臺灣博碩士論文加值系統

苦瓜素1(M1)是一種從苦瓜中分離出來的葫蘆烷型三萜次級代謝產物。苦瓜素1已被證實在高葡萄糖條件下具有抗氧化壓力和抗纖維化活性。氧化壓力是指活性氧(ROS)的產生與生物系統對ROS解毒或修復由此產生的細胞和組織損傷的能力之間的不平衡，被認為與糖尿病(Diabetes mellitus, DM)等病理狀況的發展有關。糖尿病是一種代謝紊亂綜合症，主要是由於胰島素分泌不足或胰島素阻抗導致血糖異常升高。血液中的高葡萄糖會導致氧化壓力，損害體內細胞的 DNA、酶，干擾脂質過氧化，損害穀胱甘肽代謝。此外，先前的研究表明，苦瓜素1可能是SARS-CoV-2 Mpro的潛在抑制劑，可以抑制SARS-CoV-2病毒進行複製。最近，由嚴重急性呼吸綜合症冠狀病毒2(SARS-CoV-2)引起的傳染病冠狀病毒COVID-19已經危及全球人類的健康。由SARS-CoV-2引起的症狀包含咳嗽、發燒、呼吸急促，甚至會引起肺炎和急性呼吸窘迫綜合症，目前尚未有確切的治療方法可以完全治療。主蛋白酶(Mpro)參與病毒的複製和轉錄，由於識別的序列和人類任何蛋白酶都不同，是一個具有吸引力的藥物靶點。因此，開發具有抗氧化壓力和抗 SARS-CoV-2 活性的新型 M1 化學品非常重要。
在本研究中，我們使用M1作為受體和不同的單醣作為供體，以N-乙酰己糖胺1-激酶、無機焦磷酸酶、UDP-糖焦磷酸化酶和糖基轉移酶(Bs-YjiC)作為酶，用於一鍋多酶(OPME)系統合成不同苦瓜素1-糖苷以提高苦瓜素1的生物活性。對合成的苦瓜素1-醣苷進行薄層色譜分析和液相色譜純化，並透過ESI-MS與NMR進行質譜與結構分析。最後，我們針對這些化合物進行SARS-CoV-2 Mpro的抑制活性測試，並分析了抗氧化能力，以評估它是否可以應用於減少氧化壓力。

Momordicine I (M1) is a cucurbitane-type triterpenoid secondary metabolite isolated from Momordica charantia, which has been shown to have anti-oxidative stress and anti-fibrotic effects under high-glucose conditions. Oxidative stress, the imbalance between the production of reactive oxygen species (ROSs) and the ability of biological systems to detoxify ROS or repair the resulting cellular and tissue damage, is thought to be associated with the development of pathological conditions such as diabetes mellitus (DM). DM is a metabolic disorder syndrome characterized by abnormally high blood sugar due to insufficient insulin secretion and/or insulin resistance. High glucose in the bloodstream causes oxidative stress, leading to the damage of DNA and enzyme systems, interference of lipid peroxidation, and impairment of glutathione metabolism. Besides, previous studies indicated that M1 might be a potent inhibitor of the main protease (Mpro) of SARS-CoV-2, which inhibits the SARS-CoV-2 virus in the replication-transcription machinery. SARS-CoV-2 caused the global pandemic since 2019 with the symptoms like cough, fever, shortness of breath, and even pneumonia and acute respiratory distress syndrome. There are currently very limited treatments for hospitalized patients. The main protease (Mpro) in SARS-CoV-2 is involved in viral replication and transcription and is an attractive drug target due to its recognition sequence that is different from any human protease. As a result, the development of new M1 chemicals with anti-oxidative stress and anti-SARS-CoV-2 activity is important.
In this study, we synthesized several M1-β-glycosides using M1 as the sugar acceptor and different monosaccharides as the donor, with N-acetylhexosamine 1-kinase (NahK), inorganic pyrophosphatase (PmPpA), UDP‐Sugar pyrophosphorylase (BLUSP), and glycosyltransferase (Bs-YjiC) as enzymes in a one-pot multiple-enzyme (OPME) system to enhance the biological activity of M1-glycosides. The synthesized M1-β-glycosides were analyzed their synthesis by thin-layer chromatography, purified by silica column chromatography, subjected to mass spectrometry by electrospray ionization mass spectrometry, and determined structures by nuclear magnetic resonance. Finally, the inhibitory effect of these compounds on Mpro activity was evaluated and the antioxidant capacity was analyzed to assess whether it could be used for oxidative stress reduction applications.

中文摘要 i
Abstract ii
致謝 (Acknowledgement) iv
Table of Contents vi
Table of Figures ix
Table of Tables xiii
List of Abbreviations xv
Chapter 1 Introduction 1
1.1.0 COVID-19 1
1.1.1. Main Protease 1
1.1.2. SARS-CoV-2 Mpro Inhibitor GC376 4
1.1.3. Fluorescence Resonance Energy Transfer (FRET)-Based Assay 5
1.1.4. Molecular Docking 5
1.2.0 Diabetes Mellitus (DM) 6
1.2.1.0 Oxidative Stress 6
1.2.2.0 Antioxidants 7
1.2.3. Antioxidant Capacity Determination 7
1.3.0 Momordica Charantia 8
1.3.1. Momordicine I 9
1.4.0 Glycosylation 11
1.5.0 Nucleotide Sugar Synthesis 13
1.6.0 Anomeric Sugar Kinase 13
1.6.1. Nucleotidyltransferase 14
1.6.2. Inorganic Pyrophosphatase 14
1.7.0 Glycosyltransferase 15
1.7.1. Leloir Glycosyltransferase 16
1.8.0 Research Aims 17
Chapter 2 Materials and Methods 19
2.1.0 Bacterial Strains and Expression Vectors 19
2.2.0 Protein Purification Resin 19
2.3.0 Chemicals, Equipment, Reagents and Restriction Enzymes 19
2.4.0 Molecular Cloning of Enzymes 23
2.5.0 Heterologous Protein Expression in Escherichia coil Expression System 25
2.6.0 Cell Disruption and Target Protein Purification 26
2.7.0 SDS-PAGE Analysis 27
2.8.0 Column Chromatography Analysis of M1 28
2.9.0 Whole-Cell Biosynthesis for M1-Glycosides 29
2.10. Thin-Layer Chromatography Analysis 30
2.11. Column chromatography analysis of M1-glycosides 30
2.12. ESI-MS & NMR Spectroscopy 31
2.12.1. M1 32
2.12.2. M1-23-O-glucoside 1 32
2.12.3. Momordicien I-23-O-2-deoxyglucoside 1 33
2.12.4. Momordicien I-23-O-2-deoxyglucoside 2 34
2.13. Molecular Docking 34
2.14. In vitro Inhibitory Activity Assay of Main Protease 35
2.15. Trolox Equivalent Antioxidant Capacity (TEAC) Assay 36
2.16. DPPH Assay 39
Chapter 3 Results 41
3.1.0 Protein Expression and Purification of SARS-CoV-2 Main Protease 41
3.1.1. GST-tagged SARS-CoV-2 Main Protease 41
3.1.2. GST un-tagged SARS-CoV-2 Main Protease 41
3.2.0 Purification of M1 by Column Chromatography 42
3.3.0 Whole-Cell Biosynthesis for M1-Glycosides 43
3.4.0 Purification of M1-Glycosides by Column Chromatography 43
3.5.0 Characterization of M1 and Related M1-Glycosides with ESI-MS 45
3.5.1. M1 45
3.5.2. M1-23-O-Glucoside 1 46
3.5.3. M1-23-O-Glucoside 2 47
3.5.4. M1-23-O-2-Deoxyglucoside 1 48
3.5.5. M1-23-O-2-Deoxyglucoside 2 49
3.5.6. M2 50
3.5.7. M4 50
3.5.8. Momordicoside L 51
3.6.0 NMR Spectroscopy of M1 and Related M1-Glycosides 52
3.6.1. M1 52
3.6.2. M1-23-O-Glucoside 1 54
3.6.3. M1-23-O-Glucoside 2 59
3.6.4. Momordicien 1-23-O-2-Deoxyglucoside 1 65
3.6.5. Momordicien 1-23-O-2-Deoxyglucoside 2 70
3.6.6. M2 76
3.6.7. M4 81
3.6.8. Momordicoside L 86
3.7.0 In vitro Inhibitory Activity Assay of Mpro 90
3.7.1. In vitro Inhibitory Activity of Mpro by the GC376 91
3.7.2. Molecular docking of Mpro 93
3.7.3. In vitro Inhibitory Activity of Mpro by the Compound at Fixed Concentration of 50 µM 102
3.8.0 Antioxidant 103
3.8.1. Trolox Equivalent Antioxidant Capacity (TEAC) Assay 103
3.8.2. DPPH Assay 108
Chapter 4 Discussion 111
Chapter 5 Conclusion and Future Perspectives 113
Chapter 6 References 115

1. Tripathi, P. K., Upadhyay, S., Singh, M., Raghavendhar, S., Bhardwaj, M., Sharma, P., & Patel, A. K. Screening and evaluation of approved drugs as inhibitors of main protease of SARS-CoV-2. Int J Biol Macromol, 164, 2622-2631. 2020.
2. Merarchi, M., Dudha, N., Das, B. C., & Garg, M. Natural products and phytochemicals as potential anti-SARS-CoV-2 drugs. Phytother Res, 35(10), 5384–5396. 2021.
3. Ullrich, S., & Nitsche, C. The SARS-CoV-2 main protease as drug target. Bioorg Med Chem Lett, 30(17), 127377.2020.
4. Adhikari, B., Marasini, B. P., Rayamajhee, B., Bhattarai, B. R., Lamichhane, G., Khadayat, K., Adhikari, A., Khanal, S., & Parajuli, N. Potential roles of medicinal plants for the treatment of viral diseases focusing on COVID-19: A review. Phytother Res, 35(3), 1298-1312. 2021.
5. Hung, H. C., Ke, Y. Y., Huang, S. Y., Huang, P. N., Kung, Y. A., Chang, T. Y., Yen, K. J., Peng, T. T., Chang, S. E., Huang, C. T., Tsai, Y. R., Wu, S. H., Lee, S. J., Lin, J. H., Liu, B. S., Sung, W. C., Shih, S. R., Chen, C. T., & Hsu, J. T. Discovery of M protease inhibitors encoded by SARS-CoV-2. Antimicrob. Agents Chemother., 64(9). 2020.
6. Jin, Z., Du, X., Xu, Y., Deng, Y., Liu, M., Zhao, Y., Zhang, B., Li, X., Zhang, L., Peng, C., Duan, Y., Yu, J., Wang, L., Yang, K., Liu, F., Jiang, R., Yang, X., You, T., Liu, X., Yang, X., Bai, F., Liu, H., Liu, X., Guddat, L. W., Xu, W., Xiao, G., Qin, C., Shi, Z., Jiang, H., Rao, Z., & Yang, H. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, 582(7811), 289-293. 2020.
7. Sharun, K., Tiwari, R., & Dhama, K. Protease inhibitor GC376 for COVID-19: Lessons learned from feline infectious peritonitis. Ann Med Surg (Lond), 61, 122-125. 2020.
8. Kovalevsky, A. Structural plasticity of SARS-CoV-2 3CL M(pro) active site cavity revealed by room temperature X-ray crystallography. Nat Commun, 11(1), 3202. 2020
9. Zhu, W., Xu, M., Chen, C. Z., Guo, H., Shen, M., Hu, X., Shinn, P., Klumpp-Thomas, C., Michael, S. G., & Zheng, W. Identification of SARS-CoV-2 3CL protease inhibitors by a quantitative high-throughput screening. ACS Pharmacol Transl Sci, 3(5), 1008-1016. 2020.
10. Bi, S., Zhou, H., Wu, J., & Sun, X. Micronomicin/tobramycin binding with DNA: fluorescence studies using of ethidium bromide as a probe and molecular docking analysis. J. Biomol. Struct. Dyn., 37(6), 1464-1476. 2019.
11. Oyenihi, A. B., Ayeleso, A. O., Mukwevho, E., & Masola, B. Antioxidant strategies in the management of diabetic neuropathy. Biomed Res Int, 2015, 515042. 2015.
12. Bajaj, S., & Khan, A. Antioxidants and diabetes. Indian J Endocrinol Metab, 16(Suppl 2), S267-271. 2012.
13. Pham-Huy, L. A., He, H., & Pham-Huy, C. Free radicals, antioxidants in disease and health. Int J Biomed Sci, 4(2), 89-96. 2008.
14. Johansen, J. S., Harris, A. K., Rychly, D. J., & Ergul, A. Oxidative stress and the use of antioxidants in diabetes: linking basic science to clinical practice. Cardiovasc Diabetol, 4, 5. 2015.
15. Chen, X., Guo, C., & Kong, J. Oxidative stress in neurodegenerative diseases. Neural Regen Res, 7(5), 376–385. 2012.
16. Rahimi-Madiseh, M., Malekpour-Tehrani, A., Bahmani, M., & Rafieian-Kopaei, M. The research and development on the antioxidants in prevention of diabetic complications. Asian Pac J Trop Med, 9(9), 825-831. 2016.
17. Bortolotti, M., Mercatelli, D., & Polito, L. Momordica charantia, a nutraceutical approach for inflammatory related diseases. Front. Pharmacol., 10, 486. 2019.
18. Sur, S., Steele, R., Isbell, T. S., Venkata, K. N., Rateb, M. E., & Ray, R. B. Momordicine-I, a bitter melon bioactive metabolite, displays anti-tumor activity in head and neck cancer involving c-Met and downstream signaling. Cancers (Basel), 13(6). 2021.
19. Ogidigo, J. O., Iwuchukwu, E. A., Ibeji, C. U., Okpalefe, O., & Soliman, M. E. S. Natural phyto, compounds as possible noncovalent inhibitors against SARS-CoV2 protease: computational approach. J. Biomol. Struct. Dyn., 40(5), 2284-2301. 2022.
20. Chen, P. Y., Shih, N. L., Hao, W. R., Chen, C. C., Liu, J. C., & Sung, L. C. Inhibitory effects of momordicine I on high-glucose-induced cell proliferation and collagen synthesis in rat cardiac fibroblasts. Oxid Med Cell Longev, 2018, 3939714. 2018.
21. Chou, M. C., Lee, Y. J., Wang, Y. T., Cheng, S. Y., & Cheng, H. L. Cytotoxic and anti-inflammatory triterpenoids in the vines and leaves of momordica charantia. Int J Mol Sci, 23(3). 2022
22. Desmet, T., Soetaert, W., Bojarová, P., Křen, V., Dijkhuizen, L., Eastwick-Field, V., & Schiller, A. Enzymatic glycosylation of small molecules: challenging substrates require tailored catalysts. Chemistry, 18(35), 10786–10801. 2012.
23. Lairson, L. L., Henrissat, B., Davies, G. J., & Withers, S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem, 77, 521-555. 2008.
24. Nidetzky, B., Gutmann, A., & Zhong, C. Leloir glycosyltransferases as biocatalysts for chemical production. ACS Catalysis, 8(7), 6283-6300. 2018.
25. Dai, L., Li, J., Yao, P., Zhu, Y., Men, Y., Zeng, Y., Yang, J., & Sun, Y. Exploiting the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis and its application in synthesis of glycosides. J. Biotechnol, 248, 69-76. 2017.
26. Cialoni, D., Brizzolari, A., Samaja, M., Bosco, G., Paganini, M., Pieri, M., Lancellotti, V., & Marroni, A. Nitric oxide and oxidative stress changes at depth in breath-hold diving. Front. Physiol., 11, 609642. 2021.
27. Becker, M., Nunes, G., Ribeiro, D., Silva, F., Catanante, G., & Marty, J. L. Determination of the antioxidant capacity of red fruits by miniaturized spectrophotometry assays. J Braz Chem Soc. 2019.
28. Arts, M. J. T. J., Sebastiaan Dallinga, J., Voss, H.-P., Haenen, G. R. M. M., & Bast, A. (2004). A new approach to assess the total antioxidant capacity using the TEAC assay. Food Chemistry, 88(4), 567-570. 2004.
29. Vasile, C., Sivertsvik, M., Mitelut, A. C., Brebu, M. A., Stoleru, E., Rosnes, J. T., . . . Popa, M. E. Comparative analysis of the composition and active property evaluation of certain essential oils to assess their potential applications in active food packaging. Materials (Basel), 10(1). 2017.
30. BIOVIA, Dassault Systèmes, BIOVIA discovery studio visualizer, San Diego: Dassault Systèmes, 2021.
31. Zhao, H., & Huang, D. Hydrogen bonding penalty upon ligand binding. PLoS One, 6(6), e19923. 2011.
32. Pantsar, T., & Poso, A. Binding Affinity via Docking: Fact and Fiction. Molecules, 23(8). 2018.
33. Pisoschi, A. M., & Negulescu, G. P. Methods for total antioxidant activity determination: a review. Anal. Bioanal. Chem., 01(01). 2012.
34. Reprinted from “Human Coronavirus Structure”, by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates
35. Snijder, E. J., Decroly, E., & Ziebuhr, J. The nonstructural proteins directing coronavirus RNA synthesis and processing. Adv Virus Res, 96, 59-126. 2016.
36. Hsu, K. C., Chen, Y. F., Lin, S. R., & Yang, J. M. iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis. BMC Bioinform., 12 Suppl 1(Suppl 1), S33. 2011.
37. Schrödinger, L., & DeLano, W. PyMOL. 2020. Retrieved from http://www.pymol.org/pymol