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研究生:黃瑋
研究生(外文):Wei Huang
論文名稱:設計與合成用於標記H1N1病毒目標蛋白之雙功能光親和探針
論文名稱(外文):Design and Synthesis of a Bifunctional Photoaffinity Probe for Labeling Target Protein in H1N1 Virus
指導教授:方俊民方俊民引用關係
指導教授(外文):Jim-Min Fang
口試委員:戴桓青徐丞志王宗興鄭婷仁
口試委員(外文):Hwan-Ching TaiCheng-Chih HsuTsung-Shing WangTing-Jen Cheng
口試日期:2019-07-02
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:82
中文關鍵詞:光親和探針疊氮苯點擊化學目標蛋白辨識
DOI:10.6342/NTU201901493
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甲型流感病毒經常引起季節性的呼吸道疾病,甚至導致嚴重的全球性大流行。由於核蛋白(NP)在流感病毒生命週期中扮演重要的角色,又因其基因序列不容易變異的特質,使核蛋白成為抗流感藥物的目標蛋白之一。
中研院的翁啟惠團隊於2011年透過高通量篩選系統找到一種先導化合物A,其能夠破壞NP三聚體之間的E339...R416鹽橋作用力,而迫使NP成為單體,進而抑制流感病毒的複製。以化合物A的結構為基礎,我們的團隊致力於進行結構與活性關係 (structure−activity relationship) 的研究。在本篇研究中,我們引入光親和探針 (photoaffinity probe) 進行光親合標記 (photoaffinity labeling) 實驗並配合質譜技術,進而鑑定出化合物A的目標蛋白。
我們基於SAR研究的結果而設計一個同時具有疊氮芳香基 (aryl azide) 和疊氮烷基 (alkyl azide) 的雙功能化合物作為光親和探針。疊氮芳香基的部分可以透過UV光照射而產生氮烯活性中間體,其將嵌入目標蛋白中活性位點附近的胺基酸殘基。疊氮烷基的部分位於化合物的另一端,其可以與生物素標籤進行銅(I)催化炔−疊氮化物環加成 (CuAAC) 反應,以進行蛋白富集、純化和分析。該雙功能化合物對流感病毒 (H1N1) 具有良好的抑制活性 (EC50 =9.06 μM)。光親和標記實驗和蛋白質身分鑑定的結果可以使我們進一步地探索此藥物候選者與流感病毒的結合模式。
Influenza A viruses often cause seasonal respiratory diseases, and even lead to severe global pandemics. Nucleoprotein (NP) is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle.
Wong and coworkers have found a lead compound A, which has good inhibitory activity against influenza viruses by disrupting the E339...R416 salt bridge interaction between NP monomers. Based on the structure of this lead compound, our group currently involves in the structure−activity relationship (SAR) studies. My work is to verify the target protein of H1N1 virus by using a combination of techniques including photoaffinity labeling and mass spectrometry.
Based on the SAR study, a bifunctional compound is designed to bear both aryl azide and alkyl azide moieties. The aryl azide moiety can be irradiated by UV light to produce an active nitrene intermediate, which will form covalent bonds with proximal residues in the active site of target protein. The alkyl azide moiety is located on the opposite end to react with a biotin tag through the Cu(I)-catalyzed alkyne−azide cycloaddition (CuAAC) for protein enrichment and analysis. This bifunctional compound was synthesized, and shown to possess good inhibitory activity (EC50 = 9.06 uM) against influenza virus. The photoaffinity labeling experiment and protein identification were performed and the result allows us to further explore the binding mode of drug candidate with influenza virus.
謝誌 I
摘要 II
Abstract III
Table of Contents V
Index of Figures VIII
Index of Scheme X
Index of Tables XII
Abbreviations XII
Chapter 1. Introduction 1
1.1 Influenza A virus 1
1.1.1 Epidemiology and pathophysiology of influenza 1
1.1.2 Biology of influenza viruses 2
1.1.3 Antiviral drugs: present status and future prospects 4
1.1.4 Nucleoprotein as a druggable target 8
1.2 Fishing for drug target 11
1.2.1 Activity-based protein profiling (ABPP) 11
1.2.2 Photoaffinity labeling (PAL) 12
1.2.3 Tandem photoaffinity labeling–bioorthogonal conjugation 19
1.2.4 Protein identification by mass spectrometry (MS) 20
Chapter 2. Results and Discussion 23
2.1 Research motivation 23
2.2 Strategy for designing photoaffinity probe 24
2.3 Synthesis of photoaffinity probe 28
2.4 Bioassay and UV-vis spectral analysis 30
2.4.1. Inhibitory activity of photoaffinity probe JMF4496 30
2.4.2 Ultraviolet–visible spectral analysis 31
2.5 Photoaffinity labeling experiment 33
2.5.1 Bovine serum albumin (BSA) model test 35
2.5.2 Results of PAL in virus infected cell lysate 38
2.5.3 Control experiments with influenza nucleoprotein 43
2.5.4 Revision of PAL protocol 45
Chapter 3. Conclusion 47
Chapter 4. Experimental Section 48
4.1 General part 48
4.2 Instrumentation 48
4.3 Synthetic procedure and characterization of compounds 49
4.4 Procedure of bioassay 56
4.4.1 Materials and methods 56
4.4.2 Determination of influenza virus TCID50 56
4.4.3 Determination of EC50 of NP inhibitors 57
4.5 Photoaffinity labeling related experiments 57
4.5.1 A/WSN/1933 (H1N1) infected MDCK cell lysis 57
4.5.2 Bicinchoninic acid (BCA) assay 58
4.5.3 Photoaffinity labeling (PAL) and CuAAC for BSA test 59
4.5.4 Photoaffinity labeling (PAL) and CuAAC in cell lysate 60
4.5.5 Affinity chromatography 60
4.5.6 SDS-PAGE 61
4.5.7 Western blot analysis 62
4.5.8 In-gel trypsin digestion 63
4.5.9 Zip Tip C18 protocol 64
4.5.10 NanoLC−MS/MS analysis 65
4.5.11 Mascot protocol 67
Chapter 5. References 68
Appendix 74
1. Bouvier, N. M.; Palese, P. The biology of influenza viruses. Vaccine 2008, 26, D49–D53.
2. Taubenberger, J. K.; Morens, D. M. The pathology of influenza virus infections. Annu. Rev. pathol. 2008, 3, 499–522.
3. Petrova, V. N.; Russell, C. A. The evolution of seasonal influenza viruses. Nat. Rev. Microbiol. 2017, 16, 47.
4. Muramoto, Y.; Noda, T.; Kawakami, E.; Akkina, R.; Kawaoka, Y. Identification of novel influenza A virus proteins translated from PA mRNA. J. Virol. 2013, 87, 2455.
5. Horimoto, T.; Kawaoka, Y. Influenza: lessons from past pandemics, warnings from current incidents. Nat. Rev. Microbiol. 2005, 3, 591−600.
6. Ait-Aissa, A.; Derrar, F.; Hannoun, D.; Gradi, E. A.; Scaravelli, D.; Bouslama, Z. Surveillance for antiviral resistance among influenza viruses circulating in Algeria during five consecutive influenza seasons (2009-2014). J. Med. Virol. 2018, 90, 844−853.
7. Krammer, F.; Smith, G. J. D.; Fouchier, R. A. M.; Peiris, M.; Kedzierska, K.; Doherty, P. C.; Palese, P.; Shaw, M. L.; Treanor, J.; Webster, R. G.; García-Sastre, A. Influenza. Nat. Rev. Dis. Primers. 2018, 4, 3.
8. Hurt, A. C. The epidemiology and spread of drug resistant human influenza viruses. Curr. Opin. Virol. 2014, 8, 22−29.
9. Wang, X.; Jia, W.; Zhao, A.; Wang, X. Anti-influenza agents from plants and traditional Chinese medicine. Phytother Res. 2006, 20, 335−341.
10. Watanabe, T.; Kawaoka, Y. Influenza virus-host interactomes as a basis for antiviral drug development. Curr. Opin. Virol. 2015, 14, 71−78.
11. Sheu, T. G.; Deyde, V. M.; Okomo-Adhiambo, M.; Garten, R. J.; Xu, X.; Bright, R. A.; Butler, E. N.; Wallis, T. R.; Klimov, A. I.; Gubareva, L. V. Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008. Antimicrob. Agents Chemother. 2008, 52, 3284.
12. Hutchinson, E. C.; Fodor, E. Transport of the influenza virus genome from nucleus to nucleus. Viruses 2013, 5, 2424−2446.
13. Portela, A.; Digard, P. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J. Gen. Virol. 2002, 8, 723−734.
14. Ye, Q.; Krug, R. M.; Tao, Y. J. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 2006, 444, 1078−1082.
15. Turrell, L.; Lyall, J. W.; Tiley, L. S.; Fodor, E.; Vreede, F. T. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nature Comm. 2013, 4, 1591.
16. Shen, Y.-F.; Chen, Y.-H.; Chu, S.-Y.; Lin, M.-I.; Hsu, H.-T.; Wu, P.-Y.; Wu, C.-J.; Liu, H.-W.; Lin, F.-Y.; Lin, G.; Hsu, P.-H.; Yang, A.-S.; Cheng, Y.-S. E.; Wu, Y.-T.; Wong, C.-H.; Tsai, M.-D. E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors. Proc. Natl. Acad. Sci. 2011, 108, 16515−16520.
17. Kao, R. Y.; Yang, D.; Lau, L.-S.; Tsui, W. H. W.; Hu, L.; Dai, J.; Chan, M.-P.; Chan, C.-M.; Wang, P.; Zheng, B.-J.; Sun, J.; Huang, J.-D.; Madar, J.; Chen, G.; Chen, H.; Guan, Y.; Yuen, K.-Y. Identification of influenza A nucleoprotein as an antiviral target. Nat. Biotechnol. 2010, 28, 600.
18. Gerritz, S. W.; Cianci, C.; Kim, S.; Pearce, B. C.; Deminie, C.; Discotto, L.; McAuliffe, B.; Minassian, B. F.; Shi, S.; Zhu, S.; Zhai, W.; Pendri, A.; Li, G.; Poss, M. A.; Edavettal, S.; McDonnell, P. A.; Lewis, H. A.; Maskos, K.; Mörtl, M.; Kiefersauer, R.; Steinbacher, S.; Baldwin, E. T.; Metzler, W.; Bryson, J.; Healy, M. D.; Philip, T.; Zoeckler, M.; Schartman, R.; Sinz, M.; Leyva-Grado, V. H.; Hoffmann, H.-H.; Langley, D. R.; Meanwell, N. A.; Krystal, M. Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers. Proc. Natl. Acad. Sci. U.S.A. 2011, 10, 15366−15371.
19. Pang, B.; Cheung, N. N.; Zhang, W.; Dai, J.; Kao, R. Y.; Zhang, H.; Hao, Q. Structural characterization of H1N1 nucleoprotein−nucleozin binding sites. Sci. Rep. 2016, 6.
20. Shu, L. L.; Bean, W. J.; Webster, R. G. Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. J. Virol.1993, 67, 2723−2729.
21. Wang, S.; Tian, Y.; Wang, M.; Wang, M.; Sun, G.-B.; Sun, X.-B. Advanced activity-based protein profiling application strategies for drug development. Front. Pharmacol. 2018, 9.
22. Sumranjit, J.; Chung, S. J. Recent advances in target characterization and identification by photoaffinity probes. Molecules 2013, 18, 10425−10451.
23. Smith, E.; Collins, I. Photoaffinity labeling in target- and binding-site identification. Future Med. Chem. 2015, 7, 159−183.
24. Tanaka, Y.; Bond, M. R.; Kohler, J. J. Photocrosslinkers illuminate interactions in living cells. Mol. Biosyst. 2008, 4, 473−480.
25. Sadaghiani, A. M.; Verhelst, S. H. L.; Bogyo, M. Tagging and detection strategies for activity-based proteomics. Curr. Opin. Chem. Biol. 2007, 11, 20−28.
26. Lapinsky, D. J. Tandem photoaffinity labeling–bioorthogonal conjugation in medicinal chemistry. Bioorg. Med. Chem. 2012, 20, 6237−6247.
27. Jewett, J. C.; Bertozzi, C. R. Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 2010, 39, 1272−1279.
28. Thiede, B.; Höhenwarter, W.; Krah, A.; Mattow, J.; Schmid, M.; Schmidt, F.; Jungblut, P. R. Peptide mass fingerprinting. Methods 2005, 35, 237−247.
29. Baldwin, M. A. Protein Identification by Mass Spectrometry. Mol. Cell. Proteom. 2004, 3, 1.
30. Cottrell, J. S. Protein identification using MS/MS data. J. Proteom. 2011, 74, 1842−1851.
31. Eng, J. K.; McCormack, A. L.; Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994, 5, 976−989.
32. Corderoa, F. M.; Bonanno, P.; Chioccioli, M.; Gratteri, P.; Robina, I.; Vargas, A. J. M.; Brandi, Alberto. Diversity-oriented syntheses of 7-substituted lentiginosines. Tetrahedron 2011, 67, 9555−9564.
33. Nainar, S.; Kubota, M.; McNitt, C.; Tran, C.; Popik, V. V.; Spitale, R. C. Temporal labeling of nascent RNA using photoclick chemistry in live cells. J. Am. Chem. Soc. 2017, 139, 8090−8093.
34. Evan, P. K.; Rudolph, A. A. Photolysis of alkyl azides. Evidence for a nonnitrene mechanism. J. Am. Chem. Soc. 1980, 102, 735-740.
35. Saxena, A. K.; Pandey, S. K.; Seth, P.; Singh, M. P.; Dikshit, M.; Carpy, A. Synthesis and QSAR studies in 2-(N-aryl-N-aroyl)amino-4,5-dihydrothiazole derivatives as potential antithrombotic agents. Bioorg. Med. Chem. 2001, 9, 2025–2034.
36. Hajipour, A. R.; Mohammadsaleh, F. Preparation of Aryl Azides from Aromatic Amines in N-Methyl-2-Pyrrolidonium Bisulfate. Org. Prep. Proced. Int. 2011, 43, 451–455.
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