簡易檢索 / 詳目顯示
| 研究生： |
黃汶函 Huang, Wun-Han |
|---|---|
| 論文名稱： |
開發GSK-3β抑制劑作為治療阿茲海默症新藥 Discovery of novel GSK-3β inhibitors for Alzheimer's disease |
| 指導教授： |
李冠群
Lee, Guan-Chiun |
| 學位類別： |
碩士 Master |
| 系所名稱： |
生命科學系 Department of Life Science |
| 論文出版年： | 2015 |
| 畢業學年度： | 103 |
| 語文別： | 中文 |
| 論文頁數： | 66 |
| 中文關鍵詞： | 阿茲海默症 、肝醣合成酶激酶 、酵素抑制劑 、虛擬藥物篩選 、分子動力學模擬 |
| 英文關鍵詞： | Alzheimer's disease, GSK-3β, enzyme inhibitor, virtual screening, MD simulation |
| 論文種類： | 學術論文 |
| 相關次數： | 點閱：173 下載：27 |
| 分享至: |
| 查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
阿茲海默症是目前最常見的一種失智症，為一種不可逆的神經退化性疾病，會造成記憶與行為上的缺失，以及人格的改變，有兩個主要的病徵，分別是類澱粉斑(Amyloid plaques)和神經纖維纏結(Neurofibrillary tangles, 簡稱NFTs)，於罹患阿茲海默症之病人腦部發現。GSK-3β蛋白磷酸化tau蛋白會促使NFTs形成，在阿茲海默症中扮演很重要的角色，因此抑制GSK-3β蛋白之活性會是一個治療阿茲海默症的方式。我們與本校化學系孫英傑老師合作，利用電腦輔助藥物設計及分子間作用力分析技術，開發GSK-3β抑制劑作為治療阿茲海默症新藥。為了獲得足夠的GSK-3β進行抑制劑之篩選，我們構築了含人類GSK-3β基因的質體，並於多株不同的大腸桿菌宿主中表達，然而GSK-3β卻形成了包涵體(Inclusion body)，為此，我們將蛋白表達的條件進一步優化，以及進行蛋白的重折疊(Protein refolding)，期望能獲得可溶的蛋白，然而卻依然無法提升蛋白的溶解度。因此，在本研究中，我們購買了GSK-3β蛋白進行抑制劑之篩選，在電腦模擬篩選出的三十八個藥物中，我們以抑制後蛋白殘餘活性在百分之六十五以下為篩選標準，共篩選出了三個抑制劑，這些抑制劑有潛力能成為治療阿茲海默症之藥物。
Alzheimer's disease (AD) is the most common form of dementia in the elderly. AD is a progressive and irreversible neurodegenerative disorder that results in significant memory loss and behavioral and personality changes. The two hallmarks of AD, the amyloid plaques and neurofibrillary tangles (NFTs) are found in the brain of those with a diagnosis of AD. Hyperphosphorylation of tau by GSK-3β plays a crucial role in NTF formation. As GSK-3β activation is a critical step in the cascade of detrimental events in AD, therapeutics targeted to inhibiting GSK-3β may be beneficial in the treatment of this devastating disease. In the present study, computer modeling (by Dr. Ying-Chieh Sun) and experimental analysis were used to assist discovery/design of GSK-3β inhibitors. To obtain sufficient enzyme sample for the inhibitor screening, human GSK-3β gene has been cloned and expressed in several E. coli strains. However, the recombinant GSK-3β was expressed as inclusion bodies. Conditions of protein expression and protein refolding were further optimized to express active soluble protein and still cannot improve the solubility of recombinant GSK-3β. GSK-3β assay has been established by using commercial human GSK-3β to screen for potent inhibitors. Three of the 38 compounds predicted by computer modeling have been tested to be potent inhibitors with a residual activity lower than 65% of the activity without inhibitor. These three inhibitors could be applied in the treatment of AD.
Braak, H., & Braak, E. (1991a). Demonstration of amyloid deposits and neurofibrillary changes in whole brain sections. Brain Pathol, 1(3), 213-216.
Braak, H., & Braak, E. (1991b). Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol, 82(4), 239-259.
Grimes, C. A., & Jope, R. S. (2001). The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol, 65(4), 391-426.
Ishiguro, K., Shiratsuchi, A., Sato, S., Omori, A., Arioka, M., Kobayashi, S., Uchida, T., Imahori, K. (1993). Glycogen synthase kinase 3 beta is identical to tau protein kinase I generating several epitopes of paired helical filaments. FEBS Lett,325(3), 167-72.
Lee, M. S., Kao, S. C., Lemere, C. A., Xia, W., Tseng, H. C., Zhou, Y., Neve, R., Ahlijanian, M. K., Tsai, L. H. (2003). APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol, 163(1), 83-95.
Lucas, J. J., Hernandez, F., Gomez-Ramos, P., Moran, M. A., Hen, R., & Avila, J. (2001). Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J, 20(1-2), 27-39.
Massoud, F., & Gauthier, S. (2010). Update on the pharmacological treatment of Alzheimer's disease. Curr Neuropharmacol, 8(1), 69-80.
Nakashima, H., Ishihara, T., Suguimoto, P., Yokota, O., Oshima, E., Kugo, A., Terada, S., Hamamura, T., Trojanowski, J. Q., Lee, V. M., Kuroda, S. (2005). Chronic lithium treatment decreases tau lesions by promoting ubiquitination in a mouse model of tauopathies. Acta Neuropathol, 110(6), 547-556.
Phukan, S., Babu, V. S., Kannoji, A., Hariharan, R., & Balaji, V. N. (2010). GSK3beta: role in therapeutic landscape and development of modulators. Br J Pharmacol, 160(1), 1-19.
Takashima, A. (2006). GSK-3 is essential in the pathogenesis of Alzheimer's disease. J Alzheimers Dis, 9(3 Suppl), 309-317.
ter Haar, E., Coll, J. T., Austen, D. A., Hsiao, H. M., Swenson, L., & Jain, J. (2001). Structure of GSK3beta reveals a primed phosphorylation mechanism. Nat Struct Biol, 8(7), 593-596.
Woodgett, J. R. (1990). Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J, 9(8), 2431-2438.
Wuwongse, S., Chang, R. C., & Law, A. C. (2010). The putative neurodegenerative links between depression and Alzheimer's disease. Prog Neurobiol, 91(4), 362-375.
Xu, S. F., Xu, J., & Li, M. T. (2011). Comparison of two techniques for expression and purification of glycogen synthase kinase 3beta. Nan Fang Yi Ke Da Xue Xue Bao, 31(3), 397-402.
Yoon, S. Y., Choi, J. E., Huh, J. W., Hwang, O., Nam Hong, H., & Kim, D. (2005). Inactivation of GSK-3beta in okadaic acid-induced neurodegeneration: relevance to Alzheimer's disease. Neuroreport, 16(3), 223-227.
Zegzouti, H., Zdanovskaia, M., Hsiao, K., & Goueli, S. A. (2009). ADP-Glo: A Bioluminescent and homogeneous ADP monitoring assay for kinases. Assay Drug Dev Technol, 7(6), 560-572.
