探討新穎的抗金黃色葡萄球菌藥物SC-5005

Abstract

抗甲氧苯青黴素之金黃色葡萄球菌具有對抗大多數抗生素之能力，導致治療上非常棘手，對於人類健康而言是個很嚴重的威脅，因此必須發展新穎的抗葡萄球菌藥物。在我們的研究中發現原本應用於治療癌症的酪胺酸激酶抑制劑蕾莎瓦(Nexavar; sorafenib)具有抑制抗藥性較低的金黃色葡萄球菌能力，但對於抗甲氧苯青霉素之金黃色葡萄球菌卻沒有抑制效果。為了降低蕾莎瓦對細胞之毒性且保留抑制細菌之能力，我們以蕾莎瓦衍生物抑菌能力以及細胞毒性進行篩選，發現SC-78與SC-5005不只能夠抑制臨床分離的多重抗藥葡萄球菌，對於抗萬古黴素之腸球菌以及大多數具有致病性的革蘭氏陽性菌皆有抑制效果，但無法抑制革蘭氏陰性細菌。相較於細胞毒性極高的蕾莎瓦，SC-78與SC-5005對人類細胞株的毒性降低許多，其中又以SC-5005具有較高的選擇性(抑制百分之五十細胞增生之藥物濃度/最低抑制細菌濃度)，比較適合作為抗細菌藥物。此外，我們也使用了線蟲模式評估這兩種藥物在活體內的細胞毒性，發現SC-78具有急毒性但SC-5005沒有，因此我們選擇SC-5005進行動物實驗評估藥效。我們在動物實驗結果中發現，若給予受到全身性多重抗藥葡萄球菌感染的小鼠SC-5005治療，能夠顯著地提高小鼠的存活率。目前從巨分子合成分析法得到的結果顯示SC-5005可能作用於細菌的細胞膜上，並使用螢光染色後發現加藥處理後會造成細菌的細胞膜破損，亦造成細菌胞內ATP流失。然而即使在一百二十八倍的最小抑菌濃度下仍不會造成人類紅血球產生溶血現象。

Methicillin-resistant Staphylococcus aureus (MRSA) becomes a serious threat to human health because of multidrug resistance to most antibiotics used in medical treatment. Thus, it is imperative to develop a novel anti-Staphylococcus agent. In our study, we find that sorafenib (Nexavar), a tyrosine kinase inhibitor which was used in cancer therapy, can not only suppress human cell proliferation but also moderately inhibit methicillin-sensitive Staphylococcus aureus (MSSA). However, it has no inhibitory effect on MRSA. To improve sorafenib’s antibacterial activity, an in-house sorafenib-based focused compound library was synthesized and screened followed by in vitro efficacy against S. aureus and cytotoxicity to human cell line evaluation. We found that SC-78 and SC-5005 can inhibit clinically-isolated MRSAs, vancomycin-resistant Enterococci (VRE), and most pathogenic Gram-positive bacteria. Nevertheless, Gram-negative bacteria are not susceptible to these two agents. In comparison with sorafenib that possess high cytotoxicity, SC-78 and SC-5005 are less toxic to cell, and SC-5005 is more suitable for development of antibacterial agents because of much higher selectivity ratio (antiproliferative activity /antibacterial activity) than SC-78. In addition, we also assessed in vivo cytotoxicity of these two agents by C. elegans model. The results demonstrated that SC-78 possessed acute cytotoxicity, but SC-5005 didn’t. Therefore, we choose SC-5005 to evaluate the efficacy against systemic infection by MRSA in C57BL/6J mice. The survival rate of infected mice treated with SC-5005 increases significantly in contrast to control. The findings indicated that SC-5005 has potential to fight with MRSA infection. The mode of action of SC-5005 on S. aureus was unveiled by macromolecule synthesis assay, and the result pointed out that SC-5005 might act on cell membrane. After treatment, the damage to bacterial cell membrane was observed by fluorescence microscopy. Apart from that, SC-5005 also lead to leakage of intracellular ATP in bacteria. However, the agent did not cause hemolysis on human red blood cell at the concentration of 128-fold MIC against S. aureus.