A Drug Screening Method Based on the Autophagy Pathway and Studies of the Mechanism of Evodiamine against Influenza A Virus

In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation - fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1β, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound.本研究以自噬信号通路为核心靶点，建立了一种基于双分子荧光互补-荧光共振能量转移（BiFC-FRET）技术的药物筛选方法，用于开发新型抗甲型流感病毒（IAV）药物。研究团队从83种中药材中筛选出吴茱萸（Evodia rutaecarpa Benth），并对其主要活性成分吴茱萸碱的抗IAV活性及作用机制进行了深入探究。经空斑抑制实验、甲型流感病毒vRNA启动子荧光素酶报告基因实验以及磺酰罗丹明B法检测细胞病变效应（CPE）减轻程度验证，结果显示吴茱萸碱可显著抑制IAV的复制。此外，吴茱萸碱可显著抑制LC3-II与p62的积累，以及EGFP-LC3的点状聚集。该化合物还可抑制Atg5-Atg12/Atg16异源三聚体的形成、IAV感染后Atg5、Atg7及Atg12的表达，以及肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）与白细胞介素-8（IL-8）的细胞因子释放。吴茱萸碱对甲型流感病毒诱导的自噬的抑制作用，依赖于其对AMPK/TSC2/mTOR信号通路的调控。综上，本研究成功建立了一种新型药物筛选方法，并筛选出吴茱萸碱作为极具潜力的抗甲型流感病毒候选化合物。