新型氨基吡啶类化合物抗真菌作用的分子机制研究

Invasive fungal infections are associated with significant morbidity and mortality. In the previous study we first performed antifungal drug development based on inhibiting the biosynthesis of GPI anchors in the country. We found aminopyridine derivatives could inhibit GPI-anchor biosynthesis, and possess strong antifungal activity in vitro and in vivo. We obtained new aminopyridine antifungal lead compounds by design and synthesis focus library. The active aminopyridine derivatives could not only inhibit fungal cell wall synthesis, but also increase pathogen-associated molecular patterns (PAMPs) exposure by inhibiting biosynthesis of GPI anchors. But the molecular mechanism and target of active aminopyridine derivatives against fungi remain unclear. In the present project, we first intend to continue the structure activity relation (SAR) and structural optimization study, for screening the compound with higher activity and specificity, and synthesizing affinity-based probes based on active aminopyridine derivatives with target recognition capability for further target research. Next, we intend to investigate the cellular and molecular mechanism of the active compounds against fungi, using confocal laser mass spectrometry technology combined with quantitative proteomics technology to study the biosynthesis of GPI anchors and the GPI-anchored protien attaching to cell wall after active aminopyridine derivatives treatment, and using western blot technique and PRRs gene knockout mice to detect the signal pathway and molecular mechanism of the enhanced host defense against fungi after active aminopyridine derivatives treatment. Finally, we will study the interaction between the affinity-based aminopyridine derivatives probes and fungi proteome to screen the interaction protein in fungi with the above active compounds, and then combine bioinformatic technologies to analyze the target of the active compounds against fungi. In the end, to validate the target of the novel compounds, we will knockout the target gene in fungi cell and compare the phenotype between gene deletion strain and wild type strain after incubation with the active compounds, and study the interaction between the candidate target protein and the active compounds by biacore technologies. The present project will provide theoretical basis and work foundation for new effective antifungal agent development.

侵袭性真菌感染的发病率逐年上升，病死率居高不下。我们在国内率先开展了抑制GPI锚生物合成的抗真菌药物研究，前期通过活性筛选、结构优化获得的氨基吡啶类抗真菌新先导化合物能抑制真菌GPI锚生物合成，具有较强的体内外抗真菌活性，并能促进真菌模式识别分子暴露，增强宿主免疫应答。但这类活性化合物具体的抗真菌分子机制和作用靶点尚不明确。本项目拟对活性化合物进行深入构效关系及结构优化研究，以发现具有更高活性和安全性的化合物；采用激光共聚焦结合质谱技术研究活性化合物对真菌GPI锚合成和细胞壁GPI-锚定蛋白种类和含量的影响；Western blot技术研究化合物增强真菌抗原暴露和宿主抗感染免疫应答的信号通路和分子机制；设计合成光亲和小分子探针，并结合iTRAQ定量蛋白质组学技术、Biacore技术、基因敲除/回复技术研究活性化合物与真菌特异相互作用靶点，为发现新型高效的抗真菌药物提供理论依据和工作基础。

侵袭性真菌感染的发生率逐年上升，病死率居高不下。真菌天然耐药和获得性耐药为真菌感染的治疗带来巨大困难，且近十多年来未发现基于新靶点的抗耐药真菌新药。本课题通过构效关系和结构优化研究，共设计合成氨基吡啶类化合物210多个，其中（GPI-89）11g的抗真菌MIC达0.0313-1μg/ml，并有很强地抑制真菌菌丝形成作用，减弱真菌在体内的侵袭能力，体外与氮唑类药物氟康唑有很好地协同抗耐药真菌活性。体内能剂量依赖性地保护真菌感染动物，小鼠体内（4mg/kg、 8mg/kg、16mg/kg）能剂量依赖性协同氟康唑抗耐药白念珠菌感染，同时显著降低肝脏、肾脏等重要器官的组织载菌量。进一步作用机制研究发现此类化合物作用后的白念珠菌，其细胞壁最外层的甘露糖层显著缺失，真菌细胞壁结构不完整，通过激光共聚焦显微镜观察，证明其能抑制白念珠菌糖基化磷脂酰肌醇（GPI）锚的合成。发现这类化合物能促进真菌细胞壁葡聚糖的暴露，有利于宿主对感染真菌的免疫识别，通过Dectin-1受体及NF-κB和MAPK信号通路，促进炎症细胞因子的产生，产生免疫保护作用。采用iTRAQ高分辨质谱定量蛋白质组学技术，比较GPI-89低浓度下对白念珠菌细胞壁蛋白表达的影响，发现PGA10、PGA45、RHD3、PGA4及YWP1等GPI锚定蛋白含量出现显著降低，PDI1、KAR2（在蛋白质转运至内质网过程中发挥重要作用）等蛋白含量出现显著升高。KEGG通路分析结果显示，共同检出了7种属于GPI锚合成通路的蛋白，GPI蛋白转氨基酶亚基GPI8蛋白含量均出现显著升高，在甾醇合成通路中ERG251蛋白和ERG3蛋白含量显著升高。发现GPI-89对GWT1/gwt1Δ菌株的生长和菌丝形成抑制作用更敏感，采用白念珠菌基因替换和条件表达菌库研究初步发现此类化合物对GPI的合成转运及麦角甾醇的生物合成通路有抑制作用。