Biological Evaluation of Novel Targeted Inhibitors Against SbnE, RAF1, and BRAF Class I/II/III Mutants to Overcome Drug Resistance

목차 (Table of Contents)

• List of figures v
• List of tables vii
• List of Abbreviations viii
• List of Publications ix
• Part 1. Biological evaluation of Baulamycin A-inspired compounds in methicillin-resistant Staphylococcus aureus (MRSA) １
• 1.1 Introduction １
• 1.2 Materials and Methods ４
• 1.2.1 Chemical synthesis ４
• 1.2.2 Bacterial strains and media ４
• 1.2.3 Antibiotics ４
• 1.2.4 SbnE cloning, overexpression, and purification ４
• 1.2.5 Enzymatic activity test ５
• 1.2.6 MIC measurement ５
• 1.2.7 Chrome azurol S (CAS) liquid assay ６
• 1.2.8 Time-kill assay ６
• 1.2.9 Membrane integrity assay ７
• 1.2.10 Hemolysis assay ７
• 1.2.11 Biofilm formation assay and imaging ７
• 1.3 Results and Discussion ９
• 1.3.1 Structure-Activity Relationships (SAR) Study against SbnE enzyme inhibition effect and MRSA antibacterial potency ９
• 1.3.2 Effect of baulamycin derivatives on siderophore production in S. aureus １３
• 1.3.3 Effect of BmcA derivatives on membrane disruption in S. aureus １７
• 1.3.4 Bactericidal kinetics of BmcA derivatives １９
• 1.3.4 Effect of BmcA derivatives on hemolysis ２１
• 1.3.5 Bactericidal effect study of baulamycin derivatives against selected bacteria strains ２３
• 1.3.6 Biofilm inhibitory activities of compounds ２５
• 1.4 Conclusion ２７
• Part 2. Biological evaluation of oxazole derivatives as highly selective RAF1 inhibitors ２９
• 2.1 Introduction ２９
• 2.2 Materials and Methods ３２
• 2.2.1 Chemical synthesis ３２
• 2.2.2 Cell culture & cell-based assay ３２
• 2.2.3 Cell proliferation assay ３２
• 2.2.4 Western blot ３２
• 2.3 Results and Discussion ３４
• 2.3.1 Structure-activity relationships ３４
• 2.3.2 Kinome-wide profile ３６
• 2.3.3 Signaling inhibitory activities of 2d ３８
• 2.3.4 Anti-proliferation activities of 2d ４０
• 2.4 Conclusion ４２
• Part 3. Novel and potent small molecules against melanoma harboring BRAF class I/II/III mutants for overcoming drug resistance ４３
• 3.1 Introduction ４３
• 3.2 Materials and Methods ４６
• 3.2.1 Chemical synthesis ４６
• 3.2.2 Cell culture and reagents ４６
• 3.2.3 Site-directed mutagenesis ４６
• 3.2.4 Anti-proliferation assay ４７
• 3.2.5 Western blot ４７
• 3.2.6 Flow cytometry analysis ４８
• 3.2.7 Migration and invasion assay ４８
• 3.2.8 Colony formation assay ４９
• 3.3 Results and Discussion ５０
• 3.3.1 Six derivatives strongly suppress proliferation of melanoma cells harboring class I/II/III BRAF mutation. ５０
• 3.3.2 Effects of 3d on Ba/F3 cells harboring class I/II BRAF mutation. ５３
• 3.3.3 Effects of 3d on MAPK/AKT signaling against melanoma cells harboring BRAF wt or class I/II/III mutations ５５
• 3.3.4 Effects of 3d on apoptosis induction in melanoma cells ５７
• 3.3.5 Effects of 3d on cell migration and invasion activities in melanoma cells ５９
• 3.3.6 Colony formation inhibitory activities of 3d ６１
• 3.4 Conclusion ６３
• Part 4. Biological evaluation of anticancer effects of GNF-7 against KRAS-mutant colorectal cancer cell lines ６５
• 4.1 Introduction ６５
• 4.2 Materials and Methods ６７
• 4.2.1 Chemical synthesis ６７
• 4.2.2 Cell culture & cell-based assay ６７
• 4.2.3 Cell proliferation assay ６７
• 4.2.4 Western blot analysis ６７
• 4.2.5 Flow cytometry analysis ６８
• 4.2.5 Migration assay ６８
• 4.2.7 2D and 3D colony formation assay ６９
• 4.2.8 Analysis of whole exome sequencing (WES) and RNA sequencing ６９
• 4.2.9 Combination analysis ７０
• 4.210 Statistical analysis ７０
• 4.3 Results and Discussion ７１
• 4.3.1 Anti-proliferative and signaling inhibitory activities of GNF-7 on CRC cells ７１
• 4.3.2 Anti-cancer effects of GNF-7 on HCT-116 and SW480 cells ７３
• 4.3.3 Effect of GNF-7 on the AKT signaling and identification as functional target ７７
• 4.3.4 Identification of EGFR as a functionally relevant target of GNF-7 ８１
• 4.4 Conclusion ８７
• Summary ８８
• Korean abstract ９１
• References ９４