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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 | 李振維 | zh_TW |
| dc.contributor.author | CHEN-WEI LI | en |
| dc.date.accessioned | 2023-09-07T16:57:40Z | - |
| dc.date.available | 2024-07-01 | - |
| dc.date.copyright | 2023-09-11 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-07 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89426 | - |
| dc.description.abstract | 由嚴重急性呼吸道症候群冠狀病毒2型 (SARS-CoV-2) 引起的 2019 年冠狀病毒病 (COVID-19) 已在全球奪去許多人的生命，且仍在蔓延中。 病毒在與宿主細胞表面的血管緊張素轉化酶 2 (ACE2) 受體結合後，細胞表面的跨膜絲胺酸蛋白酶2 (TMPRSS2) 會切割 SARS-CoV-2 刺突蛋白，使病毒與細胞膜融合並進入細胞。 因此，已知藥物（如 camostat 和 nafamostat），藉著不可逆地抑制 TMPRSS2，從而抑制病毒進入。 如本文所述，我使用桿狀病毒感染的昆蟲細胞表達並激活TMPRSS2 胞外域以測定我們實驗室合成的抑制劑以及選定的天然產物和 FDA 批准的藥物。 這些化合物還使用弗林蛋白酶 (Furin) 和組織蛋白酶 L (Cathepsin L) 的試劑進行了分析，Furin 是一種人類細胞表面絲氨酸蛋白酶，可以與 TMPRSS2 分別在刺突蛋白的 S1/S2 及 S2' 位點切割。Cathepsin L一種參與切割刺突蛋白的細胞內蛋白酶，從而由胞內體釋放入侵的病毒。 根據 IC50 建立針對 TMPRSS2、Furin 和/或 Cathepsin L的抑制劑的構效關係，並通過分子嵌合計算 (docking) 對結構進行模擬以提供抑制的解釋。因此，這項研究提供了可能的 TMPRSS2、Furin 和/或 Cathepsin L 抑制劑來阻止 SARS-CoV-2 進入細胞。 | zh_TW |
| dc.description.abstract | Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in global fatalities and continues to spread. Transmembrane protease, serine 2 (TMPRSS2) on the surface of infected host cells cleaves SARS-CoV-2 spike protein, enabling the primed virus to fuse with the cell membrane and enter the cells after binding with the angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, known antivirals like camostat and nafamostat irreversibly inhibit TMPRSS2 and virus entry. In this thesis, TMPRSS2 ectodomain was expressed using baculovirus-infected insect cells and activated to screen the inhibitors synthesized in our laboratory, as well as the selected natural products and FDA-approved drugs. These compounds were also assayed using the commercial kits to determine their inhibitory effects against Furin and Cathepsin L. Furin is a human cell-surface serine protease that works with TMPRSS2 to cleave the spike protein at the canonical S1/S2 and S2' site, respectively. Cathepsin L is an intracellular protease involved in cleaving the spike protein to release the invading virus from the endosome. Structure-activity relationships of the inhibitors against TMPRSS2, Furin, and/or Cathepsin L were established from the IC50 and the complex structures were modeled by the docking program to provide the structural rationale. Therefore, this study offers promising TMPRSS2, Furin, and/or Cathepsin L inhibitors to block SARS-CoV-2 entry. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-07T16:57:40Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-07T16:57:40Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 誌謝 ..............................................................................................................................v
中文摘要 .....................................................................................................................vi Abstract .......................................................................................................................vii Abbreviations .............................................................................................................viii 1. Introduction ...............................................................................................................1 2. Materials and Methods ..............................................................................................5 2.1. Materials .........................................................................................................5 2.2. TMPRSS2 ectodomain expression ..................................................................5 2.3. TMPRSS2 ectodomain activation and purification .........................................7 2.4. TMPRSS2 kinetics and inhibitory assay .........................................................8 2.5. Furin inhibitory assay ......................................................................................9 2.6. Cathepsin L inhibitory assay .........................................................................10 2.7. Molecular docking ........................................................................................10 3. Results .....................................................................................................................12 3.1. Purification and characterization of the recombinant TMPRSS2 ectodomain ..............................................................................................................................12 3.2. Test of the synthesized pyrrolidinones for inhibiting TMPRSS2 ectodomain ..............................................................................................................................13 3.3. Binding modes of the pyrrolidinone inhibitors with TMPRSS2 ....................14 3.4. Test of the synthesized pyrrolidinones for inhibiting Furin ...........................15 3.5. Binding modes of the pyrrolidinone inhibitors with Furin ...........................16 3.6. Test of the FDA-approved drugs previously shown to inhibit 3CLpro and/or PLpro as antivirals for inhibiting TMPRSS2, Furin, and Cathepsin L ....................16 3.7. Test of the selected natural products for inhibiting TMPRSS2, Furin, and Cathepsin L ..........................................................................................................18 3.8. Binding modes of the selected natural products with TMPRSS2 ...................20 3.9. Binding modes of the selected natural products with Furin ...........................21 3.10. Binding modes of the selected natural products with Cathepsin L ...............21 3.11. Analysis of the selected natural products interacting with the substrate binding pocket of TMPRSS2 or Cathepsin L ........................................................22 4. Discussion ................................................................................................................23 5. Tables .......................................................................................................................30 Table 1. Inhibitory effects of compound 2a-l against TMPRSS2, Furin and SARS-CoV-2 ..................................................................................................................30 Table 2. Inhibitory effects of compound 4a-c against TMPRSS2, Furin and SARS-CoV-2 …..............................................................................................................31 Table 3. Inhibitory effects of the FDA-approved drugs against TMPRSS2, Furin, Cathepsin L and SARS-CoV-2 ............................................................................32 Table 4. Inhibitory effects of the natural products against TMPRSS2, Furin, Cathepsin L and SARS-CoV-2 ............................................................................33 6. Figures .....................................................................................................................34 Figure 1. Chemical structures of camostat and nafamostat, and their mechanisms of action for inhibiting TMPRSS2 and virus entry. ..............................................34 Figure 2. The subdomains of TMPRSS2 ectodomain and the strategy for the expression of TMPRSS2 ectodomain. .................................................................36 Figure 3. Purification of TMPRSS2 ectodomain. .................................................38 Figure 4. Characterization of the kinetic constants of the recombinant TMPRSS2 ectodomain. ..........................................................................................................40 Figure 5. Inhibition of TMPRSS2 by the synthesized 4-carboxy-1-(4-styryl carbonylphenyl)-2-pyrrolidinones inhibitors. ......................................................42 Figure 6. Modeled binding modes of the synthesized pyrrolidinone inhibitor 81 on TMPRSS2 (PDB: 7MEQ). ...................................................................................44 Figure 7. Inhibition of Furin by the synthesized 4-carboxy-1-(4-styryl carbonylphenyl)-2-pyrrolidinones inhibitors. ......................................................45 Figure 8. Modeled binding modes of the synthesized pyrrolidinone inhibitor 81 on Furin (PDB: 4RYD). ............................................................................................47 Figure 9. The chemical structures of some selected flavonoids, flavanols, and polyphenols. .........................................................................................................49 Figure 10. The chemical structures of some selected unsaturated phenols, steroids with or without sugar moieties, etc. ......................................................................51 Figure 11. Testing the inhibition of TMPRSS2, Furin, and/or Cathepsin L using the 6 FDA-approved drugs previously been shown to inhibit 3CLpro and/or PLpro, as well as antivirals. .............................................................................................53 Figure 12. Inhibition of TMPRSS2 by the selected natural products. ...................54 Figure 13. Inhibition of Furin by the selected natural products. ............................56 Figure 14. Inhibition of Cathepsin L by the selected natural products. .................57 Figure 15. Modeled binding modes of the selected natural products with TMPRSS2 to rationalize their inhibition specificity. ............................................58 Figure 16. Modeled binding modes of the selected natural products with Furin to rationalize their inhibition specificity. ..................................................................60 Figure 17. Modeled binding modes of the selected natural products with Cathepsin L to rationalize their inhibition specificity. ...........................................................62 Figure 18. Analysis of the best inhibitors interacting with the binding pocket of TMPRSS2 or Cathepsin L. ...................................................................................63 7. Reference .................................................................................................................65 | - |
| dc.language.iso | en | - |
| dc.subject | 跨膜絲胺酸蛋白酶2 | zh_TW |
| dc.subject | 2019年冠狀病毒病 | zh_TW |
| dc.subject | 分子嵌合計算 | zh_TW |
| dc.subject | 組織蛋白酶 L | zh_TW |
| dc.subject | 嚴重急性呼吸道症候群冠狀病毒2型 | zh_TW |
| dc.subject | 弗林蛋白酶 | zh_TW |
| dc.subject | 天然物 | zh_TW |
| dc.subject | Molecular docking | en |
| dc.subject | SARS-CoV2 | en |
| dc.subject | COVID-19 | en |
| dc.subject | TMPRSS2 | en |
| dc.subject | Cathepsin L | en |
| dc.subject | Furin | en |
| dc.subject | Natural products | en |
| dc.title | 製備活化的TMPRSS2胞外域以測試SARS-CoV-2進入細胞的抑制劑 | zh_TW |
| dc.title | Preparation of the activated TMPRSS2 ectodomain for assay of the inhibitors that block SARS-CoV-2 entry | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 徐尚德;張淑媛 | zh_TW |
| dc.contributor.oralexamcommittee | Shang-Te Hsu;Sui-Yuan Chang | en |
| dc.subject.keyword | 嚴重急性呼吸道症候群冠狀病毒2型,2019年冠狀病毒病,跨膜絲胺酸蛋白酶2,組織蛋白酶 L,弗林蛋白酶,天然物,分子嵌合計算, | zh_TW |
| dc.subject.keyword | SARS-CoV2,COVID-19,TMPRSS2,Cathepsin L,Furin,Natural products,Molecular docking, | en |
| dc.relation.page | 71 | - |
| dc.identifier.doi | 10.6342/NTU202302510 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2023-08-09 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2024-07-01 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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