臺灣博碩士論文加值系統

嚴重急性呼吸道症候群(Severe Acute Respiratory Syndrome)是由新型人類冠狀病毒 (SARS-CoV) 感染所造成。此疾病在2002~2003年爆發，造成全球性的感染與恐慌。根據世界衛生組織(WHO)指出，這段期間內各國總共有8,465通報病例，而其中有801死亡病例。SARS病毒在宿主體內之成熟需藉由一主要蛋白酶(3CLpro)，其負責的工作為切剪由病毒基因轉錄而產生的蛋白鏈，進而產生病毒內部各個重要且具功能的蛋白質，讓病毒得以感染與複製。在人類小RNA病毒(picornavirus，簡稱PV)的感染機制中，也需要一個類似的蛋白酶(3Cpro)來專職上述功能。 因此，3CL與3C蛋白酶遂成為研究抗CoV與PV藥物的重要標靶。然而，目前已知的鼻病毒(rhinovirus，亦屬PV的成員之一)抑制劑，AG7088，並無法有效的抑制SARS 3CLpro活性，這顯示此兩種蛋白酶在結構上具有某程度的差異性。
本篇論文利用大腸桿菌表現SARS 3CL蛋白酶，並設計螢光胜肽受質來偵測酵素活性與催化特性，以及篩選抑制劑。配合上蛋白酶之蛋白質晶體結構，我們推論出3CL蛋白質二聚體之成熟(maturation)模式。在藥物篩選方面，我們成功的篩選出多種形式的抑制劑，並透過蛋白質共結晶技術與電腦分子模擬，深入探討這些抑制劑與酵素之間的結合模式，且進一步的將其修飾與改良。在另一方面,我們也針對抗PV藥物進行研究，包括腸病毒(enterovirus)71型與柯薩奇病毒(coxsackievirus)B3型。我們篩選出數個化合物能有效的抑制酵素活性，並證明其可以有效的保護細胞抵抗病毒的感染。此外，我們從6800個小分子中，利用高效能藥物篩選方法來篩選SARS 3CLpro的抑制劑，在篩選出的五個化合物之中，有一個化合物與其類似物可以同時抑制CoV與PV的蛋白酶。接著，我們利用電腦分子模擬運算，釐清出這些抑制劑與此兩類蛋白酶在結合模式上的差異。
另一方面，我們也探討SARS 3CL蛋白酶結構中，調控受質P1’位置專一性之重要胺基酸(Threonine)，將其突變成甘胺酸(Glycine)後，可將原本受質P1’位置上的可供辨識的絲胺酸(Serine)置換成甲硫胺酸(methionine)。這個改造過的SARS T25G 3CL蛋白酶，可將利用大腸桿菌與酵母菌所表現出來的融合蛋白(fusion protein)上的標誌蛋白(tag)，作有效率切除，頗具商業應用價值。

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV), which infected more than 8000 people during the 2003 outbreak. The viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. Accordingly, in human picornavirus (PV) family which consists of over 200 medically relevant viruses, a chymotrypsin-like protease (3Cpro) is required for viral replication through the processing the polyproteins. As a result, the 3CLpro and 3Cpro are regarded as anti-viral drug targets. However, known inhibitors (AG7088) against PV 3Cpro failed to inhibit SARS-CoV 3CLpro, indicating differences in their active-site structures.
In this study, we have prepared the recombinant SARS 3CLpro without redundant residues at both N- and C-termini and characterized its kinetic property using a fluorogenic substrate. Combined with our crystallography data, we proposed a model to illustrate the maturation process of SARS 3CLpro. In addition, we evaluated several types of inhibitors and investigated their inhibitory mechanisms.
Additionally, the recombinant 3Cpro from PV (including entervirus, coxsackievirus, and rhinovirus, abbreviated as EV, CV, and RV, respectively) were purified and characterized as well. We have identified several inhibitors which show their potencies against viral replication. Furthermore, we tested 6800 small molecules by high-throughput screening for anti-SARS agents and found one hit and its analogues could serve as the common inhibitors against CoV 3CLpro and PV 3Cpro. By computer modeling, the structural features of these compounds were elucidated to enhance our knowledge for developing anti-viral agents against PV and CoV.
In order to determine the amino acid residues essential for the substrate specificity and engineer 3CLpro as a tool for tag removal of the recombinant fusion proteins, we developed a mutant 3CLpro (T25G) which has altered substrate specificity to cleave Gln↓Met. We have also constructed E. coli and yeast vectors to express recombinant fusion proteins with the T25G 3CLpro recognition site (Ala-Val-Leu-Gln↓Met) between the tags and the target proteins for tag removal.

誌謝...i
中文摘要...vii
ABSTRACT...ix
ABBREVIATIONS...xi
1. INTRODUCTION... 1
2. MATERIALS AND METHODS... 7
Materials...7
Inhibitors...7
Fluorogenic substrate of SARS 3CLpro...7
Expression and purification of SARS 3CLpro...7
Gel filtration determination of SARS 3CLpro quaternary structure...9
Activity assay of SARS 3CLpro using the fluorogenic substrate...9
Inhibition assay: IC50 determination...10
Inhibitor assay: Ki determination... 11
Expression and purification of SARS 3CLpro mutants for maturation study...11
Kinetics of maturation assayed by SDS-PAGE...13
Analytical ultracentrifuge experiments...14
Expression and purification of EV71 3Cpro...14
Protease activity assay of EV71 3Cpro...16
Enzyme inhibition assay for EV71 3Cpro...17
Anti-viral assay for EV71...18
Western blot analysis for detecting EV71 3Cpro...18
Structural modeling of EV71 3Cpro...19
Preparation of CVB3 3Cpro, RV14 3Cpro, and CoV-229E 3CLpro...19
Examination of substrate specificity of CVB3 3Cpro...20
Protease activity assay of CVB3 3Cpro...21
Enzyme inhibition assay of CVB3 3Cpro...21
Primary screening of SARS 3CLpro inhibitors...21
IC50 determination of the inhibitors of coronavirus and picornarvirus main proteases...22
Computer modeling of the inhibitors binding with the proteases...22
Expression and purification of mutant SRAS 3CLpro for cleaving Q-M...23
Expression of the tag-cleavable fusion proteins in E. coli and in yeast...24
Tag cleavage of the fusion protein by the mutant SARS 3CLpro... 26
Substrate specificity and kinetic parameters of the mutant SARS 3CLpro...27
3. RESULTS...29
Expression and purification of SARS 3CLpro... 29
Effect of reducing agents on enzyme activity...29
Kinetic and equilibrium constants of SARS 3CLpro...30
Gel filtration experiments...30
Inhibitor assay using the fluorogenic substrate...31
Inhibition of C2-symmetric compounds against SARS 3CLpro...32
Inhibition of anilide compounds against SARS 3CLpro...33
Inhibition of α,β-unsaturated ester compounds against SARS 3CLpro...33
Inhibition of benzotriazole ester compounds against SARS 3CLpro...34
Autoprocessing of tagged SARS 3CLpro during lysate preparation...35
Facilitated processing...36
AUC analysis of wild-type and mutant SARS 3CLpro...36
Preparation of the EV71 3Cpro...37
Substrate specificity and kinetics of EV71 3Cpro...37
Synthesis of inhibitors against EV71 3Cpro...38
Evaluation of the EV71 3Cpro inhibitors...39
Substrate specificity and kinetics of CVB3 3Cpro...41
Evaluation of CVB3 3Cpro inhibitors...42
Obtaining SARS 3CLpro inhibitors by high-throughput screening...42
Inhibition potencies of the 43146 analogues...43
Computer modeling of 21155, 22723, 27548, and 48511 binding to the proteases...44
Binding modes of 43146 and its analogues to the proteases...45
Predicting the role of Thr25 of SARS 3CLpro in P1’-specificity by computer modeling...46
Preparation and characterization of T25G and T25S 3CLpro...46
Substrate specificity of T25G 3CLpro...47
Construction of E. coli and yeast vectors to express tag-cleavable fusion proteins by T25G 3CLpro...48
Comparison of the tag cleavage using TEVpro and T25G 3CLpro...50
4. DISCUSSION...51
TABLES...63
Table 1 Ki values and the structures of metal-containing inhibitors of SARS 3CLpro...63
Table 2 Ki values of three metal ions in inhibiting the SARS 3CLpro...64
Table 3 IC50 Values for Some 2-Chloro-4-nitroanilide Inhibitors against the SARS 3CLpro...65
Table 4 IC50 values of AG7088 (E1a) and the related compounds for inhibition of SARS 3CLpro...66
Table 5 IC50, Ki, kinact, and CC50 of Benzotriazole Esters...67
Table 6 IC50, EC50, and CC50 of the peptidomimetic inhibitors against the EV71 3Cpro...68
Table 7 Inhibition of peptidemimetic against CVB3 3Cpro and SARS 3CLpro...69
Table 8 Summary of IC50 values (μM) of the five hits with SARS-CoV 3CLpro, and other 3C(L) proteases...70
Table 9 IC50 values (μM) of compound 43146 analogs with SARS 3CLpro, and other 3C(L) proteases...71
FIGURES...72
Figure 1 SDS-PAGE analysis of the SARS 3CLpro at different stages of purification procedure...72
Figure 2 Measurements of kinetic parameters of SARS 3CLpro using a fluorogenic substrate...73
Figure 3 Dependence of SARS 3CLpro reaction rate on enzyme concentration...74
Figure 4 Gel filtration study of the SARS 3CLpro...75
Figure 5 The Ki measurements of a selected SARS 3CLpro inhibitor 1-hydroxypyridine-2-thione zinc...76
Figure 6 The Ki measurement for zinc ion...77
Figure 7 The Lopinavir-like structures and their IC50 against SARS 3CLpro...78
Figure 8 Summary of anilide inhibitors of the SARS 3CLpro...79
Figure 9 Molecular structures of α,β-unsaturated ester compounds...80
Figure 10 Molecular structures of inhibitors B3-B10 against SARS 3CLpro...81
Figure 11 Kinetic studies of inhibitor 4 and SARS 3CLpro...82
Figure 12 SDS-PAGE analysis of the maturation of SARS 3CLpro...83
Figure 13 Facilitated processing of Trx-10aa-C145A-10aa-GST by the active 3CLpro...84
Figure 14 AUC experiments of wild-type SARS 3CLpro...85
Figure 15 Proposed scheme of SARS 3CLpro maturation...86
Figure 16 Purification and characterization of EV71 3Cpro...87
Figure 17 Synthesis of EV71 3C protease inhibitors with α,β-unsaturated ester...88
Figure 18 Enzyme inhibition studies of 6b...89
Figure 19 Inhibition of EV71 protein accumulation in RD cells by 10d treatment...90
Figure 20 Computer modeling of the complex structures of EV71 3Cpro with 6b and 10b based on the published structure (PDB code 1CQQ) of RV protease with AG7088 bound...91
Figure 21 Substrate specificity and kinetics of CVB3 3Cpro...92
Figure 22 Ki of EPDTC against CVB3 3Cpro...93
Figure 23 Dose-response curves for the five hits against SARS 3CLpro from the screening...94
Figure 24 Dose-response curves for 43146 against 229E 3CLpro, CVB3 3Cpro, EV71 3Cpro and RV14 3Cpro...95
Figure 25 Computer modeling of the binding modes of the inhibitors in the active site of the SARS 3CLpro...96
Figure 26 Predicted structural model of SARS 3CLpro with a modified peptide (Thr-Ser-Ala-Val-Leu-Gln-Met-Phe-Arg-Lys) containing Met at P1’ site...97
Figure 27 Purification and activity measurements of SARS 3CLpro T25G mutants...98
Figure 28 Substrate specificities and kinetics of wild-type and T25G 3CLpro...99
Figure 29 Expression of UPPs fusion protein in E. coli and the tag cleavage by the 3CLpro...100
Figure 30 Expression of EGFP fusion protein in yeast Pichia and the tag cleavage by T25G 3CLpro...101
Figure 31 Comparison of the efficiency of tag cleavage using TEVpro and T25G 3CLpro...102
REFERENCES...103
PUBLICATION LIST...110
APPENDIX...113

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