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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌 | zh_TW |
| dc.contributor.advisor | Po-Huang Liang | en |
| dc.contributor.author | 鄭緁玲 | zh_TW |
| dc.contributor.author | Chieh-Ling Cheng | en |
| dc.date.accessioned | 2024-09-24T16:11:36Z | - |
| dc.date.available | 2024-09-25 | - |
| dc.date.copyright | 2024-09-24 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-07-09 | - |
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PMID: 36760740; PMCID: PMC9890616.4 Li CW, Chao TL, Lai CL, Lin CC, Pan MY, Cheng CL, Kuo CJ, Wang LH, Chang SY, Liang PH. Systematic Studies on the Anti-SARS-CoV-2 Mechanisms of Tea Polyphenol-Related Natural Products. ACS Omega. 2024 May 17;9(22):23984-23997. doi: 10.1021/acsomega.4c02392. PMID: 38854515; PMCID: PMC11154727 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95911 | - |
| dc.description.abstract | 腸病毒71型（EV71）曾在台灣引發多次疫情，過去造成數千名兒童死亡，目前仍無有效藥物治療。在EV71中，3C蛋白酶（3Cpro）是一種高度保守的半胱氨酸蛋白酶，具有非典型的Cys-His-Glu催化三聯體，對病毒複製過程中的多蛋白加工至關重要。SARS-CoV-2中的3C樣蛋白酶（3CLpro）負責將病毒多蛋白裂解為成熟的非結構蛋白（NSPs）。3CLpro因其結構類似於小RNA病毒的3C蛋白酶（3Cpro）而得名。在本論文中，我們旨在從已知的3CLpro抑制劑中識別3Cpro抑制劑，因此在本研究中，我首先建立3C蛋白酶的活性量測系統來篩選3C樣蛋白酶的抑制劑及測量他們對3C樣蛋白酶及3C蛋白酶的半抑制常數。 然而，篩選出一些含鋅配合物的胜肽類似抑制劑能同時有效抑制3CLpro和3Cpro。最後，電腦模擬用來釐清他們的結合樣式。 | zh_TW |
| dc.description.abstract | Enterovirus 71 (EV71) that caused outbreaks in Taiwan and killed thousands of children in the past, and currently, there is no effective drug against it. In EV71, 3C protease (3Cpro) is a highly conserved cysteine protease, featuring a non-canonical Cys-His-Glu catalytic triad essential for polyprotein processing during virus replication. The 3C-like protease (3CLpro) in SARS-CoV-2 is responsible to cleave the viral polyproteins into mature non-structural protein (NSPs). 3CLpro got its name as analogous to the picornavirus 3C protease (3Cpro) in structures. In this thesis, we aimed to identify 3Cpro inhibitors from the known 3CLpro inhibitors. I set up assay for 3Cpro to screen known 3CLpro inhibitors and measured their IC50 values against 3CLpro and 3Cpro. However, I screen out some peptide-like inhibitors with Zinc complexes that could effectively inhibit both 3CLpro and 3Cpro. Finally, computer modeling was used to rationalized their binding modes | en |
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| dc.description.tableofcontents | 口試委員會審定書…………………………………………………………i
誌謝…………………………………………………………………………ii 中文摘要……………………………………………………………………iii Abstract……………………………………………………………………xi Abbreviations………………………………………………………………xii 1. Introduction………………………………………………………………1 2. Materials and Methods……………………………………………………3 2.1 Materials……………………………………………………………3 2.2 EV71 3Cpro plasmid construction………………………………………4 2.3 SARS-CoV-2 3CLpro plasmid construction………………………………5 2.4 EV71 3Cpro expression and purification…………………………………5 2.5 SARS-CoV-2 3CLpro expression and purification………………………6 2.6 Kinetic and inhibition assays of EV71 3Cpro and SARS-CoV-2 3CLpro……8 2.7 Centrifugation experiments…………………………………………10 2.8 Molecular docking…………………………………………………10 3. Results…………………………………………………………………12 3.1 Expression and purification of the recombinant EV71 3Cpro……………12 3.2 Expression and purification of the recombinant SARS-CoV-2 3CLpro……12 3.3 Measurements of the kinetic constants for SARS-CoV-2 3CLpro and EV71 3Cpro…………………………………………………………………13 3.4 3CLpro peptidomimetic inhibitor nirmatrelvir failed to inhibit EV71 3Cpro…………………………………………………………………13 3.5 Inhibition of SARS-CoV-2 3CLpro by the compounds from Dr. Jiun-Jie Shie with aldehyde or α,β-unsaturated ester warhead……………………………14 3.6 Inhibition of SARS-CoV-2 3CLpro by the compounds from Dr. Jiun-Jie Shie with β-keto ester warhead………………………………………………15 3.7 Inhibition of SARS-CoV-2 3CLpro by the compounds from Dr. Jiun-Jie Shie with zinc cyclic complex ………………………………………………………16 3.8 Inhibition of EV71 3Cpro by the compounds from Dr. Jiun-Jie Shie with different warheads…………………………………………………16 3.9 Binding modes of JJS394 with EV71 3Cpro and SARS-CoV-2 3CLpro……17 3.10 Time-dependent inhibition of JJS394 against EV71 3Cpro and SARS-CoV-2 3CLpro…………………………………………………………………17 3.11 Activities of EV71 3Cpro and SARS-CoV-2 3CLpro after adding the inhibitor and being centrifuged……………………………………………………18 3.12 Screening of the FDA-approved drugs on inhibiting SARS-CoV-2 3CLpro and/or EV71 3Cpro………………………………………………………19 3.13 Binding mode of Tepotinib with EV71 3Cpro…………………………20 3.14 Identification of SARS-CoV-2 3CLpro inhibitors by virtual screening……21 3.15 Screening the polyphenols and the related natural products for inhibiting SARS-CoV-2 3CLpro and/or EV71 3Cpro…………………………………21 3.16 Binding mode of Theaflavin-3'-gallate with the substrate-binding pockets of EV71 3Cpro and SARS-CoV-2 3CLpro……………………………………23 3.17 Comparing binding modes of Theaflavin-3'-gallate, Theaflavin-3-gallate and TF3 with the substrate-binding pocket of SARS-CoV-2 3CLpro………………24 3.18 Screening other natural products on inhibiting SARS-CoV-2 3CLpro and/or EV71 3Cpro……………………………………………………………24 4. Discussion………………………………………………………………25 5. Tables……………………………………………………………………30 Table 1. IC50 value of nirmatrelvir (a positive control) against SARS-CoV-2 3CLpro but not EV71 3Cpro………………………………………………30 Table 2. IC50 values of the synthetic compounds from Dr. Jiun-Jie Shie against SARS-CoV-2 3CLpro and/or EV71 3Cpro…………………………………32 Table 3. The percentages of EV71 3Cpro/ SARS-CoV-2 3CLpro activities left after adding JJS394 before and after centrifugation . …………………………………33 Table 4. None of the FDA-approved drugs previously identified to inhibit SARS-CoV-2 3CLpro against EV71 3Cpro..………………………………………………34 Table 5. IC50 values of FDA-approved drugs from GOLDEN_LIB database after virtual screening against SARS-CoV-2 3CLpro or EV71 3Cpro …………………35 Table 6. Inhibition of SARS-CoV-2 3CLpro by other compounds obtained by virtual screening . ………………………………………………………………36 Table 7. IC50 values of tea polyphenols and related natural products against SARS-CoV-2 3CLpro and/or EV71 3Cpro ………………………………………………37 Table 8. IC50 values of the natural products against SARS-CoV-2 3CLpro ………39 6.Figures …………………………………………………………………40 Figure 1. Cloning plasmid and the amino acid sequence of the recombinant EV71 3Cpro…………………………………………………………………40 Figure 2. Construction of the SARS-CoV-2 3CLpro expression plasmid………43 Figure 3. The polyprotein compositions of 3Cpro and 3CLpro…………………44 Figure 4. The assay protocol for monitoring activities of 3Cpro and 3CLpro……46 Figure 5. Illustration of the centrifuge experimental procedure………………47 Figure 6. The crystal structure of EV71 3Cpro (PDB: 5BPE) …………………48 Figure 7. The crystal structure of SARS-CoV-2 3CLpro (PDB: 7P35) …………49 Figure 8. SDS-PAGE analysis for the NiNTA purification of the recombinant EV71 3Cpro……………………………………………………………50 Figure 9. Gel filtration column chromatography of EV71 3Cpro………………51 Figure 10. SDS-PAGE analysis for purification of the recombinant SARS-CoV-2 3CLpro…………………………………………………………………52 Figure 11. SDS-PAGE analysis for the purification of the tag-free SARS-CoV-2 3CLpro…………………………………………………………………53 Figure 12. Gel filtration column chromatography of SARS-CoV-2 3CLpro……54 Figure 13. Determination of the kinetic constants for the recombinant EV71 3Cpro and SARS-CoV-2 3CLpro…………………………………………………55 Figure 14. The chemical structure of nirmatrelvir and its SARS-CoV-2 3CLpro inhibitory profile………………………………………………………57 Figure 15. Inhibition of SARS-CoV-2 3CLpro by the inhibitors from Dr. Jiun-Jie Shie……………………………………………………………………58 Figure 16. Inhibition potency for SARS-CoV-2 3Cpro and structures of JJS compounds……………………………………………………………61 Figure 17. Inhibition of EV71 3Cpro by the compounds from Dr. Jiun-Jie Shie…63 Figure 18. No time-dependent inhibition of JJ394 on 3Cpro and 3CLpro…………64 Figure 19. No time-dependent inhibition of JJ386 on 3CLpro…………………66 Figure 20. The remained activity of EV71 3Cpro inhibited by 1 μM JJS394……67 Figure 21. The remained activity of SARS-CoV-2 3CLpro inhibited by 400 nM JJS394…………………………………………………………………68 Figure 22. The remained activity of SARS-CoV-2 3CLpro inhibited by 50 nM JJS386…………………………………………………………………69 Figure 23. Molecular Docking of JJS394 in EV71 3Cpro and SARS-CoV-2 3CLpro…………………………………………………………………70 Figure 24. Sennoside B inhibited SARS-CoV-2 3CLpro……………………71 Figure 25. Tepotinib inhibited EV71 3Cpro…………………………………72 Figure 26. Binding modes of Tepotinib……………………………………73 Figure 27. Inhibition of SARS-CoV-2 3CLpro by other compounds obtained from virtual screening………………………………………………………75 Figure 28. Chemical structures of the selected natural products tested ………77 Figure 29. Inhibition of SARS-CoV-2 3CLpro by the tea polyphenol-related nature products………………………………………………………………81 Figure 30. Inhibition of EV71 3Cpro by Theaflavin-3’-gallate………………82 Figure 31. Molecular docking of Theaflavin-3'-gallate in EV71 3Cpro and SARS-CoV-2 3CLpro and 2D graphs of the binding modes of Theaflavin-3'-gallate, Theaflavin-3-gallate, and TF3 in 3CLpro……………………………………83 Figure 32. Inhibition of SARS-CoV-2 3CLpro by other nature products…………85 Reference …………………………………………………………………86 | - |
| dc.language.iso | en | - |
| dc.title | 從新冠病毒的3C樣蛋白酶抑制劑來發現71型腸病毒的3C蛋白酶抑制劑 | zh_TW |
| dc.title | Identification of enterovirus 71 3C protease inhibitors from SARS-CoV-2 3C-like protease inhibitors | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 張淑媛;謝俊結 | zh_TW |
| dc.contributor.oralexamcommittee | Sui-Yuan Chang;Jiun-Jie Shie | en |
| dc.subject.keyword | 新冠病毒,71型腸病毒,3C樣蛋白酶,3C蛋白酶,抑制劑, | zh_TW |
| dc.subject.keyword | Coronavirus,Human enterovirus 71,3C-like protease,3C protease,inhibitor, | en |
| dc.relation.page | 98 | - |
| dc.identifier.doi | 10.6342/NTU202401554 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2024-07-09 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2027-07-31 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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| ntu-112-2.pdf 未授權公開取用 | 4.39 MB | Adobe PDF | 檢視/開啟 |
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