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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張震東 | |
dc.contributor.author | Cheng-Han Yu | en |
dc.contributor.author | 余承翰 | zh_TW |
dc.date.accessioned | 2021-06-16T03:50:27Z | - |
dc.date.available | 2020-01-28 | |
dc.date.copyright | 2015-01-28 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-01-22 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55185 | - |
dc.description.abstract | 藥物的發展一直以來都是一個重要但又令人卻步的領域,但是還是吸引了許多科學家前仆後繼的進入這個領域來研究。這幾年來,一個以活性機制為基礎的藥物目標蛋白建檔的方法出現,這是一個有邏輯性的方法去找尋具有選擇性且在生物體內是有效的酵素藥物的篩選方法。然而,這個方法受限於必需去對藥物進行進一步的修飾,才能加上一些官能基讓我們來偵測或是純化。因此我們在這裡提供且驗證了一個以抗體為基礎的方法,不需要進行進一步的化籌修飾就可以針對藥物研究提供一個新方式。首先,我們針對我們有興趣的藥物製做抗體,我們挑選了苯乙烯碸 (Phenyl Vinyl Sulfone) 和妥復克 (Afatinib) 來製作會特別辨認他們的抗體。苯乙烯碸是酪氨酸磷酸酶 (Tyrosine Phosphatase) 的抑制劑,具有一個比較簡單的結構;妥復克是一個上皮細胞生長因子受體 (EGFR) 的抑制劑,一種酪氨酸激酶 (Tyrosine Kinase)。這樣會辨認特殊藥物的抗體,在我們的實驗裡證明是可以被用在西方墨點法的針測,或是可以被用在螢光染色法上來用顯微鏡觀察。此外,我們可以進一步利用免疫沉澱法和質譜分析的方法來確認在細胞裡面這些共價藥物的目標蛋白的身份,經由這些目標蛋白身份的確認,我們也許可以提供一些藥物上的機制來說明藥物為何針對某些症狀或是某些疾病有效,甚至我們可以透過這些目標蛋白的身份來開發這個藥物的新的適應症。在這裡,我們發現苯乙烯碸和它的類似藥物在試管中或是在細胞中可以被用來抑制精氨酸甲基轉移酶一號的活性,同時,在試管中也可以被用來抑制麩胱甘肽還原酶的活性。另一方面,我們發現妥復克和它的非共價類似藥物,艾瑞莎 (Gefitinib) 和得舒緩 (Erlotinib),不只是上皮細胞生長因子受體的抑制劑,更有潛力是核醣核苷酸還原酶的抑制劑。在這些實驗中,我們在針對這些共價藥物上的偵測有更近一步的發展,我們可以針對一個特殊有興趣被挑選蛋白來偵測它和藥物的複合體的結合,而不需要透過按鍵化學法來達成,在這裡我們舉苯乙烯碸和酪氨酸磷酸酶為例子。最後我們想強調的是,以這種以抗體為基礎的方法,提供了有力的且直接正向的方法來針對這些共價藥物的目標蛋白進行身份上的確認。 | zh_TW |
dc.description.abstract | Drug development is always a vital and daunting field for many scientists to participate in one after another. Recently, activity-based protein profiling has emerged as a logical tool to discover selective and in vivo active inhibitors for enzymes. However, it has been limited by the need of further chemical modification to these covalent inhibitors with the handle for detection and enrichment. Thus, we have offered and confirmed an antibody-based approach herein named target identification by specific tagging and antibody detection (TISTA) to the chemocentric approach without chemically further modifying these covalent inhibitors. First, we have successfully raised the antisera against the phenyl vinyl sulfone (PVS) and afatinib that PVS is a covalent tyrosine phosphatase inhibitor with simple structure and afatinib is a well-known covalent receptor tyrosine kinase EGFR inhibitor with more complex structure. The data showed that the antiserum can be used in the immunoblotting and immunofluorescence staining. In addition, we can use the immunoprecipitation method to identify the in cellulo target proteins and explain the mechanisms behind the chosen inhibitor and to find a new therapeutic indication based on the candidates identified by the Mass spectrometry following the immunoprecipitation. Here, we found that the PVS and its analogs can be used to inhibit protein arginine methyltranferase 1 and glutathione reductase, and afatinib and its non-covalent analogs, gefitinib and erlotinib, are not only inhibitors of EGFR but also potential inhibitors of ribonucleotide reductase. In these experiments, we also have an advance in raising an antiserum against PVS-tagged catalytic cysteine of PTP1B to individually monitor the redox status of cysteine in the PTP1B. Finally, we must to emphasize the importance that TISTA is a powerful approach and a positive selection method to identify the target proteins of covalent drugs and their non-covalent analogs. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T03:50:27Z (GMT). No. of bitstreams: 1 ntu-104-D97b46002-1.pdf: 3451179 bytes, checksum: 1ccb539e54380b78ca9bd24a3b5243b4 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | Contents
中文摘要…………………………………………………………………………………i Abstract…………..………………………………………………………….…….…….ii Lists of Figures and Tables……………………………………….……………………..vi Lists of Appendixes………………………………………………………………….…..x Lists of Abbreviations………….....................................................................................xii 1. Introduction………………………………………………...………..………………..1 1.1 Development of covalent drugs…………………………………...………..………..1 1.2 An example of protein tyrosine phosphatase ( A simple drug)………...…………….3 1.3 An example of protein tyrosine kinase ( A sophisticated drug)………...……………4 2. Materials and Methods…………………………..……………………………………7 2.1 Materials………………………….………………………………………………….7 2.2 Methods……………………………………………………………..……………….8 3. Results…………………………………………………………………….…………17 3.1 Phenyl vinyl sulfone (A porotein tyrosine phosphatase inhibitor)…………………17 3.1.1 Production and Characterization of anti-PVS antiserum…………………………17 3.1.2 Application of anti-PVS antiserum……………………………………………….19 3.1.3 Characterization and applications of anti-PVS-PTP1B antiserum……………….20 3.1.4 Use of anti-PVS-PTP1B in monitoring the redox status of PTP1B active site cysteine…………………………………………………………………………………21 3.1.5 PVS-and PVSN-tagged proteins in HeLa cells…………………………………..22 3.1.6 PVS, PVSN and Bay 11-7082 as PRMT1 inhibitors……………………….…….25 3.1.7 Applications of PVS labeling in monitoring cellular redox status…….…………27 3.2 Afatinib (A protein tyrosine kinase inhibitor)………………………………………29 3.2.1 Production and characterization of anti-afatinib antiserum………………………29 3.3.2 Phenotypic observation of cell cycle arrest………………………………………31 3.3.3 Afatinib-tagged proteins in PC14 cells……………………….………...………...31 3.3.4 Ribonucleotide reducatase as a target protein of afatinib……….………………..32 3.3.5 Decline of ribonucleotide reductase protein level in cells treated with afatinib.…32 3.3.6 Effect of afatinib on cell cycle regulation and DNA damage response….……….34 3.3.7 Treatment of afatinib in combination with another cancer drug…………………35 3.3.8 Identification of afatinib-tagged sites of ribonucleotide reductase………………37 4. Discussion……………………………………………………………………………40 4.1 Phenyl vinyl sulfone (A porotein tyrosine phosphatase inhibitor)…………………40 4.2 Afatinib (A protein tyrosine kinase inhibitor)………………………………………43 References…………………………………..………………………………………...106 Lists of Figures and Tables Figure 1. PVS as a covalent protein tyrosine phosphatase inhibitor in cellulo could be recognized by anti-PVS antiserum…………………………………….……………….47 Figure 2. Competition of PVS labeling by EVS blocking in cellulo…………………...48 Figure 3. Competition of PVS labeling by a PTP inhibitor pervanadate (PVD) in cellulo (A) and in vitro (B)………………………………………………………………..……49 Figure 4. PVS tagging to the proteins in cellulo in a time-dependent manner………....51 Figure 5. Competition of PVS labeling by PTP inhibitors BVT 948 (A) and NSC 95357 (B) in vitro and in cellulo…………………………………………………………...….52 Figure 6. Applications of anti-PVS antiserum in immunofluorescence staining and immunoprecipitation experiments……………………………………………………...54 Figure 7. Use of the anti-PVS antiserum in recognition of PVSN and Bay 11-7082 adducts…………………………………………………………………………...……..55 Figure 8. Applications of anti-PVS antiserum in immunoprecipitation experiment with PVSN labelling………………..………………………………………………………..56 Figure 9. Characterization of anti-PVS-PTP1B antibody…………………...…………57 Figure 10. In vitro tagging of PVS to recombinant PTP1B…………………….………58 Figure 11. Changes in redox status of PTP1B following insulin (A) and EGF treatment (B) in HeLa cells………………………………………………………………………..59 Figure 12. Confirmation of PVS and PVSN tagging by immunoprecipitation followed by immunoblotting…………………………………………………..…………………60 Figure 13. Effects of PVS, PVSN, Bay 11-7082 and AMI-1 on the in vitro enzyme activity of protein arginine methyltransferase 1 (PRMT1, A) and anti-PVS detection of PVS, PVSN and Bay 11-7082 tagging of PRMT1 (B)…………………………………13 Figure 14. LC-MS analysis of reaction products of PVS, PVSN and Bay 11-7082 with cysteine…………………………………………………………………………………14 Figure 15. Mechanism of PVS, PVSN, and BAY 11-7082 reacting to cysteine……….65 Figure 16. Effects of PVS, PVSN, BAY 11-7082 and AMI-1 on the cellular arginine asymmetric dimethylation levels……………………………………………………….66 Figure 17. Structure comparison between Bay 11-7082 and C-7280948………………67 Figure 18. Effects of PVS, PVSN, and Bay 11-7082 on the in vitro enzyme activity of glutathione reductase…………………………………………………...………………68 Figure 19. Detection of anti-PVS antiserum of PVS, PVSN and Bay 11-7082 tagging of glutathione reductase (GR)……………………………………………………..……69 Figure 20. PVS used in monitoring the redox statue in cellulo…………...……………70 Figure 21. Characterization of afatinib labeling in dose-dependent and time-dependent manners………………………...……………………………………………………….71 Figure 22. Effect of pH on afatinib labeling………………………………………..…..72 Figure 23. Characterization of anti-afatinib antiserum on afatinib-like covalent drugs..73 Figure 24. Patterns of afatinib labeling in several lung cell lines………………………74 Figure 25. Cell cycle arrest induced by afatinib………………………………………..75 Figure 26. Applications of anti-afatinib antiserum in immunoprecipitation experiment………………………………………………………………………….…..76 Figure 27. Effect of afatinib and its non-covalent analog erlotinib on ribonucleotide reductase and EGFR in PC14 cells……………………………………………………..77 Figure 28. Effect of treatment of afatinib on cell cycle in PC14 cells………………….79 Figure 29. Effect of treatment of afatinib on DNA damage response in PC14 cells…...80 Figure 30. MTT assay of treatment of afatinib in PC14 cells………………………….81 Figure 31. MTT assay of treatment of cisplatin in PC14 cells………………………....82 Figure 32. MTT assay of combined treatment of cisplatin and afatinib in PC14 cells...83 Figure 33. MTT assay of treatment of doxorubicin in PC14 cells……………………..84 Figure 34. MTT assay of combined treatment of doxorubicin and afatinib in PC14 cells……………………………………………………………………………………..85 Figure 35. MTT assay of combined treatment of gemcitabine and afatinib in PC14 cells……………………………………………………………………………………..86 Figure 36. Effect of combined treatment of gemcitabine and afatinib on ribonucleotide reductase in PC14 cells…………………………………………………………………87 Figure 37. Effect of combined treatment of gemcitabine and afatinib on ribonucleotide reductase in HeLa cells…………………………………………………………………88 Figure 38. In vitro tagging of afatinib to recombinant ribonucleotide purified from human cell line………………………………………………………………………….89 Figure 39. Effect of ADP competition for substrate-binding site with afatinib………...90 Figure 40. Effect of Gemcitabine competition for substrate-binding site with afatinib..91 Table 1. Identified PVS-and PVSN-tagged proteins in HeLa cells…………………….92 Table 2. Identified Modification Sites of PVS-tagged Proteins………………………..98 Table 3. Identified Modification Sites of PVSN-tagged Proteins……………………..100 Table 4. PVS-modified peptides of Hela cell lysate treated with GSNO……………..102 Table 5. PVS-modified peptides of Hela cell lysate treated with SNAP……………...103 Table 6. Identified afatinib-tagged proteins in PC14 cells……………………………104 Table 7. Identified modification sites of afatinib-tagged ribonucleotide reductase…...105 Lists of Appendixes Appendix A1. Comparison of affinity chromatography and drug affinity responsive target stability…………………………………………...…………………………….114 Appendix A2. Timeline of covalent drugs…………………………………………….115 Appendix A3. Representation of protein-targeting strategies…………………………115 Appendix A4. Mechanism of covalent inhibitor………………………………………115 Appendix A5. Reactions of click chemistry…………………………………………..116 Appendix A6. Structure of four sulfone-based phosphatase inhibitors……………….116 Appendix A7. Mechanism of YopH phosphotase inactivation by PVSN……………..116 Appendix A8. Structure of erlotinib and gefitinib…………………………………….117 Appendix A9. Structure of afatinib, canertinib, dacomitinib, and neratinib………….117 Appendix B1. Competition of PVS labeling by PRL-3 inhibitor in vitro and in cellulo…………………………………………………………………………………118 Appendix B2. Competition of PVS labeling by PTP1B inhibitor CinnGEL 2Me in cellulo…………………………………………………………………………………119 Appendix B3. Competition of PVS labeling by PP2A inhibitor Cantharidin in cellulo…………………………………………………………………………………120 Appendix B4. Competition of PVS labeling by PP1 and PP2A inhibitor okadaic acid in cellulo…………………………………………………………………………………121 Appendix B5. Competition of PVS labeling by PTP1B inhibitor RK-682 in cellulo...122 Appendix B6. Competition of PVS labeling by PTP1B and SH-PTP1 inhibitor dephostatin in cellulo………………………………………………………………….123 Appendix B7. PVS labeling in NGF-induced PC12 cell differentiation……………...124 Appendix B8. Model of recognization of anti-PVS antiserum against PVS, PVSN and Bay 11-7082………………………………………………………………………...…125 Appendix B9. Model of recognization of anti-PVS-PTP1B antiserum……….………126 Appendix B10. Applications of anti-afatinib antiserum in immunofluorescence staining…………………………………………………………………...……………127 | |
dc.language.iso | en | |
dc.title | 利用 Phenyl Vinyl Sulfone 和 Afatinib 的抗體來尋找細胞中的目標蛋白及其應用 | zh_TW |
dc.title | Using the Antisera Against Phenyl Vinyl Sulfone and Afatinib to Identify the Target Proteins in the Cells and Their Applications | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 李明亭,蕭超隆,陳宏文,張茂山 | |
dc.subject.keyword | 苯乙烯?,妥復克, | zh_TW |
dc.subject.keyword | Phenyl Vinyl Sulfone,Afatinib, | en |
dc.relation.page | 127 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-01-22 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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