Title Page
Contents
ABSTRACT 9
Ⅰ. Introduction 11
1.1. The potential risks associated with COVID-19 11
1.2. Identity between other Coronaviruses 11
1.3. The rules of the main protease in the life cycle of SARS-CoV-2 11
1.4. The dimerization of main protease as drug target 12
Ⅱ. Results 13
2.1. Mpro purification[이미지참조] 13
2.2. Real-time fluorometric turn-on assay system 17
2.3. Mpro inhibition of GC376[이미지참조] 20
2.4. Dimer interaction surface of Mpro[이미지참조] 22
2.5. Activity inhibition by N-terminal peptide 29
2.6. Disruption of dimer conformation by N-terminal peptide 33
Ⅲ. Discussion 34
Ⅳ. Materials and Methods 40
4.1. Reagents 40
4.2. Cloning and expression of the main protease 40
4.3. Expression and purification of Mpro[이미지참조] 40
4.4. Size exclusion chromatography for dimer confirmation 41
4.5. Fluorogenic substrate preparation for assay 42
4.6. FRET-based Mpro proteolytic assay[이미지참조] 42
4.7. Calculations of solvent accessible surface area (SASA) of Mpro[이미지참조] 42
4.8. The N-terminal ten amino acids peptide of Mpro[이미지참조] 42
4.9. Inhibition assay 43
References 44
Abstract (in Korean) 47
Table. 1. Interacting surface area of N10 in Mpro dimer[이미지참조] 23
Table. 2. Dimer interface area of protomer A in Mpro dimer[이미지참조] 28
Figure. 1. Purified Mpro proteins in SDS-page[이미지참조] 14
Figure. 2. Size exclusion chromatography of Mpro alone (blue) and supplemented with N10 (red)[이미지참조] 15
Figure. 3. The calibration graph of size exclusion chromatography for Mpro alone and Mpro supplemented with N10[이미지참조] 16
Figure. 4. Scheme of the real-time fluorometric turn-on assay system of Mpro[이미지참조] 18
Figure. 5. Substrate concentration-dependent Mpro activity in the real-time fluorometric turn-on assay system[이미지참조] 19
Figure. 6. The inhibition of the Mpro activity by GC376 and N10 in the real-time fluorometric turn-on assay system[이미지참조] 21
Figure. 7. The crystal structure of Mpro dimer[이미지참조] 24
Figure. 8. The dimer interface surface of Mpro dimer[이미지참조] 25
Figure. 9. The N-terminal ten amino acids exist at the center of the dimer interface between two protomers 26
Figure. 10. The enlarged view of the N-terminal part (yellow) bound in protomer B (cyan) 27
Figure. 11. The purity and identification of N10 peptides 30
Figure. 12. The inhibition of Mpro activity of GC376 and N10[이미지참조] 31
Figure. 13. Chemical structures of GC376 and N10 and the inhibitory effects 32
Figure. 14. Potential inhibitory mechanisms of N10 36
Figure. 15. Mutated residues in SARS-CoV-2 Mpro variants[이미지참조] 37
Figure. 16. The amino acid substitutions 38
Figure. 17. Structure and sequence comparison of Mpro among SARS-CoV-2, SARS60 CoV, and MERS[이미지참조] 39