2021-04-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/673072"俱多重抗藥性致病菌如沙門氏菌的感染發生率不斷上升，已對全世界公眾健康造成 一個嚴重的威脅。因此，發展一種創新方式來控制這些多重抗藥致病菌的感染，已 成為臨床醫療上一個迫切的需求。異體吞噬(Xenophagy)為一種具選擇性的巨自噬 作用(macroautophagy)且在細胞內在防衛中扮演一個重要的角色，有研究指出細 胞可透過活化單磷酸腺苷活化蛋白質激酶(AMPK)去誘發異體吞噬以清除入侵的細 菌。我們的初期研究亦發現一個俱活化AMPK活性的小分子藥物Nilotinib（原做為 抗癌藥物），對鼠巨噬細胞內的鼠傷寒桿菌具高度的抑制活性，卻對在液態培養基 中的細菌生長沒有任何影響，暗示其可能為一個以宿主細胞為標的之抗菌藥物。鑒 於Nilotinib的獨特抗菌活性，我們計畫以Nilotinib結構為基礎，去合成一個 Nilotinib的結構衍生物庫，並利用以影像為基礎的高內涵分析系統，同時分析 Nilotinib衍生物的抗菌活性與細胞毒性。若小分子藥物在低於微莫爾濃度下即有高 度抑菌活性，且有大於40倍以上的選擇性(抗菌活性除以細胞毒性)，則會更進一步 被挑選於小鼠沙門氏菌感染模式中去分析其抗菌藥效。此外，我們亦會使用免疫墨 點和免疫螢光等技術去評估個別藥物誘發自體吞噬的能力，並利用藥理和遺傳方法 去確認AMPK和其下游自體吞噬路徑在Nilotinib優化衍生物的抗菌活性中的角色。 我們預期這個研究計畫將可以開發出新的抗細菌藥物，用於治療俱多重抗藥性沙門 氏菌所造成的感染。" "The increasing incidences of infections caused by multiple-antibiotic resistant bacteria, including Salmonella enterica, have become a serious threat to public health worldwide. Thus, the development of an innovative therapeutic approach for the control of such infections is highly needed. Xenophagy is a type of selective macroautophagy and plays an important role in cellular innate defense. It has been shown that activation of AMP-activated protein kinase (AMPK) can initiate xenophgay to eradicate invading bacteria. In our pilot study, we found that an AMPK activator Nilotinib, originally used as an anticancer drug, exhibited antibacterial activity against intracellular S. enterica serovar Typhimurium in RAW264.7 murine macrophage but have no suppressive effect on bacteria in the growth medium. In light of its unique antibacterial activity, we propose to synthesize a Nilotinib focused compound library and to utilize an image-based high-content assay to simultaneously assess the anti-intracellular Salmonella activity and the cytotoxic effect of Nilotinib derivatives. Next, the in vivo efficacy of compounds that possess potent in vitro bacteria inhibiting activity at sub-M and have a selectivity of >40 (antibacterial activity over cytotoxicity) will be further evaluated in murine salmonellosis model. As nilotinib has been shown to induce autophagy via elevating of AMPK activity, we will assess the autophagy-inducing activity of individual compound by using immunoblotting and immunofluorescence techniques, and investigate the role of AMPK and downstream autophagy pathway in the antibacterial activity of Nilotinib’s optimal derivatives by using pharmacologic and genetic approaches. We expect this project will identify a novel host-targeted agent for the treatment of diseases caused by intracellular Salmonella, especially those with resistance to multiple antibiotics."自體吞噬高內涵分析以宿主為標的抗菌藥物AutophagyHigh-content assayHost-targeted antibacterial drug