以新穎二苯氧氮平類化合物去削弱巨噬細胞內沙門氏菌透過外排幫浦的抗氧化能力

Abstract

可入侵宿主細胞內進行感染的病原菌是全球一個嚴重的威脅。在侵入宿主細胞後，胞內細菌會藉由表現致病因子躲避細胞的先天性免疫防衛而得以存活。因此，針對胞內菌致病因子為標的之透過新穎殺菌機制的抗菌藥物開發是不可或缺的。我們之前的研究發現抗精神病用藥―樂賜平，具有抗小鼠巨噬細胞內沙門氏菌存活的效果。為了保留樂賜平的抗菌活性並去除其作為抗精神病用藥的活性，我們設計合成一系列的樂賜平衍生物，再利用高內涵影像分析平台來評估衍生物的抗菌活性及細胞毒性。經過一系列的篩選，我們找到一個新的化合物―SW14具有比樂賜平更為良好的抗胞內沙門氏菌的活性。除此之外，SW14也對胞內的福氏志賀氏菌、單核球增多性李斯特菌、抗甲氧苯青黴素金黃色葡萄球菌、小腸結腸耶氏菌，還有多重抗藥性的沙門氏菌具有良好的抗菌活性，並且在小鼠的沙門氏菌感染模型中SW14也展現其活性。機制方面，我們發現抗氧化劑可以反轉SW14對胞內沙門氏菌的抑制，並證實SW14具有抑制細菌外排幫浦的活性，且會增加細菌對於氧化壓力的感受性，此些發現表明SW14可能是藉由抑制細菌透過外排幫浦的抗氧化能力來達到降低胞內感染的細菌量，後續可發展為新一類的抗菌藥物。

Pathogenic bacteria capable of invading and proliferating inside host cells have been a serious threat worldwide. After entering host cells, these intracellular pathogens can express a variety of virulence factors to evade the innate immunity of host cells. In light of this, the development of a novel virulence-targeted antibacterial drug has been a potential strategy to control the infections of intracellular bacteria. Previously, we showed that an antipsychotic drug, loxapine, exhibited potent antibacterial activity against intracellular Salmonella enterica in murine RAW264.7 macrophagic cells. To improve loxapine’s antibacterial activity, a series of loxapine derivatives were designed and synthesized. The antibacterial activity and cytotoxicity of these compounds were simultaneously evaluated using an image-based high-content assay. The screening identified a new compound SW14 which showed a multifold increase in antibacterial activity against intracellular multiple-antibiotic-resistant S. enterica, Shigella flexneri, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and Yersinia enterocolitica. Moreover, SW14 is also efficacious in the murine salmonellosis model. Subsequent mechanism studies indicated that the ROS scavengers could reverse the antibacterial effect of SW14 against intracellular Salmonella. Also, SW14 exhibited suppressive activity on the bacterial efflux pump and increase the susceptibility of bacteria to oxidative stresses. Together, our data suggested that SW14 might suppress intracellular bacteria via impeding the efflux pump-mediated resistance to oxidative stresses. The structure of SW14 represents a promising scaffold for the development of a new virulence-targeted antibacterial agent against intracellular bacteria.