基于数据挖掘发现加味补中益气汤中AChE抑制剂的研究

发布时间：2019-05-22 21:52

【摘要】：目的：国医大师邓铁涛教授临床运用加味补中益气汤治疗重症肌无力(MG)几十年,其治疗效果十分显著。本研究以加味补中益气汤方为例,采用数据挖掘、确定靶标、建立数据库和虚拟筛选的现代计算机方法,研究加味补中益气汤对MG的治疗作用。并在君臣佐使中,找到主要针对乙酰胆碱酯酶(AChE)靶标的草药；对于主要的抗AChE的草药,发现抑制AChE化合物的主要骨架结构；以及发现新的AChE抑制剂；从传统中药复方得到启发,快速找到活性化合物的新方法。方法：1.研究MG发病机制,确定AChE作为研究靶标,从蛋白数据库中获得AChE的晶体蛋白结构。2.通过数据挖掘建立中药复方君臣佐使的化学成分数据库和已知的AChE抑制剂的数据库。3.本研究采用了利平斯基五规则,进行初步的化合物的类药性筛选。4.采用分子二维(2D)、三维(3D)相似度叠合技术和Glide分子对接技术,对中药复方化学成分进行基于AChE抑制剂配体的虚拟筛选。5.骨架分析,得到潜在的抗AChE活性化合物新骨架,从商业数据库中购买相同的骨架的化合物。6.酶的活性实验,以donepezil为阳性对照品,测定24个化合物的IC50值,通过Ellman酶分析实验,找到了5个活性化合物。7用分子动力学模拟的方法进行验证,验证基于分子对接虚拟筛选预测活性化合物的可靠性。结果：通过对MG发病机制的研究,预测AChE该病的主要研究靶标。研究发现加味补中益气汤中的抗AChE活性成分主要是黄酮类的化合物,同时,还发现了一类新的抗AChE的化合物骨架,通过实验验证,找到了5个新的AChE抑制剂X4, X10, X11, X19和X21,IC50分别为6.5±0.23μM,11.4±2.72μM,7.1±1.34μM,10.9±0.48μM,2.3±0.15μm。分子动力学模拟表明,预测的结合自由能△G值与新的AChE抑制剂的生物活性的数据是一致的,呈线性关系。说明通过分子对接筛选活性化合物的技术的可靠性。这项研究表明,新的活性化合物的发现可以从中药材的活性成分骨架中得到启发。结论：本研究的结论如下：(1)加味补中益气汤对重症肌无力具有治疗作用,其中君药和使药发挥了主要作用,臣药和佐药对重症肌无力的治疗可能具有不同的功能。(2)主要的活性成分是黄酮衍生物类AChE抑制剂。(3)本文发现了5个新骨架的AChE抑制剂。(4)本文提供新的研究方法,从传统的中医药经典处方,结合数据挖掘方法研究中药复方活性成分,找到活性化合物。
[Abstract]:Objective: professor Deng Tietao, a master of Chinese medicine, has been using Jiawei Buzhong Yiqi decoction in the treatment of myasthenia gravis (MG) for decades, and its therapeutic effect is very remarkable. In this study, taking Jiawei Buzhong Yiqi decoction as an example, the therapeutic effect of Jiawei Buzhong Yiqi decoction on MG was studied by using data mining, determining target, establishing database and virtual screening. Among the envoys, the herbs targeting acetylcholinesterase (AChE) were found, the main skeleton structures of AChE compounds were found for the main anti-AChE herbs, and the new AChE inhibitors were found. Inspired by traditional Chinese medicine compound, a new method of finding active compounds quickly. Methods: 1. To study the pathogenesis of MG and determine AChE as the research target, the crystal protein structure of AChE was obtained from the protein database. 2. The chemical composition database and known AChE inhibitor database of traditional Chinese medicine compound Junchen Zoran were established by data mining. 3. In this study, the Lipinski five rules were used to screen the properties of the compounds. 4. Two dimensional (2D), three dimensional (3D) similarity superposition technique and Glide molecular docking technique were used to screen the chemical components of traditional Chinese medicine based on AChE inhibitor ligands. 5. Skeleton analysis, a new skeleton of potential anti-AChE active compounds was obtained, and the same skeleton compounds were purchased from commercial databases. 6. The IC50 values of 24 compounds were determined by donepezil as positive reference, and 5 active compounds were found by Ellman enzyme analysis. 7 the results were verified by molecular dynamics simulation. The reliability of predicting active compounds based on virtual screening of molecular docking was verified. Results: through the study of the pathogenesis of MG, the main research targets of AChE were predicted. It was found that the anti-AChE active components in Jiawei Buzhong Yiqi decoction were mainly flavonoids. At the same time, a new kind of anti-AChE compound skeleton was also found. Five new AChE inhibitors X4, X10, X11 were found by experiments. The IC _ (50) of X19 and X21 are 6.5 卤0.23 渭 M, 11.4 卤2.72 渭 M, 7.1 卤1.34 渭 M, 10.9 卤0.48 渭 M and 2.3 卤0.15 渭 M, respectively. Molecular dynamics simulation shows that the predicted binding free energy G value is consistent with the data of the biological activity of the new AChE inhibitor, and there is a linear relationship between the predicted binding free energy G value and the biological activity of the new binding free energy G. It shows the reliability of the technology of screening active compounds through molecular docking. This study shows that the discovery of new active compounds can be inspired by the skeleton of active components of Chinese medicinal materials. Conclusion: the conclusions of this study are as follows: (1) Jiawei Buzhong Yiqi decoction has therapeutic effect on myasthenia gravis, in which monarch medicine and make medicine play a major role. Subjects and adjuvants may have different functions in the treatment of myasthenia gravis. (2) the main active components are flavonoids derivative AChE inhibitors. (3) five new skeleton AChE inhibitors have been found in this paper. (4) in this paper, five new skeleton AChE inhibitors have been found. (4) the main active components are flavonoids derivative AChE inhibitors. To provide new research methods, From the traditional classical prescription of traditional Chinese medicine, combined with data mining method, the active components of traditional Chinese medicine compound were studied, and the active compounds were found.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R249;R277.7

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