王惠鈞臺灣大學：生化科學研究所許敏峯Hsu, Min-FengMin-FengHsu2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52795嚴重急性呼吸道症候群為由一新型人類冠狀病毒 (SARS-CoV) 所感染且快速傳染的疾病。此病毒在宿主體內之成熟需藉由一主要蛋白酶—3C-like (3CL)蛋白酶。3CL蛋白酶主要負責的工作為剪切病毒基因所轉錄出來的蛋白鍊並促使3CL蛋白酶本身之熟成 (二聚體的形成)進而啟動病毒基因的複製作用。因此3CL蛋白酶成為對抗病毒之藥物設計時的重要標的物。本論文包含了此蛋白酶之蛋白質晶體結構及一與產物相結合之C145A突變株之結構。藉由此產物結合之結構與一系列研究方法，推論出3CL蛋白質二聚體形成之模式，同時透過基質序列之專一性作為對抗此一冠狀病毒之藥物設計基礎。最後，利用rhinovirus藥物-AG7088作為開端進行對抗SARS 藥物設計。本論文結合了生物資訊之基質專一性資料，酵素抑制及病毒抑制細胞實驗加上四個抑制劑與蛋白酶之蛋白質結晶結構設計出一個能有效對抗SARS病毒之抑制劑-TG-0205221。最後，建立了一個快速、有效、且節省成本之以結構為基礎之藥物設計模式。Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. In this study, the dimeric 3-D structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. This product-bound structure also provides insights into the maturation process of the SARS 3CLpro from the polyprotein and design of new structure-based inhibitors. For structure-based drug design, AG7088 (an anti-rhinivirus drug) is the lead compound for the design of anti-SARS 3CL protease inhibitor. Substrate based bioinformatics, enzyme based, anti-viral cell-based assay and four inhibitor-protease complex crystal structures established a potent anti-SARS protease inhibitor-TG-0205221. Finally, fast, efficient and cost-saving chemical synthesis strategy for structure based anti-viral protease drug design is established.CONTENTS CHINESE ABSTRACT……….……………….…………………………i ENGLISH ABSTRACT…….……………...............…………………….ii ACKNOWLEDGEMENTS…..………..………………..……………… CONTENTS….…………………………......…….…………..…………iii LIST OF FIGURES………………….......………………..…………… vii LIST OF TABLES……………….......…………………….……………..x ABBREVIATIONS…...………………...………………….……………xi CHAPTER 1 Introduction.………………………….................……………. 1 1.1 Virus…….………………..…………..…………………1 1.2 Virus classification.....…...………….…………………..1 1.3 RNA virus……………….....………..…........………….3 1.4 Coronaviridae…………………………………………..4 1.4.1 Classification of Coronaviridae……………….……4 1.4.2 Morphology of coronavirus………….……………..5 1.4.3 Genome of coronavirus……………….…………….6 1.4.4 Replication of coronavirus………………….………7 1.4.5 Diseases of coronavirus……………………………8 1.4.6 Human coronavirus………………………………...9 1.5 Severe Acute Respiratory Syndrome (SARS)………...10 1.5.1 Introduction of SARS……………….…………….10 1.5.2 Where did the SARS virus come from?...................11 1.6 3C and 3C-like proteases……………………………...13 1.7 Virus protease maturation……………………………..16 1.8 3C(L)protease drug design……………………………16 2 Materials and methods……..……………….……..……….18 2.1 Materials..... ….………………….……..…..…………18 2.2 Cloning……...…….………………...……….………..18 2.3 Protein expression and purification….…..……….…...18 2.4 Crystallization of wild-type and C145A SARS 3CLpro.21 2.4.1 Crystallization of wild-type SARS 3CLpro………..21 2.4.2 Crystallization of SARS 3CCLpro C145A………...21 2.5 Data collection and process of wild-type and C145A 3CLpro…………………………………………………21 2.6 Structure solutions and refinement of wild-type and C145A3CLpro…………………………………….……22 2.7 Maturation assay………………………………………22 2.8 Analytical Ultracentrifuge Experiments………………23 2.9 Chemical synthesis……………………………………24 2.10 Inhibition assay………………………………………...24 2.11 Co-crystallization of SARS 3CLpro-inhibitor complex...25 2.12 Data and structure of SARS 3CLpro-inhibitor complex..25 2.13 Structure solution and refinement of SARS 3CLpro- inhibitor complex…………………………………….26 2.14 Human coronavirus HCoV 229E viral load reduction Assay…………………………………………………..26 2.15 SARS anti-viral activity measurements..………………27 2.16 Graphics………………………………………………..28 3 Result………………………………………………………..30 3.1 Overall structure of the SARS-CoV wild-type……..…30 3.2 Overall structure of SARS-CoV C145A 3CLpro……....30 3.3 Autoprocessing of tagged SARS-CoV 3CLpro during lysate preparation……………………………………...31 3.4 Facilitated processing…………………………………32 3.5 Analysis of wild-type and mutant proteases…………..32 3.6 Processing intermediate-like, product-bound C145A Structure………………………………………………..33 3.7 Comparison of 3CLpro structures……………………...34 3.8 Substrate specificity initializes the peptide-like inhibitor Design…………………………………………………35 3.9 AG7088 as the lead compound for SARS 3CLpro inhibitor design……………………………………….36 3.10 TG-0203770 in complex with SARS 3CL protease……36 3.11 Structure based drug design evidenced by protease- inhibitor complex structure……………………………37 3.12 A potent anti-SARS inhibitor-TG-0205221……………38 3.13 Comparison of product-bound form and TG-0205221- SARS 3CLpro structures………………………………..41 3.14 Human coronavirus HCoV 229E viral load reduction Assay………………………………………………….42 3.15 SARS anti-viral activity measurements……………….43 3.16 Strategy for anti-viral protease inhibitor design……….44 4 Discussion…..….....................................................………….45 REFERENCES…........…………………….…………………...……….80 APPENDIXES……………………..........................................................91 Appendix 1….................................................................................92 Appendix 2….................................................................................93 LIST OF FIGURES Figure 1.1 Classification of RNA virus………………………………..3 Figure 1.2 Morphology of coronavirus………………………………..5 Figure 1.3 Structure of the coronavirus viron…………………………6 Figure 1.4 Replication of coronavirus…………………………………8 Figure 1.5 EM of SARS-CoV…………………………………………11 Figure 1.6 SARS is from palm civets…………………………………12 Figure 1.7 Evolution tree of coronavirus……………………………...12 Figure 1.8 Genome of SARS-CoV……………………………………13 Figure 1.9 Catalytic mechanism of 3CL protease…………………….15 Figure 3.1 Sequences surrounding the N-terminal and C-terminal cutting sites of 3CLpro in different coronaviruses…...…..58 Figure 3.2 Crystal structure of the wild-type of 3CLpro………….……59 Figure 3.3 Crystal structure of the C145A 3CLpro…………………….60 Figure 3.4 Product-bounded 3CLpro structure…………………………61 Figure 3.5 SARS-CoV recombinant proteases………………………..62 Figure 3.6 SDS-PAGE analysis of the maturation of SARS coronavirus recombinant proteases……………………………………..63 Figure 3.7 Facilitated processing of Trx-10aa-C145A-10aa-GST by the active 3CL protease………………………………………..64 Figure 3.8 AUC experiments of wild-type SARS 3CLpro…………….65 Figure 3.9 Stereo view of the electron density map of C145A 3CLpro..66 Figure 3.10 Molecular interactions of the active site residues of promoter B with the C-terminal residues of promoter B’……………67 Figure 3.11 Superposition of 3CLpro active sites and inhibitors………..68 Figure 3.12 Proposed scheme of SARS 3CLpro maturation…………….69 Figure 3.13 Inhibitor structures of SARS 3CLpro……………………….70 Figure 3.14 Stereo view of the electron density map of the inhibitors in the active site residues of 3CLpro…………………………..71 Figure 3.15 The SARS 3CL protease dimer structure complexed with TG-0205221…………………….………………………...72 Figure 3.16 Stereo view of the TG-0205221 inhibitor bound with the protease shown by elkectrostatic potential………………...73 Figure 3.17 Superposition of 3CLpro-TG-005221 and 3CLpro-product structures…………………………………………………...74 Figure 3.18 Schematic representation of key interactions between SARS 3CLpro and TG-0205221………………………………......75 Figure 3.19 Viral load reduction on HCoV 229E by TG-0205221…….76 Figure 3.20 Plaque forming assay on HCoV 229E by TG-0205221……77 Figure 3.21 Viral load reduction on SARS-CoV by TG-0205221……...78 Figure 3.22 Anti-SARS coronavirus activity from CPE (cytopathic effect) on Vero E6 cells……………………………………………79 LIST OF TABLES Table 3.1 Data collection and refinement statistics for wild-type and C145A3CLpro……………………………….…………….50 Table 3.2 Inter- and inra-interactions of SARS 3CLpro dimmers……...51 Table 3.3 Substrate specificity of SARS-CoV 3CL protease…………52 Table 3.4 X-ray data collection and refinement statistics for the four protease-inhbiitor complexes………………………………53 Table 3.5 List of peptide-mimetic compounds with 3CLpro enzyme- and cell-based activity and HCoV-229E cell-based activity..54 Figure 3.6 Activity of a 3CL protease inhibitor on SARS coronavirus, TG-0205221, and the analogue of a 3C protease inhibitor on rhinovirus, AG708855………………………………………55 Figure 3.7 Plasma stability of TG-0205221……………………………56en-US嚴重急性呼吸道症候群蛋白酶藥物設計晶體結構SARS3CL proteasestructure based drug designcrystal structure[SDGs]SDG3other