蛋白赖氨酸去酰化酶SIRT6和SIRT5的抑制剂研究

发布时间：2018-02-10 07:24

本文关键词： sirtuin SIRT6 SIRT5 去酰化抑制剂　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：Sirtuin是一类依赖于β-NAD+的去除蛋白酰化赖氨酸侧链酰基的去酰化酶,与其他去酰化酶不同,sirtuin催化的酰化赖氨酸侧链去酰化不是一个简单的酰胺水解反应,而是从烟酰胺腺嘌呤二核苷酸(β-NAD+或NAD+)上裂解一分子烟酰胺以生成三个产物(即去酰化的蛋白、烟酰胺和2’-O-AADP-核糖)的一个过程。现在有越来越多的研究证明sirtuin催化的去酰化反应能调节体内多种重要的生物过程,同时,该催化反应也被认为是多种人类疾病治疗的潜在靶点。SIRT6是哺乳类动物及人类中发现的7个sirtuin(SIRT1-7)之一,主要存在于细胞核中,除了催化去乙酰化反应,SIRT6具有更强的去脂肪酰(比如十四酰)的活性。SIRT6可以催化脱去组蛋白H3K9和H3K56上的乙酰基从而调控基因表达并且维持染色质端粒。此外,SIRT6催化的去酰化反应还与很多疾病息息相关,如心血管疾病,癌症,神经退行性疾病和糖尿病等。SIRT5是一个主要存在于线粒体的酶,相比于催化去乙酰化活性,SIRT5具有更强的去丙二酰、去丁二酰和去戊二酰的活性。有研究发现丁二酰化广泛存在于线粒体能量代谢调控酶中,这些酶参与调控氨基酸代谢、脂肪酸代谢和三羧酸循环等多种重要的代谢通路,所以SIRT5可以通过催化去丁二酰来调节这些代谢调控酶的活性以达到调节代谢过程的目的。由于sirtuin催化的去酰化反应与生命体的健康和疾病有着千丝万缕的关系,sirtuin抑制剂的开发在近几年引起了学者们极大的兴趣,这不仅会为相关疾病提供新的治疗方法,也为探索sirtuin的未知世界提供了一把新的钥匙。本课题针对SIRT6和SIRT5各设计了一系列的环肽类化合物,设计中运用了硫脲类sirtuin抑制弹头。主要通过固相合成的方式制得目标化合物,将化合物分离纯化后对其进行sirtuin各亚型的体外抑制测试、蛋白酶水解稳定性测试和免疫印迹测试。经过一系列的综合评估,我们发现在针对SIRT6设计的化合物中,含有Nε-十二烷基(或十四烷基)-硫代氨甲酰赖氨酸弹头的环肽类化合物对SIRT6都表现出了强效的抑制活性。此外,我们还发现其中一个化合物虽然对SIRT1也保持着很强的抑制力度,但是相对于SIRT2/3/5,其仍具有较好的SIRT6抑制选择性(分别为~20倍、~11倍和940倍)。同时我们发现该化合物还具备很好的蛋白酶水解稳定性和较好的细胞通透性。在针对SIRT5设计的化合物中,我们发现了一个强效且具有选择性的SIRT5环肽类抑制剂(相对于SIRT1/2/3/6),该化合物也被发现比其相应的直链肽化合物具有更强的稳定性。这些发展SIRT6和SIRT5抑制剂的研究为以后发展更强且具有选择性、酶解稳定且具有细胞膜通透性的代谢安全的抑制剂奠定了一个好的基础。
[Abstract]:Sirtuin is a kind of 尾 -NAD dependent deacylated lysine side chain deacylase, which is not a simple amidolysis reaction, which is different from other deacylase catalyzed by acylated lysine side chain deacylation. Instead, it cleans a molecule of nicotinamide from nicotinamide adenine dinucleotides (尾 -NAD or NAD) to produce three products, the deacylated protein, There is a growing body of research that suggests that sirtuin catalyzed deacylation can regulate many important biological processes in the body. The catalytic response is also considered to be a potential target for the treatment of multiple human diseases. SIRT6 is one of the seven sirtuin SIRT1-7 found in mammals and humans, and is mainly present in the nucleus. In addition to catalytic deacetylation, SIRT6 has a stronger activity of degreasing fatty acyl (such as 14 acyl). SIRT6 can catalyze the removal of acetyl from histone H3K9 and H3K56, which regulates gene expression and maintains chromatin telomeres. In addition, SIRT6 catalyzes the removal of acetyl groups from histone H3K9 and H3K56. The deacylation reaction is also linked to many diseases, SIRT5, such as cardiovascular disease, cancer, neurodegenerative diseases and diabetes mellitus, is an enzyme that mainly exists in mitochondria, and has stronger demalonyl than catalytic deacetylation activity. Studies have found that succinylation is widely found in mitochondrial energy metabolism regulatory enzymes, these enzymes are involved in the regulation of amino acid metabolism, fatty acid metabolism and tricarboxylic acid cycle and other important metabolic pathways. Therefore, SIRT5 can regulate the activity of these metabolic regulatory enzymes by catalyzing desuccinyl. Because sirtuin catalyzed deacylation is closely related to the health and disease of life, sirtuin is closely related to the metabolism process. The development of inhibitors has aroused great interest among scholars in recent years. This will not only provide a new treatment for related diseases, but also provide a new key to explore the unknown world of sirtuin. A series of cyclic peptide compounds have been designed for SIRT6 and SIRT5 respectively. Thiourea sirtuin inhibitory warhead was used in the design. The target compounds were prepared by solid phase synthesis. The compounds were separated and purified and tested for the inhibition of sirtuin subtypes in vitro. Protease hydrolysis stability tests and Western blotting tests. After a series of comprehensive evaluations, we found that in compounds designed for SIRT6, Cyclopeptide compounds containing N 蔚 -#number0# alkyl (or 14 alkyl-carbamyl lysine warhead) showed strong inhibitory activity against SIRT6. In addition, we also found that one of the compounds maintained a strong inhibition on SIRT1. But compared with SIRT2 / 3 / 5, it still has good SIRT6 inhibition selectivity (11 times and 940 times) respectively, and we also found that the compound also has good proteolytic stability and good cell permeability. In the compounds designed for SIRT5, We have found a potent and selective SIRT5 cyclic peptide inhibitor (compared with SIRT 1 / 2 / 3 / 6), which is also found to be more stable than its corresponding straight chain peptide compounds. In the future, the development is stronger and more selective. The enzyme-stable and membrane-permeable metabolite-safe inhibitors have laid a good foundation.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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