淡江大學覺生紀念圖書館 (TKU Library)
進階搜尋


下載電子全文限經由淡江IP使用) 
系統識別號 U0002-2501201801224900
中文論文名稱 嚴重急性呼吸道症候群冠狀病毒之似木瓜素蛋白水解酶之野生種與C271A突變種之結構與功能之對比分析
英文論文名稱 Comparative Structural and Functional Analysis of Wild-Type and C271A Mutant Forms of Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 106
學期 1
出版年 107
研究生中文姓名 穆則鳴
研究生英文姓名 David Charles Moses
學號 604185016
學位類別 碩士
語文別 英文
口試日期 2018-01-22
論文頁數 50頁
口試委員 指導教授-陳曜鴻
委員-周記源
委員-陳銘凱
中文關鍵字 嚴重急性呼吸道症候群  似木瓜素蛋白水解酶 
英文關鍵字 SARS  disulfiram  protease  PLpro 
學科別分類 學科別自然科學化學
中文摘要 嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)和中東呼吸系統症候群冠狀病毒(MERS-CoV)的似木瓜素蛋白水解酶(PLpro),由於其在病毒成熟過程中重要的角色,為抗病毒藥物的理想目標。最近的研究發現,酒精厭惡療法藥物disulfiram以競爭性的方式抑制SARS-CoV PLpro,但以非競爭性的方式抑制MERS-CoV PLpro,而且最近刊出的文章中的docking model顯示disulfiram從4.0Å的距離和SARS-CoV PLpro blocking loop 上的C271胺基酸互動。因為MERS-CoV PLpro同功能的位置不是Cys而是Ala,我們針對SARS-CoV PLpro 野生種與C271A突變種進行了一系列的對比性的測試以研究此抑制方式的差別是否跟此位置的胺基酸的差別有關聯。酵素動力學分析顯示,C271A突變種的disulfiram IC50值是野生種的250%,而且CD和AUC測試沒有顯示任何可以說明此IC50值的差別的二級或四級結構的差別。這些結果顯示,SARS-CoV PLpro有C271,而MERS-CoV PLpro同功能的位置有Ala,可能可以說明disulfiram針對SARS-CoV PLpro和MERS-CoV PLpro不同方式的抑制。期待晶體結構提供更可靠的證據。
英文摘要 The papain-like proteases of the SARS and MERS coronaviruses (SARS-CoV and MERS-CoV PLpros) are vital to viral maturation and therefore are promising antiviral drug targets. A recent study suggests that the alcohol-aversion drug disulfiram inhibits SARS-CoV PLpro by a competitive mechanism but MERS-CoV PLpro by a non-competitive mechanism, and a recently published docking model shows blocking-loop residue C271 of SARS-CoV PLpro interacting with disulfiram at a distance of 4.0 Å. Because MERS-CoV PLpro has an alanine rather than a cysteine residue at the analogous position, we performed a series of comparative assays of wild-type and C271A mutant forms of SARS-CoV PLpro to investigate whether this difference in modes of inhibition might be related to this difference in residues at the relevant position. Steady-state kinetic analysis showed a 2.5-fold increase in IC50 value for disulfiram for the mutant in comparison to the wild-type form of the enzyme, and circular dichroism and analytical ultracentrifugation assays showed no difference in secondary or quaternary structure that would account for the difference in IC50 values. We conclude that the presence of a cysteine residue at position 271 in SARS-CoV PLpro in contrast to its absence at the analogous position in MERS-CoV PLpro may explain disulfiram’s different modes of inhibition of the two enzymes. Further confirmation of these findings awaits crystallographic evidence.
論文目次 Table of Contents
1 Introduction 1
1.1 SARS and MERS 1
1.2 Rationale for continued anti-SARS drug discovery efforts 2
1.3 The search for commercially available drugs to use against the SARS and MERS coronaviruses 3
1.4 General characteristics of coronaviruses 4
1.5 Life cycle of SARS-CoV and role of SARS-CoV PLpro 5
1.6 Possible targets of anti-SARS and anti-MERS drugs 6
1.7 Structure and mechanism of SARS-CoV PLpro 7
1.8 Disulfiram as an anti-SARS-CoV drug 9
1.9 Disulfiram and BL2 blocking loop residue C271 10
1.10 Motivation of the present study 11
2 Materials 13
2.1 Chemical reagents 13
2.2 Instruments 13
2.3 Buffers used in production of competent cells 14
2.4 Buffers used in protein purification 14
2.5 Solutions used for SDS-PAGE 16
2.6 Buffer used for agarose gel electrophoresis 16
2.7 Buffer used for protein activity assays 16
3 Methods 17
3.1 Culturing broths and plates 17
3.2 Competent cell preparation 17
3.3 Transformation 18
3.4 Plasmid preparation and purification 19
3.5 Agarose gel electrophoresis 20
3.6 Recombinant protein production and purification 20
3.6.1 Recombinant protein production 20
3.6.2 Lysis and resuspension 21
3.6.3 Nickel ion affinity chromatography 21
3.6.4 Gel-filtration chromatography 22
3.6.5 Bradford assay of protein concentration 23
3.6.6 SDS-PAGE 23
3.7 Steady-state kinetic analysis 24
3.8 Inhibition assays 25
3.9 Circular dichroism (CD) assays of secondary structure 26
3.10 Circular dichroism (CD) assays of thermostability 26
3.11 Analytical ultracentrifugation (AUC) assays of quaternary structure 26
3.12 Crystallization screening 27
4 Results and Discussion 28
4.1 Protein production and purification 28
4.2 Steady-state kinetic analysis 29
4.3 Inhibition assay 32
4.4 Crystallization screening 33
4.5 Circular dichroism (CD) assay of secondary structure 34
4.6 Circular dichroism (CD) assay of thermal stability 36
4.7 Analytical ultracentrifugation (AUC) assay of quaternary structure 38
5 Conclusion 40
6 References 43
7 Appendices 47
7.1 Appendix I: Flow chart summarizing logic of experiments 47
7.2 Appendix II: Steady-state kinetic assays 48
7.3 Appendix III: Inhibition assays 49
7.4 Appendix IV: DICHROWEB results 50

List of Figures
Fig. 1: SDS-PAGE gel (left panel) and 280 nm UV spectrum (right panel) from protein purification process 28
Fig. 2: Saturation curves for SARS-CoV PLpro WT and C271A mutant forms in relation to Dabcyl–FRLKGGAPIKGV–EDANS peptidyl substrate. 31
Fig. 3: Inhibition by disulfiram of SARS-CoV PLpro WT and C271A. 33
Fig. 4: CD spectra of SARS-CoV PLpro WT and C271A mutant forms 35
Fig. 5: Thermal stability of SARS-CoV PLpro C271A with and without disulfiram 36
Fig. 6: SEDFIT analysis of AUC data 38

List of Tables
Table 1: Enzymatic parameters of SARS-CoV PLpro WT and C271A mutant forms 31
Table 2: IC50 values for inhibition by disulfiram of SARS-CoV PLpro WT and C271A mutant forms 33
Table 3: Secondary structure information for SARS-CoV PLpro WT and C271A forms obtained by analysis of circular dichroism data on DICHROWEB website 36
Table 4: TM (“melting temperature”) values of SARS-CoV PLpro WT and C271A forms obtained by circular dichroism 36
Table 5: Quaternary structure of SARS-CoV PLpro WT and C271A from SEDFIT analysis of AUC data 39
參考文獻 Adedeji AO, Severson W, Jonsson C, et al., (2013). Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus Entry That Act by Three Distinct Mechanisms. J Virol. 87(14): 8017–8028.
Adedeji AO and Sarafianos SG, (2014). Antiviral Drugs Specific for Coronaviruses in Preclinical Development. Curr Opin Virol. 0: 45–53.
Ahmed-Belkacem A, Guichou J-F, Brillet R, et al., (2014). Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention. Nucleic Acids Research 42(14): 9399–9409.
Barretto N, Jukneliene D, Ratia K, et al., (2005). The Papain-Like Protease of Severe Acute Respiratory Syndrome Coronavirus Has Deubiquitinating Activity. Journal of Virology 79(24): 15189–15198.
Berg JM, Tymoczko JL, and Stryer L, (2002). Biochemistry, 5th edition. New York: WH Freeman.
Cheng KW, Cheng SC, Chen WY, et al., (2015). Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus. Antiviral Research 115: 9–16.
Cheng SC, Chang GG, and Chou CY, (2010). Mutation of Glu-166 Blocks the Substrate-Induced Dimerization of SARS Coronavirus Main Protease. Biophysical Journal 98(7): 1327–1336.
Chou CY, Chien CH, Han YS, et al., (2008). Thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus. Biochemical Pharmacology 75(8): 1601–9.
Chou CY, Hsieh YH, Chang GG, (2011). Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses. Methods 54(1): 76–82.
Chou YW, Cheng SC, Lai HY, et al., (2012). Differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease. Archives of Biochemistry and Biophysics 520(2):74–80.
Chou CY, Lai HY, Chen HY, et al., (2014). Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease. Acta Crystallogr D Biol Crystallogr 70(Pt 2): 572–81.
Díaz-Sánchez AG, Alvarez-Parrilla E, Martínez-Martínez A, et al., (2016). Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans. Molecules 21(12), 1628.
Du L, He Y, Zhou Y, et al., (2009). The spike protein of SARS-CoV: a target for vaccine and therapeutic development. Nature Reviews Microbiology 7(3): 226–36.
Durai P, Batool M, Shah M, et al., (2015). Middle East respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control. Exp Mol Med. 47: e181.
Fehr AR and Perlman S, (2015). Coronaviruses: An Overview of Their Replication and Pathogenesis. Methods Mol Biol 1282: 1–23.
Flint, J, Racaniello V, Rall G, et al., (2015). Principles of Virology, 4th Edition, Vol. 1, American Society for Microbiology Press.
Galkin A, Kulakova L, Lim K, et al., (2014). Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram. J Biol Chem 289(15): 10502–10509.
Greenfield NJ, (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols 1(6): 2876–2890.
Guo J, Xu C, Li X, et al., (2014). A Simple, Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection. PLoS One 9(12): e114124.
Han YS, Chang GG, Juo CG, et al., (2005). Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition. Biochemistry 44(30): 10349–59.
Hilgenfeld R, (2014). From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. The FEBS Journal 281(18): 4085–96.
Hilgenfeld R, Peiris M, (2013). From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses. Antiviral Research 100: 286–295.
Ho BL, Cheng SC, Shi L, et al., (2015). Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease. PLoS One 10(12): e0144865.
Kelly SM, Jess TJ, Price NC, (2005). How to study proteins by circular dichroism. Biochimica et Biophysica Acta 1751: 119–139.
Kragh H, (2008). From disulfiram to antabuse: the invention of a drug. Bull Hist Chem 33(2).
Lei J, Mesters JR, Drosten C, et al., (2014). Crystal structure of the papain-like protease of MERS coronavirus reveals unusual, potentially druggable active-site features. Antiviral Research 109: 72-82.
Lin M-H, Moses DC, Hsieh C-H, et al., (2018). Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes. Antiviral Research 150: 155–163.
Lua G, Bluemlinga GR, Collopa P, et al., (2017). Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays. Antimicrobial Agents and Chemotherapy 61(3): e01967-16.
Paranjpe A, Zhang R, Ali-Osman F, (2014). Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage. Carcinogenesis 35(3):692–702.
Ratia K, Kilianski A, Baez-Santos YM, et al., (2014). Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease. PLOS Pathogens 10(5): e1004113.
Ratia K, Saikatendu KS, Santarsiero BD, et al., (2006). Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme. Proc Natl Acad Sci U S A 103(15): 5717–22.
Rota PA, Oberste MS, Monroe SS, et al., (2003). Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. Science 300(5624): 1394–1399.
Schuck P, (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophysical Journal 78(3): 1606–1619.
Tarantino D, Cannalire R, Mastrangelo E, et al., (2016). Targeting flavivirus RNA dependent RNA polymerase through a pyridobenzothiazole inhibitor. Antiviral Research 134: 226–235.
Sreerama N, Woody RW, (2000). Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287(2): 252–260.
Whitmore L, Wallace BA, (2004). DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Research 32(Web Server issue): W668–673.
Xu X, Liu Y, Weiss S, et al., (2003). Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design. Nucleic Acids Research 31(24): 7117–7130.
Yang H, Yang M, Ding Y, et al., (2003). The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc Natl Acad Sci U S A. 100(23): 13190–5.
論文使用權限
  • 同意紙本無償授權給館內讀者為學術之目的重製使用,於2018-01-30公開。
  • 同意授權瀏覽/列印電子全文服務,於2018-01-30起公開。


  • 若您有任何疑問,請與我們聯絡!
    圖書館: 請來電 (02)2621-5656 轉 2486 或 來信