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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 方俊民(Jim-Min Fang) | |
| dc.contributor.author | Yi-Hsuan Chen | en |
| dc.contributor.author | 陳怡瑄 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:40:20Z | - |
| dc.date.available | 2022-02-21 | |
| dc.date.copyright | 2017-02-21 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-12-21 | |
| dc.identifier.citation | 1. Pommier, Y. Drugging topoisomerases: lessons and challenges. ACS Chem. Biol. 2013, 8, 82−95.
2. Bailly, C. Contemporary challenges in the design of topoisomerase II inhibitors for cancer chemotherapy. Chem. Rev. 2012, 112, 3611−3640. 3. Tacar, O.; Sriamornsak, P.; Dass, C. R. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J. Pharm. Pharmacol. 2013, 65, 157−170. 4. Thorn, C. F.; Oshiro, C.; Marsh, S.; Hernandez-Boussard, T.; McLeod, H.; Klein, T. E.; Altman, R. B. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011, 21, 440−446. 5. San Filippo, J.; Sung, P.; Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 2008, 77, 229−257. 6. Hartlerode, A. J.; Scully, R. Mechanisms of double-strand break repair in somatic mammalian cells. Biochem. J. 2009, 423, 157-168. 7. Hu, C. M.; Yeh, M. T.; Tsao, N.; Chen, C. W.; Gao, Q. Z.; Chang, C. Y.; Lee, M. H.; Fang, J. M.; Sheu, S. Y.; Lin, C. J.; Tseng, M. C.; Chen, Y. J.; Chang, Z. F. Tumor cells require thymidylate kinase to prevent dUTP incorporation during DNA repair. Cancer Cell 2012, 22, 36−50. 8. Reynes, J. P.; Tiraby, M.; Baron, M.; Drocourt, D.; Tiraby, G. Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus. J. Bacteriol. 1996, 178, 2804−2812. 9. Ostermann, N.; Schlichting, I.; Brundiers, R.; Konrad, M.; Reinstein, J.; Veit, T.; Goody, R. S.; Lavie, A. Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate. Structure 2000, 8, 629−642. 10. Reichard, P. Interactions between deoxyribonucleotide and DNA synthesis. Annu. Rev. Biochem. 1988, 57, 349−374. 11. Mathews, C. K. Deoxyribonucleotide metabolism, mutagenesis and cancer. Nat. Rev. Cancer 2015, 15, 528−539. 12. Ayusawa, D.; Shimizu, K.; Koyama, H.; Takeishi, K.; Seno, T. Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells. J. Bio. Chem. 1983, 258, 12448−12454. 13. Ahmad, S. I.; Kirk, S. H.; Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 1998, 52, 591−625. 14. Whittingham, J. L.; Carrero-Lerida, J.; Brannigan, J. A.; Ruiz-Perez, L. M.; Silva, A. P.; Fogg, M. J.; Wilkinson, A. J.; Gilbert, I. H.; Wilson, K. S.; Gonzalez-Pacanowska, D. Structural basis for the efficient phosphorylation of AZT-MP (3'-azido-3'-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase. Biochem. J. 2010, 428, 499−509. 15. Cui, Q.; Shin, W. S.; Luo, Y.; Tian, J.; Cui, H.; Yin, D. Thymidylate kinase: an old topic brings new perspectives. Curr. Med. Chem. 2013, 20, 1286−1305. 16. Haouz, A.; V anheusden, V .; Munier-Lehmann, H.; Froeyen, M.; Herdewijn, P .; Van Calenbergh, S.; Delarue, M. Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism. J. Biol. Chem. 2003, 278, 4963−4971. 17. Cerqueira, N. M.; Fernandes, P. A.; Ramos, M. J. Ribonucleotide reductase: a critical enzyme for cancer chemotherapy and antiviral agents. Recent. Pat. Anticancer. Drug Discov. 2007, 2, 11−29. 18. Aye, Y.; Li, M.; Long, M. J.; Weiss, R. S. Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies. Oncogene 2015, 34, 2011−2021. 19. Colicelli, J. Human RAS superfamily proteins and related GTPases. Sci. STKE. 2004, 2004 (250), RE13. 20. Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 2003, 3, 11−22. 21. Johnson, L.; Mercer, K.; Greenbaum, D.; Bronson, R. T.; Crowley, D.; Tuveson, D. A.; Jacks, T. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 2001, 410, 1111−1116. 22. Kranenburg, O. The KRAS oncogene: past, present, and future. Biochim. Biophys. Acta. 2005, 1756, 81−82. 23. Shackelford, D. B.; Shaw, R. J. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat. Rev. Cancer 2009, 9 (8), 563−575. 24. Liu, Y.; Marks, K.; Cowley, G. S.; Carretero, J.; Liu, Q.; Nieland, T. J.; Xu, C.; Cohoon, T. J.; Gao, P.; Zhang, Y.; Chen, Z.; Altabef, A. B.; Tchaicha, J. H.; Wang, X.; Choe, S.; Driggers, E. M.; Zhang, J.; Bailey, S. T.; Sharpless, N. E.; Hayes, D. N.; Patel, N. M.; Janne, P. A.; Bardeesy, N.; Engelman, J. A.; Manning, B. D.; Shaw, R. J.; Asara, J. M.; Scully, R.; Kimmelman, A.; Byers, L. A.; Gibbons, D. L.; Wistuba, II; Heymach, J. V.; Kwiatkowski, D. J.; Kim, W. Y.; Kung, A. L.; Gray, N. S.; Root, D. E.; Cantley, L. C.; Wong, K. K. Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer. Cancer Discov. 2013, 3, 870−879. 25. Su, J. Y.; Sclafani, R. A. Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast. Nucleic Acids Res. 1991, 19, 823−827. 26. Gardberg, A.; Shuvalova, L.; Monnerjahn, C.; Konrad, M.; Lavie, A. Structural basis for the dual thymidine and thymidylate kinase activity of herpes thymidine kinases. Structure 2003, 11, 1265−1277. 27. Chen, Y. H.; Hsu, H. Y.; Yeh, M. T.; Chen, C. C.; Huang, C. Y.; Chung, Y. H.; Chang, Z. F.; Kuo, W. C.; Chan, N. L.; Weng, J. H.; Chung, B. C.; Chen, Y. J.; Jian, C. B.; Shen, C. C.; Tai, H. C.; Sheu, S. Y.; Fang, J. M. Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation. J. Med. Chem. 2016, 59, 9906−9918. 28. Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput. Chem. 2010, 31, 455−461. 29. Liang, P.; Averboukh, L.; Zhu, W.; Haley, T.; Pardee, A. B. Molecular characterization of the murine thymidylate kinase gene. Cell Growth Differ. 1995, 6, 1333−1338. 30. Hu, C. M.; Chang, Z. F. A bioluminescent method for measuring thymidylate kinase activity suitable for high-throughput screening of inhibitor. Anal. Biochem. 2010, 398, 269−71. 31. Huang, S. H.; Tang, A.; Drisco, B.; Zhang, S. Q.; Seeger, R.; Li, C.; Jong, A. Human dTMP kinase: gene expression and enzymatic activity coinciding with cell cycle progression and cell growth. DNA Cell Biol. 1994, 13, 461−471. 32. Vanheusden, V.; Munier-Lehmann, H.; Froeyen, M.; Dugue, L.; Heyerick, A.; De Keukeleire, D.; Pochet, S.; Busson, R.; Herdewijn, P.; Van Calenbergh, S. 3'-C-branched-chain-substituted nucleosides and nucleotides as potent inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase. J. Med. Chem. 2003, 46 , 3811−3821. 33. Channg, C. H. Synthesis of human TMPK inhibitors. Master thesis, National Taiwan University, 2014. 34. Yeh, M. T. Synthesis and identification of novel chemosensitizers against human thymidylate kinase. Master thesis, National Taiwan University, 2011. 35. Huang, M. R. Design and Synthesis of Inhibitors against human thymidylate kinase. Master thesis, National Taiwan University, 2013. 36. Velazquez-Campoy, A.; Ohtaka, H.; Nezami, A.; Muzammil, S.; Freire, E. Isothermal titration calorimetry. Curr. Protoc. Cell Biol. 2004, Chapter 17, Unit 17 18. 37. Leavitt, S.; Freire, E. Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 2001, 11, 560−566. 38. Sumranjit, J.; Chung, S. J. Recent advances in target characterization and identification by photoaffinity probes. Molecules 2013, 18 (9), 10425−10451. 39. Dorman, G.; Prestwich, G. D. Using photolabile ligands in drug discovery and development. Trends Biotechnol. 2000, 18, 64−77. 40. Hamouda, A. K.; Jayakar, S. S.; Chiara, D. C.; Cohen, J. B. Photoaffinity labeling of nicotinic receptors: diversity of drug binding sites! J. Mol. Neurosci. 2014, 53, 480−486. 41. Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.; Aoyama, H.; Hosoya, A.; Iwata, T.; Maruyama, K.; Endo, M.; Suzuki, M. Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units. Org. Biomol. Chem. 2004, 2, 637−641. 42. Tanaka, Y.; Bond, M. R.; Kohler, J. J. Photocrosslinkers illuminate interactions in living cells. Mol. Biosyst. 2008, 4, 473−480. 43. Weng, J. H.; Liang, M. R.; Chen, C. H.; Tong, S. K.; Huang, T. C.; Lee, S. P.; Chen, Y. R.; Chen, C. T.; Chung, B. C. Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration. Nat. Chem. Biol. 2013, 9, 636−642. 44. Vaidya, A. S.; Karumudi, B.; Mendonca, E.; Madriaga, A.; Abdelkarim, H.; van Breemen, R. B.; Petukhov, P. A. Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2. Bioorg. Med. Chem. Lett. 2012, 22, 5025−5030. 45. Smith, E.; Collins, I. Photoaffinity labeling in target- and binding-site identification. Future Med. Chem. 2015, 7, 159−183. 46. Li, G.; Liu, Y.; Yu, X.; Li, X. Multivalent photoaffinity probe for labeling small molecule binding proteins. Bioconjug. Chem. 2014, 25, 1172−1180. 47. Taldone, T.; Patel, P. D.; Patel, H. J.; Chiosis, G. About the reaction of aryl fluorides with sodium sulfide: investigation into the selectivity of substitution of 176 fluorobenzonitriles to yield mercaptobenzonitriles via SNAr displacement of fluorine. Tetrahedron Letters 2012, 53, 2548−2551. 48. Hamada, Y.; Suzuki, K.; Nakanishi, T.; Sarma, D.; Ohta, H.; Yamaguchi, R.; Yamasaki, M.; Hidaka, K.; Ishiura, S.; Kiso, Y. Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P(2) position. Bioorg. Med. Chem. Lett. 2014, 24, 618−623. 49. Andersen, J.; Madsen, U.; Bjorkling, F.; Liang, X. F. Rapid synthesis of aryl azides from aryl halides under mild conditions. Synlett. 2005, 14, 2209−2213. 50. Boyle, M. M.; Gassensmith, J. J.; Whalley, A. C.; Forgan, R. S.; Smaldone, R. A.; Hartlieb, K. J.; Blackburn, A. K.; Sauvage, J. P.; Stoddart, J. F. Stereochemistry of molecular figures-of-eight. Chemistry 2012, 18, 10312−10323. 51. Zhu, W.; Ma, D. Synthesis of aryl azides and vinyl azides via proline-promoted CuI-catalyzed coupling reactions. Chem. Commun. (Camb.) 2004, 7, 888−889. 52. Markiewicz, J. T.; Wiest, O.; Helquist, P. Synthesis of Primary Aryl Amines Through a Copper-Assisted Aromatic Substitution Reaction with Sodium Azide. J. Org. Chem. 2010, 75, 4887−4890. 53. MacDonald, J. P.; Badillo, J. J.; Arevalo, G. E.; Silva-Garcia, A.; Franz, A. K. Catalytic stereoselective synthesis of diverse oxindoles and spirooxindoles from isatins. ACS Comb. Sci. 2012, 14, 285−93. 54. Hajipour, A. R.; Mohammadsaleh, F. Preparation of Aryl Azides from Aromatic Amines in N-Methyl-2-Pyrrolidonium Bisulfate. Org. Prep. Procedure. Int. 2011, 43, 451−455. 55. Li, Y.; Chase, A. R.; Slivka, P. F.; Baggett, C. T.; Zhao, T. X.; Yin, H. Design, synthesis, and evaluation of biotinylated opioid derivatives as novel probes to study opioid pharmacology. Bioconjug. Chem. 2008, 19, 2585−2589. 56. Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006, 1, 2856−2860. 57. Laugesen, S.; Roepstorff, P. Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis. J. Am. Soc. Mass Spectrom. 2003, 14, 992−1002. 58. Ghafouri, B.; Karlsson, H.; Mortstedt, H.; Lewander, A.; Tagesson, C.; Lindahl, M. 2,5-Dihydroxybenzoic acid instead of alpha-cyano-4-hydroxycinnamic acid as matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for analyses of in-gel digests of silver-stained proteins. Anal. Biochem. 2007, 371, 121−123. 59. Holmberg, A.; Blomstergren, A.; Nord, O.; Lukacs, M.; Lundeberg, J.; Uhlen, M. The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 2005, 26, 501−510. 60. Helppolainen, S. H.; Nurminen, K. P.; Maatta, J. A.; Halling, K. K.; Slotte, J. P.; Huhtala, T.; Liimatainen, T.; Yla-Herttuala, S.; Airenne, K. J.; Narvanen, A.; Janis, J.; Vainiotalo, P.; Valjakka, J.; Kulomaa, M. S.; Nordlund, H. R. Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family. Biochem. J. 2007, 405, 397−405. 61. Henrikson, K. P.; Allen, S. H.; Maloy, W. L. An avidin monomer affinity column for the purification of biotin-containing enzymes. Anal. Biochem. 1979, 94, 366−370. 62. Stapels, M. D.; Barofsky, D. F. Complementary use of MALDI and ESI for the HPLC-MS/MS analysis of DNA-binding proteins. Anal. Chem. 2004, 76, 5423−5430. 63. Baell, J.; Walters, M. A. Chemistry: Chemical con artists foil drug discovery. Nature 2014, 513, 481−483. 64. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320−330. 65. Leeson, P. Drug discovery: Chemical beauty contest. Nature 2012, 481, 455−456. 66. Chen, J.; Yuan, T.; Hao, W.; Cai, M. Simple and efficient CuI/PEG-400 system for hydroxylation of aryl halides with potassium hydroxide. Catalysis Communications 2011, 12, 1463−1465. 67. Yang, K.; Li, Z.; Wang, Z.; Yao, Z.; Jiang, S. Highly efficient synthesis of phenols by copper-catalyzed hydroxylation of aryl iodides, bromides, and chlorides. Org. Lett. 2011, 13, 4340−4343. 68. Tao, C. Z.; Liu, W. W.; Sun, J. Y. Copper-catalyzed synthesis of phenols from aryl halides and 4-methoxylbenzyl alcohol. Chinese Chemical Letters 2009, 20, 1170−1174. 69. Hartwig, J. F. Evolution of a fourth generation catalyst for the amination and thioetherification of aryl halides. Acc. Chem. Res. 2008, 41, 1534−1544. 70. Xu, J.; Wang, X.; Shao, C.; Su, D.; Cheng, G.; Hu, Y. Highly efficient synthesis of phenols by copper-catalyzed oxidative hydroxylation of arylboronic acids at room temperature in water. Org. Lett. 2010, 12, 1964−1967. 71. Xie, W.; Herbert, B.; Ma, J.; Nguyen, T.M.; Schumacher, R.; Gauss, C.M.; Tehim, A. Indoles, 1H-indazoles, 1,2-benzisoxazoles, 1,2-benzoisothiazoles, and preparation and uses thereof. Patent US2005250808 (A1), Nov 10, 2005. 72. Zlotin, S. G.; Kislitsin, P. G.; Podgursky, A. I.; Samet, A. V.; Semenov, V. V.; Buchanan, I. A.; Gakh, A. A. Synthetic utilization of polynitroaromatic compounds. 2. Synthesis Of 4,6-dinitro-1,2-benzisothiazol-3-ones and 4,6-dinitro-1, 2-benzisothiazoles from 2-benzylthio-4,6-dinitrobenzamides. J. Org. Chem. 2000, 65, 8439−8443. 73. Sattler, M.; Griffin, J. D. Mechanisms of transformation by the BCR/ABL oncogene. Int. J. Hematol. 2001, 73, 278−291. 74. Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781−1802. 75. MacKerell, A. D.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 1998, 102, 3586−3616. 76. Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular dynamics. J Mol Graph 1996, 14, 33−38, 27−38. 77. Ngamwongsatit, P.; Banada, P. P.; Panbangred, W.; Bhunia, A. K. WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line. J. Microbiol. Methods 2008, 73, 211−215. 78. Goddard-Borger, E. D.; Stick, R. V. An efficient, inexpensive, and shelf-stable diazotransfer reagent: imidazole-1-sulfonyl azide hydrochloride. Org. Lett. 2007, 9, 3797−3800. 79. Shimizu, M.; Sugano, Y.; Konakahara, T.; Gama, Y.; Shibuya, I. An effective synthesis of N-substituted 2-sulfenamoylbenzoates and 1,2-benzisothiazolin-3-ones that uses 1,2-benzisothiazolin-3-one as a leaving group. Tetrahedron 2002, 58, 3779−3783. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78037 | - |
| dc.description.abstract | 胸苷酸激酶可以催化將胸苷單磷酸轉成胸苷雙磷酸之反應，而此反應則是細 胞內合成及修復去氧核糖核酸所必須之步驟。由於核糖核苷酸還原酶的活性於癌 細胞被提高，因此為了平衡地提供四種核苷三磷酸，胸苷酸激酶扮演一個重要的 角色，抑制人類胸苷酸激酶可以弱化甚至殺死癌細胞。於先前的研究中我們找到 一個新類型的人類胸苷酸激酶抑制劑 YMU1(化合物 1a)，因此，本篇研究的目的 在於了解其分子機制，並且進一步開發更有效的人類胸苷酸激酶抑制劑。
在此篇研究中，等溫滴定量熱法之分析確認此類融合芳烴異噻唑酮抑制劑會 與人類胸苷酸激酶形成一比一的複合物。根據分子模擬，YMU1 傾向結合在催化 部位(B 位)然而化合物 2b 及 15 則可結合在 B 位及 D 位並展示更強的抑制能力。 光親和探針 24 被設計帶有一個烯丙基疊氮基的光反應性部分及一個生物素標記。 此光親和標示實驗順利的標記出人類胸苷酸激酶活性部位有反應的氨基酸殘基。 由質譜法鑑定得到探針修飾的胜肽大多數處於 D 位，此結果與我們分子模擬的實 驗預測一致。根據探針分析及分子模擬結果，我們設計及合成一系列 YMU1 的衍 生物以達到對人類胸苷酸激酶具有更好的抑制能力。其中，十五個化合物具有比 YMU1 更佳的抑制活性。 | zh_TW |
| dc.description.abstract | Thymidylate kinase (TMPK) catalyzes the reaction of dTMP to form dTDP, which is an essential step for cellular dTTP supply for DNA synthesis and repair. Because ribonucleotide reductase (RNR) activity is elevated in cancer cell, TMPK plays an important role to balance dNTPs supply. It is known that inhibition of human thymidylate kinase (hTMPK) will weaken and even kill cancer cells. We have previously identified an hTMPK inhibitor YMU1 (compound 1a) as a new class inhibitor of hTMPK. Therefore, the purpose of this study is to understand its molecular mechanism and develop advanced potent hTMPK inhibitors.
In this study, the isothermal titration calorimetry analysis confirms that the arene-fused isothiazolone inhibitor forms a 1:1 complex with hTMPK. According to molecule simulation, YMU1 prefers to bind at the catalytic site (B-site) of hTMPK, whereas compounds 2b and 15 can bind at both the B-site and ATP-binding site (D-site) and show stronger inhibitory activity. The photoaffinity probe 24 is designed to bear a photoreactive moiety of arylazide and a biotin tag. The photoaffinity labeling experiment works well to tag the interacting amino acid residues in the active sites of hTMPK. The probe-modified peptides are identified by mass spectrometric method to show that most of the interacting peptides occur at the D-site as predicted by our molecular docking experiment. According to the probe study and MD result, we designed and synthesized series of YMU1 derivatives to achieve better inhibitory activity against hTMPK. Among them, 15 compounds demonstrated better inhibitory activity than YMU1. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:40:20Z (GMT). No. of bitstreams: 1 ntu-105-D01223204-1.pdf: 41697024 bytes, checksum: 2ce853c115dcacae3e63ec631ea94208 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 摘 要 ................................................................................................................. I
Abstract ........................................................................................................... II Index of Contents..............................................................................................IV Index of Figures ............................................................................................... IX Index of Table .................................................................................................. XII Index of Schemes ............................................................................................ XIV Abbreviations .................................................................................................. XVI Chapter 1. Introduction ..................................................................................... 1 1.1 Cancer ......................................................................................................... 1 1.2 DNA DSB repair .......................................................................................... 3 1.3 The role of thymidylate kinase in nucleotide biosynthesis........................... 4 1.4 Key role of hTMPK in cancer therapy .......................................................... 7 1.5 Structure of hTMPK ................................................................................... 9 1.6 TMPK enzyme assays................................................................................. 15 1.7 YMU1 as a new class inhibitor against hTMPK............................................ 17 1.8 Isothermal titration calorimetry ................................................................. 22 1.9 Photoaffinity labeling ................................................................................ 25 1.10 Aim of this study ..................................................................................... 28 Chapter 2. Results and Discussion ................................................................. 30 2.1 Isothermal titration calorimetry (ITC)......................................................... 30 2.1.1 Synthesize compound 15 ........................................................................ 31 2.1.2 Isothermal titration calorimetry reveals that the hTMPK–inhibitor complex is formed in 1:1 stoichiometry ............................................................................ 32 2.2 Construction of binding models ............................................................... 34 2.2.1 Synthesis of compound 15...................................................................... 34 2.2.2 Molecular docking analyses predict 15 and 2b bind to both ATP-binding and catalytic sites of hTMPK ................................................................................. 35 2.2.3 hTMPK inhibition assay supports that compounds 2b and 15 are stronger inhibitors than YMU1 ...................................................................................... 38 2.3 Photoaffinity labeling ................................................................................ 39 2.3.1 Design and synthesis of photoaffinity probe 16 ...................................... 40 2.3.2 Design and synthesis of photoaffinity probe 24 ..................................... 43 2.3.3 Synthesis of photoaffinity probe 24........................................................ 45 2.3.4 Inhibitory activity of photoaffinity probe 24. .......................................... 56 2.3.5 Photoaffinity labeling ............................................................................. 57 2.3.6 MS analysis............................................................................................. 68 2.3.7 Molecular docking predicted the possible binding sites of probe 24. .... 72 2.3.8 Binding modes of arene-fused isothiazolone inhibitors...........................72 2.4 Design and synthesis of other hTMPK inhibitors........................................ 74 2.4.1 Structural modification at B part ............................................................. 74 2.4.2 YMU1 is not a PAIN ................................................................................. 76 2.4.3 Structural modification at C part ............................................................ 79 2.4.4 Structure modification at A part .............................................................. 81 2.4.4.1 Rationale of structural modification at A part......................................... 81 2.4.4.2 Synthesis of compounds having oxygen or nitrogen substituents at C6 position ............................................................................................................. 83 2.4.4.3 hTMPK inhibition of compounds having oxygen or nitrogen substituents at C6 position........................................................................................................ 92 2.4.4.4 Synthesis of compounds containing halogen atoms at C6 poistion ...... 93 2.4.4.5 hTMPK inhibition of compounds containing halogen atoms at C6 poistion .......................................................................................................................... 94 2.4.4.6 hTMPK inhibition of compounds containing fluorine atoms at C4, C5, and C7 positios. .......................................................................................................................... .97 2.4.4.7 Cell viability test of compound 50 and 87.............................................. 101 2.4.3.8 Mergence of the best A and C parts to compound 102. ...................... 102 2.5 Conclusion ................................................................................................. 105 Chapter 3. Experimental Section ...................................................................... 107 3.1. General. ..................................................................................................... 107 3.2. Instrumentation. ........................................................................................ 107 3.3. Molecular modeling and computation......................................................... 108 3.4. Construction of plasmids, expression and purification of enzymes ........... 109 3.5. Isothermal titration calorimetry .................................................................. 110 3.6. Luciferase-coupled TMPK assay ................................................................ 110 3.7. 32P-Phosphate transfer assay. .................................................................... 111 3.8. Photoaffinity labeling and Western blot analysis ......................................... 112 3.9. Proteolytic digestion of labeled hTMPK ....................................................... 113 3.10. Monomeric avidin affinity enrichment of biotinylated peptides................... 114 3.11. Proteolytic digestion of labeled hTMPK without denaturation and reduction............................................................................................................. 115 3.12. ESI/LC–MS/MS analysis .............................................................................. 115 3.13. Tyrosine kinase assay ................................................................................. 112 3.14. Cell viability assay ...................................................................................... 113 3.15. Synthetic procedures and compound characterization............................... 118 References.......................................................................................................... 169 Appendix-1 1H and 13C NMR Spectra ................................................................ 183 Appendix-2 HPLC Chromatograms..................................................................... 227 | |
| dc.language.iso | en | |
| dc.title | 設計與合成光親和探針以開發人類胸苷酸激酶之抑制劑 | zh_TW |
| dc.title | Design and Synthesis of Photoaffinity Probe for Developing
Inhibitors against Human Thymidylate Kinase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 張智芬,汪根欉,許世宜,王宗興,戴桓青 | |
| dc.subject.keyword | 人類胸?酸激?,光親和探針,芳烴異?唑酮,等溫滴定量熱法, | zh_TW |
| dc.subject.keyword | Human thymidylate kinase,photoaffinity probe,arene-fused isothiazolone,isothermal titration calorimetry, | en |
| dc.relation.page | 236 | |
| dc.identifier.doi | 10.6342/NTU201603831 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-12-21 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| 顯示於系所單位： | 化學系 | |
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| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-D01223204-1.pdf 目前未授權公開取用 | 40.72 MB | Adobe PDF |
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