新型Caspase-1小分子抑制剂的发现

发布时间：2018-03-14 23:26

本文选题：炎症Caspase　切入点：Caspase-1　出处：《华东师范大学》2006年硕士论文　论文类型：学位论文

【摘要】：炎症Caspase(Cystine proteases with aspartic acid substrate specificity)是Caspases家族中一类与炎症反应重要相关的蛋白酶，其主要功能是产生成熟的促炎症细胞因子IL-1β和IL-18。成熟的IL-1β和IL-18具有广泛的生物活性，并与多种疾病有关，如各种病毒、细菌、真菌等微生物的感染、外伤、心肌梗塞、肝炎、胰腺炎、关节炎、神经炎症和神经退行性疾病。炎症Caspase包括Caspase-1、-4、-5、-11、-12。其中最具代表性的是Caspase-1，是切割IL-1β和IL-18的高效特异的蛋白酶。体外和体内实验结果表明抑制炎症Caspase，尤其是Caspase-1的活性，能缓解炎症过度引起的各种疾病，如关节炎，牛皮癣、神经损伤等。因此，Caspase-1成为治疗炎症过度性疾病的重要靶点，寻找Caspase-1高效高选择性的抑制剂为治疗这类疾病提供了新的途径。本论文的目的在于表达和纯化Caspase-1重组蛋白，研究其酶学性质，并在此基础上建立Caspase-1抑制剂分子水平的高通量筛选模型，并通过对国家化合物样品库的筛选，，寻找高亲和力的Caspase-1抑制剂，通过对其性质进行研究，为进一步的化合物结构改造以及细胞和动物水平的药理学、药效学研究打下基础。本文的研究内容包括用RT-PCR方法克隆Caspase-1全长基因，并根据文献报道将Caspase-1的第381位氨基酸突变使重组蛋白更稳定。将Caspase-1的催化区域克隆到表达载体pET21b上，并在重组蛋白的N端加上(His)_6标签。将重组质粒转化大肠杆菌BL21-CodonPlus(DE3)后经IPTG诱导表达。通过Ni~(2+)亲和层析柱纯化，获得较纯净的活性形式Caspase-1重组蛋白。通过对Caspase-1的一系列酶学特性，包括DTT的影响、pH依赖性、动力学分析及阳性抑制剂抑制作用分析，优化Caspase-1的活性检测方法，建立了Caspase-1抑制剂高通量筛选的标准化操作流程。通过对国家化合物样品库中的47360个样品进行筛选，得到了16个IC_(50)低于10μM抑制活性较好的化合物。其中一活性化合物MXQ-1的IC_(50)为163nM，为新型的Caspase-1抑制剂。通过分子水平酶动力学方法对其进行抑制性质研究，发现该化合物为可逆的、慢结合型广谱Caspase抑制剂，其与Caspase-1之间的相互作用模式为简单型相互作用，并不发生酶的异构化过程。这一新型Caspase-1抑制剂的发现和相关抑制性质的研究为其进一步结构优化和药理学、药效学研究打下坚实的基础。
[Abstract]:Inflammatory Caspase(Cystine proteases with aspartic acid substrate specificity (Caspase(Cystine proteases with aspartic acid substrate specificity) is a kind of protease related to inflammation in Caspases family. Its main function is to produce mature inflammatory cytokines IL-1 尾 and IL-18.The mature IL-1 尾 and IL-18 have a wide range of biological activities and are related to many diseases. Such as viruses, bacteria, fungi and other microbial infections, trauma, myocardial infarction, hepatitis, pancreatitis, arthritis, Inflammation and neurodegenerative diseases. Inflammatory Caspase includes Caspase-1, caspase-1, caspase-1, caspase-1, a highly efficient and specific protease that cleans IL-1 尾 and IL-18. In vitro and in vivo experiments have shown that it inhibits the activity of inflammatory caspase, especially Caspase-1. It can relieve various diseases caused by excessive inflammation, such as arthritis, psoriasis, nerve damage, etc. Therefore, Caspase-1 has become an important target for the treatment of excessive inflammatory diseases. The aim of this paper is to express and purify the recombinant Caspase-1 protein and study its enzymatic properties. On the basis of this, a high throughput screening model of Caspase-1 inhibitors at molecular level was established, and a high affinity Caspase-1 inhibitor was found by screening the national compound sample bank, and its properties were studied. It provides a basis for further structural modification of compounds and pharmacological and pharmacodynamic studies at cellular and animal levels. In this paper, the full-length Caspase-1 gene was cloned by RT-PCR, and the 381st amino acid mutation of Caspase-1 was reported to make the recombinant protein more stable. The catalytic region of Caspase-1 was cloned into the expression vector pET21b. The recombinant plasmid was transformed into Escherichia coli BL21-CodonPlusDE3 and expressed by IPTG. The recombinant protein was purified by Ni~(2 affinity chromatography. A series of enzymatic properties of Caspase-1 were obtained. Including the effect of DTT on pH dependence, kinetic analysis and inhibition analysis of positive inhibitors, optimization of Caspase-1 activity detection method, A standardized procedure for high throughput screening of Caspase-1 inhibitors was established. 47360 samples from the national sample bank were screened. 16 compounds with better inhibitory activity than 10 渭 M were obtained, in which one active compound, MXQ-1, was 163nM, which was a new type of Caspase-1 inhibitor. The inhibitory properties of the compound were studied by enzyme kinetic method at molecular level, and it was found that the compound was reversible. A slow binding broad-spectrum Caspase inhibitor whose interaction mode with Caspase-1 is simple. There is no isomerization process of the enzyme. The discovery of this new Caspase-1 inhibitor and the study of its related inhibitory properties lay a solid foundation for its further structural optimization pharmacological and pharmacodynamic studies.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【引证文献】