as a target, and it has been reported that part of the function of HDAC is dependent on the function of the cell. More importantly, there has been a study of the synergistic effects of NO and HDACI in the treatment of myocardial hypertrophy and wound healing. This provides sufficient evidence for the combination of some diseases. The molecular hybridization strategy is one of the most important methods for the discovery of multiple target drugs in the development of new drugs. This paper has been studied in this paper. The study is mainly based on the principle of designing multi target drugs and integrating the NO donor into HDACI by molecular hybridization strategy. It is reasonable designed and directed to synthesize a new structure of NO-HDACI, so as to expect to get better antitumor activity in vivo and in vivo, more targeted and selective, and to develop our own autonomy for further development. Two, design, synthesis and antitumor activity of benzsulfonacylfurazono nitrous oxide nitric oxide donor histone deacetylase inhibitor, benzsulfonacylfurazine oxide is a classical nitric oxide donor of 1,2,5- evil two azole oxide, in various tissues and organs. The release of a large number of NO. without the action of enzymes has been used for the coupling of a variety of drug molecules and most of the efficacy of the drug as a result of its relative stability to the acid base. In view of this, this subject is preferred as a NO donor in the design and synthesis of nitric oxide donor HDACI, based on benzsulfonyl. The structural characteristics of nitrous oxide and the structural characteristics of HDACI have been designed and synthesized. 15 new compounds have been designed and synthesized. All target compounds have been evaluated for the inhibitory activity of Hela cell extracts (mainly including HDAC1 and HDAC2) and 8 tumor cells. The results show that most of the compounds have obvious inhibitory activity. The inhibitory effect, especially the target compound 5C, is particularly prominent compared with the positive drug SAHA. The sensitive tumor cell line is HEL cells. And the target compound 5C can release a large number of NO by determination of the release content of NO, and the inhibitory activity of 5C after the NO scavenger Hemoglobin (Hb) is detected and its inhibitory activity to the proliferation of HEL cells is found. The concentration of Hb increases with a gradient decreasing, indicating that the inhibition of tumor cell proliferation is a common effect of releasing NO and inhibiting HDACs enzyme. Further mechanism studies show that relative to positive drug SAHA, the target compound 5C can induce stronger apoptosis in HEL cells, and mainly in the G1 phase of the cell cycle and nude mice. The results of the internal experiment showed that 5C had the same effect as the positive drug SAHA in the same dosage, the same way and the time of administration, which was obviously superior to SAHA in inhibiting the growth of the tumor and almost no body toxicity. In addition, the target compound 5C showed a distinct selectivity to the HDACs subtype HDAC6 at the molecular level. This discovery could be found. As an important basis for further design and exploration of selective inhibitors of HDAC6. Three, design, synthesis and antitumor activity of benzooxide furazono nitric oxide nitric oxide donor deacetylase inhibitor, benzo oxide furazan, another widely used NO donor fragment of 1,2,5- oxazolide -2- oxide, because it can be used. The release of a large number of NO was applied to the development of cardiovascular diseases and antitumor drugs. So, another part of this paper was to integrate the fragment as a NO donor in the design of the target compound, and a total of 10 new NO-HDACI. bioactivity evaluations showed that most of the compounds showed significant inhibition of enzyme activity. According to the preliminary structure-activity relationship, it can be found that the length of the connecting arm and the position of benzo oxidizing furazan have an important effect on the inhibitory activity of the inhibitor. Through the MTT experiment, we can see that the target compound 10d and 15d are obviously superior to the positive drug SAHA. in inhibiting the proliferation of HCT116 tumor cell lines, as a NO donor. In the HDACI, the target products 10d and 15d can produce a large number of NO in the cell, and the effect of the NO scavenger Hemoglobin concentration increases with the increase of the NO scavenger Hemoglobin concentration. It shows that the target compounds we designed and synthesized have the inhibition of HDACs enzyme activity and the release of NO in vitro. It has a positive guiding significance for the design and synthesis of multi target antitumor drugs. Four. The full text summary and prospect is based on the discovery of multi target antitumor drugs as the premise, the integrated use of pharmaceutical chemistry, chemical biology, Computer Chemistry and other frontier disciplines, the use of molecular hybridization strategy to integrate NO donor to histone deacetylation In the design of the enzyme inhibitor (HDACI), a total of two series of 25 new NO-HDACI. skeleton structures were synthesized, and all the target compounds were screened at molecular level. The results showed that the anti tumor effect of the NO donor was obviously improved when the HDACI molecule was incorporated into the molecule, which was obviously superior to the drug SAHA in both in vivo and in vitro, both in the body and in vitro, at the same time, at the same time. The molecular level also shows significant selectivity. The preclinical pharmacological activity of compound 5C is still further evaluated, and its further structural optimization and activity screening are also in progress. The presentation of this paper has laid a good foundation for the design of multiple target drugs or for the study of selective HDACI. Foundation.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R914;R96

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