臺灣博碩士論文加值系統

本研究是討論如何利用電腦發展的分子結構的應用軟體，與生物科技或分子醫學相關的配合及應用，探討如何利用SARS 冠狀病毒（Coronavirus）複製時所需的主要蛋白酶（main proteinase）1Q2W，與文獻中找到的四種化合物（Compound）AG7088、Fig_4C、Niclosamide與Promazine，及1Q2W解結晶時使用的Ligand用Autodock對虛擬藥物MPD、AG7088、Fig_4C、Niclosamide、Promazine，於MPD結合位置（Binding site ）位置的抑制情形做分析，結果得到Ki，以Ki來說這五個藥物結合狀況都不錯，這部份以Fig_4C為最佳，與Fig_4C是由生物資訊（Bioinformatics）方式取得產生有關，且利用藥物對Ligand結合位置的殘基覆蓋遮蔽的狀況來說，AG7088、Fig_4C有較佳的阻斷性，此結果與兩者取得方式有關，在結合位置作用的Ligand與PRO-108作用為電腦分析所得，之後經過電腦模擬是否與PRO-108有氫鍵作用，則為本研究分析參考判斷之一。利用分析結合位置與抑制劑間作用關係，隨著未來的更完整的分子科學與量子力學等各方面進步下科學，相信在醫療或其他領域下的研發使用會有更迅速的突破。

In this study, the developments of applications of the molecular structure software, using computers with biotechnology or molecular medicines, were discussed. Four compounds, such as AG7088, Fig_4C, Niclosamide, and Promazine, which have been found in the literature, may inhibit the main proteinase of SARS coronavirus duplications. The crystal structure （the PDB code 1Q2W） of SARS main proteinase including ligand MPD through the Autodock analysis revealed the four hypothesized medicines at the binding site of MPD with the position suppressions. From the values of Ki, all of these five medicines binding conditions are quite good, whereas Fig_4C is the best. Based on the binding position to a ligand with a cover camouflage situation, AG7088 and Fig_4C have a good block in the nature, which has a good agreement found in the literature. The function of PRO-108 from the Autodock analysis was obtained. Whether the results obtained from the computer simulation have the hydrogen bonds with PRO-108 is one of reference judgments. The analysis unifies the relationships between the position and inhibitors. I believed that this method may have a more rapid breakthrough to find potential drugs or the developments of other domain researches.

目次

摘要...................................................1
致謝...................................................3
目次...................................................4
第一章 序論.............................................7
1.1 文獻回顧............................................7
1.1.1 冠狀病毒 Coronavirus..............................7
1.1.2 Coronavirus種的分類...............................7
1.1.3 Sars coronavirus.................................8
1.1.4 冠狀病毒生活史（Coronavirus Life Cycle） ..........10
1.1.5 3CL proteinase...................................12
1.2 研究動機............................................13
1.3 研究目的............................................14
第二章 材料.............................................15
2.1 材料準備............................................15
2.1.1 目標蛋白酶 3CL_CoV................................15
2.1.2 Substrate........................................15
2.2 軟體及工具..........................................18
第三章 方法.............................................23
3.1 基因演算法..........................................23
3.1.1染色體編碼.........................................23
3.1.2 目標函數與適應函數.................................24
3.1.3 選擇.............................................24
3.1.4 基因操作..........................................24
3.1.4.1 交配............................................24
3.1.4.2 突變............................................25
3.1.4.2 重組............................................25
3.1.5 操作配比設定.......................................26
3.1.6 替換..............................................26
3.2 Ki值的使用...........................................27
3.3 AutoDock 設定參數....................................27
3.3.1 Grid參數意義.......................................27
第四章 結果與討論..........................................31
4.1 實驗數據.............................................31
4.2 目標蛋白與substrate之間交互作用的狀況...................35
第五章 結論...............................................50
參考資料..................................................51
附件.....................................................60
附件 A: 以Fig_4C為例＊.GPF檔格式...........................60
附件 B: 以Fig_4C為例＊.DPF檔格式...........................62

[1]Ziebuhr J, Snijder EJ, Gorbalenya AE. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81 （Pt 4）: 853-79, 2000.
[2]Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen MH, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TC, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Rasmussen M, Fouchier R, Gunther S, Osterhaus AD, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300 （5624）: 1394-9, 2003.
[3]Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348 （20）: 1967-76, 2003.
[4]http://www.fda.gov/fdac/features/2003/403_sars.html
[5]http://www.ds-shanghai.org.cn/Uebersicht/Gesundheit.html
[6]Xiong B, Gui CS, Xu XY, Luo C, Chen J, Luo HB, Chen LL, Li GW, Sun T, Yu CY, Yue LD, Duan WH, Shen JK, Qin L, Shi TL, Li YX, Chen KX, Luo XM, Shen X, Shen JH, Jiang HL. A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening. Acta Pharmacol Sin 24 （6）: 497-504, 2003.
[7]Qin L, Xiong B, Luo C, Guo ZM, Hao P, Su J, Nan P, Feng Y, Shi YX, Yu XJ, Luo XM, Chen KX, Shen X, Shen JH, Zou JP, Zhao GP, Shi TL, He WZ, Zhong Y, Jiang HL, Li YX. Identification of probable genomic packaging signal sequence from SARS-CoV genome by bioinformatics analysis. Acta Pharmacol Sin 24 （6）: 489-96, 2003.
[8]Ziebuhr J, Snijder EJ, Gorbalenya AE. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81 （Pt 4）: 853-79, 2000.
[9]Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main proteases. J Gen Virol 83 （Pt 3）: 595-9, 2002.
[10]Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main proteases. J Gen Virol 83 （Pt 3）: 595-9, 2002.
[11]Ziebuhr J, Snijder EJ, Gorbalenya AE. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81 （Pt 4）: 853-79, 2000.
[12]Raamsman MJ, Locker JK, de Hooge A, de Vries AA, Griffiths G, Vennema H, Rottier PJ. Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol 74 （5）: 2333-42, 2000.
[13]Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main proteases. J Gen Virol 83 （Pt 3）: 595-9, 2002.
[14]Ziebuhr J, Siddell SG. Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab. J Virol 73 （1）: 177-85, 1999.
[15]Bonilla PJ, Hughes SA, Weiss SR. Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59. J Virol 71 （2）: 900-9, 1997.
[16]Ziebuhr J, Snijder EJ, Gorbalenya AE. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81 （Pt 4）: 853-79, 2000.
[17]Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main proteases. J Gen Virol 83 （Pt 3）: 595-9, 2002.
[18]Holmes KV. SARS coronavirus: a new challenge for prevention and therapy. J Clin Invest 111 （11）: 1605-9, 2003.
[19]Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348 （20）: 1953-66, 2003.
[20]Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen MH, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TC, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Rasmussen M, Fouchier R, Gunther S, Osterhaus AD, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300 （5624）: 1394-9, 2003.
[21]Xiong B, Gui CS, Xu XY, Luo C, Chen J, Luo HB, Chen LL, Li GW, Sun T, Yu CY, Yue LD, Duan WH, Shen JK, Qin L, Shi TL, Li YX, Chen KX, Luo XM, Shen X, Shen JH, Jiang HL. A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening. Acta Pharmacol Sin 24 （6）: 497-504, 2003.
[22]Anand K, Palm GJ, Mesters JR, Siddell SG, Ziebuhr J, Hilgenfeld R. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. Embo J 21 （13）: 3213-24, 2002.
[23]Hegyi A, Friebe A, Gorbalenya AE, Ziebuhr J. Mutational analysis of the active centre of coronavirus 3C-like proteases. J Gen Virol 83 （Pt 3）: 581-93, 2002.
[24]Yan L, Velikanov M, Flook P, Zheng W, Szalma S, Kahn S. Assessment of putative protein targets derived from the SARS genome. FEBS Lett 554 （3）: 257-63, 2003.
[25]Ziebuhr J, Siddell SG. Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab. J Virol 73 （1）: 177-85, 1999.
[26]Ziebuhr J, Heusipp G, Siddell SG. Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase. J Virol 71 （5）: 3992-7, 1997.
[27]Ziebuhr J, Herold J, Siddell SG. Characterization of a human coronavirus （strain 229E） 3C-like proteinase activity. J Virol 69 （7）: 4331-8, 1995.
[28]Herold J, Gorbalenya AE, Thiel V, Schelle B, Siddell SG. Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate. J Virol 72 （2）: 910-8, 1998.
[29]Zhang XW, Yap YL. Old drugs as lead compounds for a new diseases Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs. Bioorg Med Chem 12 （10）: 2517-21, 2004.
[30]Veselovsky AV, Medvedev AE, Tikhonova OV, Skvortsov VS, Ivanov AS. Modeling of substrate-binding region of the active site of monoamine oxidase A. Biochemistry （Mosc） 65 （8）: 910-6, 2000.
[31]Yu XJ, Luo C, Lin JC, Hao P, He YY, Guo ZM, Qin L, Su J, Liu BS, Huang Y, Nan P, Li CS, Xiong B, Luo XM, Zhao GP, Pei G, Chen KX, Shen X, Shen JH, Zou JP, He WZ, Shi TL, Zhong Y, Jiang HL, Li YX. Putative hAPN receptor binding sites in SARS_CoV spike protein. Acta Pharmacol Sin 24 （6）: 481-8, 2003.
[32]Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348 （20）: 1953-66, 2003.
[33]World Health Organisation, Cumulative Number of Reported Probable Cases of SARS , http://www.who.int/csr/sars/country/en/; （b） Centre for Disease Control and Prevention, Severe Acute Respiratory Syndrome , http://www.cdc.gov/ncidod/sars/.
[34]Bouadma L, Noel V, Schortgen F. Managing SARS. N Engl J Med 349 （7）: 707-8; author reply 707-8, 2003.
[35]Xiong B, Gui CS, Xu XY, Luo C, Chen J, Luo HB, Chen LL, Li GW, Sun T, Yu CY, Yue LD, Duan WH, Shen JK, Qin L, Shi TL, Li YX, Chen KX, Luo XM, Shen X, Shen JH, Jiang HL. A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening. Acta Pharmacol Sin 24 （6）: 497-504, 2003.
[36]RCSB PDBhttp://www.rcsb.org/
[37]Enhanced NCI Database Browser Release 2 http://cactus.nci.nih.gov/ncidb2/
[38]Patick AK, Binford SL, Brothers MA, Jackson RL, Ford CE, Diem MD, Maldonado F, Dragovich PS, Zhou R, Prins TJ, Fuhrman SA, Meador JW, Zalman LS, Matthews DA, Worland ST. In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease. Antimicrob Agents Chemother 43 （10）: 2444-50, 1999.
[39]Yan L, Velikanov M, Flook P, Zheng W, Szalma S, Kahn S. Assessment of putative protein targets derived from the SARS genome. FEBS Lett 554 （3）: 257-63, 2003.
[40]Shu-Sin Chng, Truong-Giang Hoang, Wei-Woon Wayne Lee, Mun-Pun Tham,Hui Yvonne Ling and Teck-Peng Loh. Synthetic studies towards anti-SARS agents: application ofan indium-mediated allylation of a-aminoaldehydes as the key. Tetrahedron Letters 45: 9501–9504, 2004.
[41]Zalman LS, Brothers MA, Dragovich PS, Zhou R, Prins TJ, Worland ST, Patick AK. Inhibition of human rhinovirus-induced cytokine production by AG7088, a human rhinovirus 3C protease inhibitor. Antimicrob Agents Chemother 44 （5）: 1236-41, 2000.
[42]Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. Coronavirus main proteinase （3CL_pro） structure: basis for design of anti-SARS drugs. Science 300 （5626）: 1763-7, 2003.
[43]Chou KC, Wei DQ, Zhong WZ. Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS. Biochem Biophys Res Commun 308 （1）: 148-51, 2003.
[44]Xiong B, Gui CS, Xu XY, Luo C, Chen J, Luo HB, Chen LL, Li GW, Sun T, Yu CY, Yue LD, Duan WH, Shen JK, Qin L, Shi TL, Li YX, Chen KX, Luo XM, Shen X, Shen JH, Jiang HL. A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening. Acta Pharmacol Sin 24 （6）: 497-504, 2003.
[45]Wu CJ, Jan JT, Chen CM, Hsieh HP, Hwang DR, Liu HW, Liu CY, Huang HW, Chen SC, Hong CF, Lin RK, Chao YS, Hsu JT. Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide. Antimicrob Agents Chemother 48 （7）: 2693-6, 2004.
[46]Zhang XW, Yap YL. Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs. Bioorg Med Chem 12 （10）: 2517-21, 2004.
[47]The Dundee PRODRG2 Server http://davapc1.bioch.dundee.ac.uk/programs/prodrg/
[48]Autodock 3.0 （Scripps） http://www.scripps.edu/mb/olson/doc/autodock/index.html
[49]Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK. Olson AJ. Automated Docking Using a Lamarckian Genetic Algorithm and and Empirical Binding Free Energy Function. Computational Chemistry J 19: 1639-1662, 1998.
[50]Autodocktools （Scripps） http://www.scripps.edu/mb/olson/doc/autodock/tools.html
[51]Pymol http://pymol.sourceforge.net/
[52]Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267 （3）: 727-48, 1997.
[53]Man, K. F., et. al., Genetic Algorithms, Springer-Verlag, London1999