肌醇需求酶-1抑制剂发现及其脂代谢调控研究

发布时间：2018-01-16 03:19

本文关键词：肌醇需求酶-1抑制剂发现及其脂代谢调控研究　出处：《华东师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：内质网是真核细胞中负责蛋白折叠和装配的场所,对细胞内的蛋白质稳态平衡非常重要。内质网应激是由于Ca2+稳态平衡的紊乱、分泌蛋白合成的增加、错误折叠蛋白质的表达、葡萄糖饥饿、蛋白质糖基化的抑制或胆固醇合成超载等胁迫条件,干扰蛋白质的合成,导致内质网内积累大量的未折叠蛋白质,细胞所做出的应激反应,主要激发包括PERK-elF2α,IRE1α-XBP1及ATF6等3条未折叠蛋白反应(UPR)信号通路,通过激活CHOP、ATF4、XBP1以及ATF6等多个b-zip转录因子活性,调控相关基因的表达调控。肌醇需求酶-1(inositol-requiring enzyme-1α, IRE-lα)是最保守的UPR信号通路感应器,单次跨膜定位在内质网膜上,具备丝氨酸/苏氨酸激酶和核酸酶双重功能。IRE1α通过剪切底物XBP1mRNA,激活一系列UPR相关基因的转录,从而缓解内质网压力；IRE1还可以通过不依赖XBP1的途径,激活JNK (c-junk N-terminal kinase),引发细胞的凋亡。IRE1α-XBP1通路参与众多病理和生理过程：IRElα激酶促进突变性亨廷顿蛋白的聚集,从而加剧亨廷顿综合症；IRE1α是肿瘤血管生成的关键因子；脂肪生成依赖于IRE1α/XBP1通路的激活,等等。但目前IRE1α激酶与核酸酶之间的相互调控关系及其与疾病关系尚不明晰。发现IRE1α激酶以及核酸酶的抑制剂,对IRE1α激酶与核酸酶之间的关系研究、IRE1α信号通路调控机制以及相关药物开发具有重要意义。我们在体外运用昆虫表达体系成功地表达纯化出人源重组IRE1α蛋白,通过对底物浓度、DMSO浓度的优化,我们建立了合格的IRE1α激酶抑制剂高通量筛选模型,该模型的Z’因子为0.58,CV值为7.5%,并对包含天然产物在内的12683个化合物进行了高通量筛选,最终获得了20个有效的IRE1α激酶抑制剂。随后,通过对底物浓度、DMSO浓度以及线性时间等条件进行优化,我们建立了分子水平的IRE1α核酸酶抑制剂筛选模型,并对包括细胞水平上的XBP1剪切抑制剂、NFκB-Luciferase抑制剂和分子水平上的IRE1α激酶抑制剂共810个化合物进行筛选,最终获得5个IRE1核酸酶直接抑制剂。 IRElα-XBP1通路与脂代谢调控有着密切关系,但目前相关化合物应用主要集中在对多发性骨髓瘤的治疗评价,在脂代谢调控方面尚未见报道。本文首次对在XBP1剪切细胞模型上发现的活性化合物进行代谢调控的生物学活性评价,发现多个化合物具有抑制脂肪细胞分化和抑制肝细胞甘油三酯含量。综合考虑化合物对IRE1α核酸酶、激酶以及细胞水平对XBP1剪切细胞模型抑制活性,我们重点考察17#化合物对于脂代谢紊乱的调节作用。该化合物在分子水平上抑制IRE1核酸酶的活性,其IC50为14.92μM,在细胞水平上也显著抑制XBP1剪切,IC50约在10μM左右。功功能实验结果显示,17#化合物能明显抑制脂肪细胞3T3-L1的分化,而且抑制XBP1s以及FAS、ACC1、SCD1等相关基因表达：此外显著降低HepG2细胞中甘油三酯含量,而且抑制XBP1s、FAS、 ACC1、SCD1以及sREBP-c1等相关基因表达。综上所述,本论文的研究工作建立了IREl a激酶抑制剂分子模型以及IRE1α核酸酶的高通量筛选模型。IRE1α激酶以及核酸酶小分子抑制剂的发现为研究IRE1α激酶与核酸酶的关系提供了工具,也为IRE1α相关信号通路研究打下了基础；17#化合物的发现也为IRE1α/XBP1抑制剂在抗脂肪异常代谢类药物的开发上提供了新思路。
[Abstract]:The endoplasmic reticulum is responsible for protein folding and assembly sites in eukaryotic cells, the protein homeostasis in cells is very important. The endoplasmic reticulum stress is due to Ca2+ homeostasis disorder, secretory protein synthesis increased expression of misfolded proteins, glucose starvation, protein glycosylation or inhibition of cholesterol synthesis overload stress condition, disturbance of protein synthesis, leading to accumulation of unfolded proteins in the endoplasmic reticulum stress reaction of cells, mainly including the excitation of PERK-elF2 alpha, alpha -XBP1 and IRE1 ATF6 3 unfolded protein response (UPR) signaling pathway through the activation of CHOP, ATF4, XBP1, ATF6 and other B-ZIP transcription factors activity, expression and regulation of genes.
-1 (inositol-requiring enzyme-1 inositol requiring enzyme alpha, alpha IRE-l) UPR signaling pathway sensor is the most conservative, a single transmembrane localization in the endoplasmic reticulum, a serine / threonine kinase functions and nuclease.IRE1 alpha by shear substrate XBP1mRNA, transcriptional activation of a series of UPR related genes, to alleviate ER stress; IRE1 but also through the way which is not dependent on XBP1, activation of JNK (c-junk N-terminal kinase), triggering apoptosis of.IRE1 alpha -XBP1 pathway is involved in many physiological and pathological processes: IREl alpha kinase promotes aggregation of mutant Huntington egg white, thus exacerbating the Huntington syndrome; IRE1 alpha is a key factor in tumor angiogenesis; adipogenesis is dependent on IRE1 alpha activation of the /XBP1 pathway, and so on. But at present between IRE1 alpha kinase and nuclease regulatory interaction and its relationship with diseases is still not clear. IRE1 to alpha kinase And inhibitors of nuclease are of great significance for the study of the relationship between IRE1 alpha kinase and nuclease, the regulation mechanism of IRE1 alpha signaling pathway and the development of related drugs.
We use the insect expression system in vitro successfully expressed and purified recombinant IRE1 protein, the optimal substrate concentration, DMSO concentration, we established IRE1 alpha kinase inhibitor high-throughput screening qualified model, the model Z factor was 0.58, CV was 7.5%, and the 12683 compounds containing natural the product, a high throughput screening, finally obtained 20 effective IRE1 alpha kinase inhibitors. Then, based on the substrate concentration, DMSO concentration and linear time were optimized, we established a screening model of IRE1 nucleic acid enzyme inhibitor at the molecular level, and to include the XBP1 shear cell level IRE1 inhibitors. Alpha kappa B-Luciferase kinase inhibitor NF and inhibitor at the molecular level, a total of 810 compounds were screened, obtained 5 IRE1 nuclease inhibitors directly.
The IREl alpha -XBP1 pathway and lipid metabolism are closely related, but the related compounds mainly used in the evaluation of treatment for multiple myeloma, has not been reported in the regulation of lipid metabolism. To evaluate the biological activity for the first time on the metabolism regulation of active compounds found in XBP1 cells on the shear model, found that more than one compounds can inhibit adipocyte differentiation and inhibit liver cell triglyceride content. Considering the compounds of IRE1 alpha nuclease, inhibit the activity of kinase and cell level on the XBP1 shear cell model, we study the 17# compounds in the regulation of lipid metabolism. The compound inhibits IRE1 nuclease activity at the molecular level, which is 14.92 IC50. M, at the cellular level also significantly inhibited XBP1 shear, about IC50 at about 10 M. The function of the experimental results show that 17# can obviously inhibit fatty compounds Cell 3T3-L1 differentiation, and inhibit XBP1s and FAS, ACC1, SCD1 and other related gene expression: in addition, significantly reduce the content of triglyceride in HepG2 cells, and inhibit XBP1s, FAS, ACC1, SCD1 and sREBP-c1 and other related gene expression.
In summary, this paper established the IREl model and IRE1 molecular a kinase inhibitor alpha nuclease high-throughput screening model of.IRE1 alpha kinase and nuclease inhibitor discovery to study the relationship between IRE1 kinase and alpha nuclease provides tools for IRE1 a study related signal pathway foundation; 17# for compounds found in IRE1 alpha /XBP1 inhibitors in development of anti fat abnormal metabolism of drugs provides new ideas.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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