臺灣博碩士論文加值系統

抗甲氧苯青黴素金黃色葡萄球菌因其具有對抗大多數抗生素治療的能力，使其成為全球性的健康議題。現今的處境更加凸顯發展新穎抗金黃色葡萄球菌藥物的迫切性。在我們先前的研究中篩選出一個化合物稱為SC-5005，它是抗癌藥物蕾莎瓦(Nexavar；sorafenib)的衍生物。SC-5005具有高度抗菌能力同時保持較低的細胞毒性，但其選擇性(最低抑菌濃度/細胞毒性)經計算後約39.4，與一般開發藥物所求選擇性需100稍有差距。因此我們透過修飾SC-5005的結構得到一系列的化合物，試著從這些化合物中找到更好的化合物進行開發，另一方面我們也積極尋找更適合SC-5005的藥物載體。在我們的篩選結果中找到SC-5035，但SC-5035在動物感染模式中無法表現其抗菌能力去治療小鼠的感染症而宣告失敗。而在藥物載體方面，我們找到1,2-丙二醇可以替換原本使用的藥物載體PEG-400，不會影響其藥效且可配製成較高濃度的藥劑，並減緩原載體對小鼠產生的副作用。另外我們也發現HRCA這個化合物可以大幅提升SC-5005的抗菌效果，因此更新藥物載體後將SC-5005結合HRCA進行動物實驗，結果顯示此藥物組合的確大幅提升小鼠存活率。此外我們實驗室先前的研究結果中顯示SC-5005可能藉由破壞細菌細胞膜達到抗菌的效果。在本篇研究接續發現SC-5005與SC-5035確實破壞細菌細胞膜而使得大分子螢光染劑可進入細菌體內，且藥物處理可造成金黃色葡萄球菌細胞內ATP大量流失，並在穿透是穿透式電顯實驗結果中亦明確觀察到細菌細胞膜上有孔洞產生，而SC-5005在細菌細胞膜上穿孔經Calcein leakage assay初步判定並非直接與膜上脂肪作用達成。總和實驗結果，SC-5005確可以透過修改化合物的結構得到更具開發性的藥物與合併HRCA去提升其抗菌能力並提升受MRSA感染小鼠存活率。而SC-5005以目前實驗結果推測的確會使金黃色葡萄球菌的細胞膜產生孔洞而達到抑菌的效果。

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious health problems worldwide due to its’ resistance to most antibiotic. This situation highlights the urgent need of developing novel anti-Staphylococcus agent. In our previous study, we identified a compound called SC-5005, a derivative of anti-cancer drug sorafenib (Nexavar). SC-5005 exhibited high antibacterial activity and low cytotoxicity. However, the selective ratio (antiproliferative activity / antibacterial activity) of SC-5005 is 39.4 only, which is lower than the ideal selective ratio value, 100. Thus, an in-house synthesized compound library of SC-5005 derivatives was screened to identify a better compound. Meanwhile, we keep optimizing the drug vehicle. During the screening, SC-5035 was identified to exhibit higher potency, but it is a pity that SC-5035 failed in in vivo assay. For the drug vehicle optimization, we found SC-5005 has higher solubility in 1,2-Propanediol which could replace our original PEG-400 based vehicle without affecting the antibacterial activity of SC-5005 and avoid PEG-400 induced inflammatory response. .Moreover, we also found a chemical called HRCA that can significantly enhance the antibacterial activity of SC-5005. On the other hand, we also try to figure out how SC-5005 act on S. aureus. Previously, our lab members found that SC-5005 might act on bacterial cell membrane. So we utilized fluorescent Live/Dead staining to observe whether bacterial cell membrane was damage after SC-5005 and SC-5035 treatment. Our result showed that our compounds indeed damage bacterial cell membrane, resulting an orange-colored bacteria. We also tested ATP leakage of S. aureus and observed that intracellular ATP level dropped dramatically after SC-5005 and SC-5035 treatment. The transmission electron microscopy image showed the pore on S. aureus cell membrane after SC-5005 treatment, but the calcein leakage assay result pointed out that the pore formation ability of SC-5005 is not through directly acting on the membrane lipids. In summary, first, we can modify the structure of SC-5005 to enhance its antibacterial activity. Second, 1,2-Propanidiol is a better vehicle for SC-5005 without the side effects of PEG400. Third, SC-5005’s antibacterial activity is by forming pores on bacterial cell membrane.

致謝 i
中文摘要 ii
Abstract iii
Contents v
1. Introduction 1
1.1 Staphylococcus aureus 2
1.2 Severe antibiotic resistance problem of Staphylococcus aureus 3
1.3 Difficulties in treatment for MRSA infections 4
1.4 SC-5005, a derivative from Sorafenib 5
1.5 Specific aims 5
2. Materials and methods 6
2.1 Bacteria strains and culture condition 7
2.2 Cell line and culture condition 7
2.3 Chemicals and reagents 7
2.4 Antibacterial assays 8
2.5 Antiproliferative assay 9
2.6 LDH Release assay 10
2.7 In vivo efficacy against S. aureus infection in C. elegans studies 11
2.7 In vivo MRSA bacteremia and sepsis study 12
2.8 In vivo MRSA bacteremia/sepsis study with optimized vehicle 13
2.9 ATP leakage assay 14
2.10 Fluorescence microscopy 15
2.11 Calcein leakage assay 15
2.12 Transmission Electron Microscopy for observation of S. aureus cell membrane 17
3. Results 20
3.1 Antibacterial activity of SC-5005 derivatives against Staphylococcus aureus 21
3.2 Antiproliferative activity of SC-5005 derivatives 21
3.3 Efficacy of SC-5005 against MRSA infection in C. elegans 22
3.4 Efficacy of SC-5005 in different solvents against MRSA systemic infection in 23
3.4 Evaluation of SC-5005’s antibacterial activity in a series of commercial screening vehicles 24
3.5 Efficacy of SC-5005 in HRC #3, #5, and #7 vehicles against MRSA systemic infection in C57BL/6J mice 24
3.6 Investigate which ingredient in HRC vehicle could promote the antibacterial activity of SC-5005 25
3.7 Efficacy of SC-5005 combined HRCA against MRSA systemic infection in C57BL/6J mice 26
3.8 Evaluation of membrane integrity of SC-5005 treated S. aureus 28
3.9 Calcein leakage assay of SC-5005 29
3.10 Transmission electron microscopy image of S. aureus after SC-5005 treatment 30
4. Discussion 32
4.1 SC-5035 failed in curing MRSA systemic infection in C57BL/6J mice 33
4.2 HRC screening vehicle could cause severe inflammatory response in C57BL/6J mice 33
4.3 The lipid composition of S. aureus cell membrane 35
4.4 The mechanism of HRCA enhancing SC-5005’s antibacterial activity 36
4.5 The application of SC-5005 36
5. Reference 38
6. Tables 45
Table 1. Antibacterial activity of compounds against Staphylococcus aureus 46
Table 2. Anti-Staphylococcus (MIC) versus antiproliferative (IC50) activities of test agents 47
Table 3. Antibacterial activity of SC-5005 in HRC Screening Vehicles against Staphylococcus aureus 48
Table 4. Antibacterial activity of SC-5005 combined HRC Screening Vehicle Ingredient (HRCA) against Staphylococcus aureus 49
Table 5. Antibacterial activity of SC-5005 combined HRC Screening Vehicle Ingredient (HRCA + HRCB) against Staphylococcus aureus 50
Table 6. Antibacterial activity of SC-5005 combined HRC Screening Vehicle Ingredient (HRCA+HRCB+HRCC) against Staphylococcus aureus 51
Table 7. Antibacterial activity of SC-5005 in different Vehicle against Staphylococcus aureus 52
Table 8. Antibacterial activity of SC-5005 combined different concentration of HRCA against Staphylococcus aureus 53
7. Figures 54
Figure 1. Antiproliferative activities of SC-5005 and SC-5035 56
Figure 2. Efficacy of SC-78 and SC-5005 against MRSA infection in C. elegans. 57
Figure 3. Efficacy of SC-5005 in different solvent against MRSA systemic infection in C57BL/6 mice. 59
Figure 4. Efficacy of SC-5005 in HRC Screening vehicle against MRSA systemic infection in C57BL/6 mice 61
Figure 5. Investigate ingredients in HRC screening vehicle #3, #5, and #7 63
Figure 6. Antiproliferative activities of SC-5005 combined HRCA 64
Figure 7. Efficacy of SC-5005 combined HRCA in bacteremia / systemic infection mice model 66
Figure 8. Efficacy of SC-5005 combined HRCA in bacteremia / systemic infection mice model retest 68
Figure 9. S. aureus NCTC 8325 intracellular ATP level 69
Figure 10. Pore formation on S. aureus after SC-5005 and SC-5035 treatment 71
Figure 11. calcein leakage assay of SC-5005 73
Figure 12. Transmission electron microscopy image of S. aureus after SC-5005 treatment 75

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