以酪氨酸激酶抑製劑為基礎去開發新穎可抗巨噬細胞中俱抗藥性的鼠傷寒沙門氏菌

Abstract

細菌抗藥性已成為全球嚴重的公衛問題，抗生素治療下能有效控制宿主細胞外的細菌感染，但對於胞內細菌有限的治療效果會造成細菌反覆感染，且危及病患生命，因此找尋替代方法治療胞內細菌感染是非常迫切的。 自噬作用是一種細胞內循環系統且高度受控的降解過程，可被一系列細胞壓力所激活，包括營養缺乏、蛋白毒性聚集體和細胞內病原體感染。有研究指出，細胞一旦檢測到病原細菌，就會刺激自噬途徑的上游活化-單磷酸腺苷活化蛋白質激酶（AMPK），從而減少哺乳動物雷帕黴素靶蛋白(mTORC1)介導的自噬途徑的抑制。然而，此由AMPK激活的自噬作用並不伴隨著大量非專一自噬的誘導，而是能對於細胞內病原體具有選擇性的和特異性的自噬作用，又稱為異種吞噬。為了開發新型AMPK激活劑以誘導異種吞噬消除細胞內細菌，我們與藥物化學專家合作修改了AMPK激活劑尼洛替尼(nilotinib)的結構，並使用影像為基礎的高內涵篩選同時評估細胞毒性和尼洛替尼衍生物對抗胞內沙門氏菌的活性。到目前為止，我們發現了幾種比尼洛替尼活性更高的衍生物。我們還證實尼洛替尼衍生物對胞內具抗藥性的沙門氏菌也有效。另外，這些衍生物與臨床使用的抗生素合併使用下發揮更好的效果。目前以AMPK抑制劑不能逆轉衍生物對細胞內沙門氏菌的抑制作用。在經過衍生物處理的細胞中，我們的結果表明自噬通量被阻斷。 此外，衍生物的功效不受自噬相關蛋白減量的影響。這些結果表明，衍生物對抗細胞內細菌的機制可能不是透過自噬作用。

Diseases caused by pathogenic bacteria have always been a serious threat to public health worldwide. For control of bacterial infection, antibiotics have been used to eliminate bacteria for decades, but most of them are less effective on intracellular bacteria which are a major cause of reinfection. Therefore, finding an alternative approach to treat diseases caused by intracellular bacteria is urgently needed. Autophagy, an intracellular recycling system, is a highly regulated degradative process which can be activated by a range of cellular stressors, including nutrient starvation, proteotoxic aggregates, and intracellular pathogen infection. Reports have showed that AMP-activated protein kinase (AMPK), an upstream activator of the autophagy pathway, is stimulated upon the detection of pathogenic bacteria, leading to remission of mTORC1-mediated repression of the autophagy pathway. This selective and specific autophagic degradation of intracellular pathogens is called xenophagy. To develop a new AMPK-activator for eradicating intracellular bacteria via induction of xenophagy, we worked with medicinal chemistry experts to modify the structure of an AMPK activator, nilotinib, and used an image-based high-content assay to simultaneously evaluate the cytotoxicity and anti-intracellular Salmonella activity of nilotinib derivatives. So far, we had discovered several derivatives with better activity than nilotinib. We also showed that the suppressive effect of nilotinib derivatives were effective against intracellular antibiotic-resistant Salmonella. Additionally, these derivatives had combined effects with antibiotics in clinic to exert better effect. Recently, the suppressive effects of derivatives on intracellular Salmonella cannot be reversed by AMPK inhibitors. In derivatives treated cells, our results show that the blockage of autophagy flux. Moreover, the efficacy of derivatives was not affected by autophagy-related protein knockdown. These findings show that the mechanism of derivatives against intracellular bacteria may not mediate by autophagy.