基于靶向亲和-高分辨活性轮廓分析的中药流感病毒RdRp抑制剂筛选策略研究

由于抗原漂移和转变，流感病毒耐药性问题日益严重，新型抗流感药物的开发成为疾病防控的核心段。流感病毒RNA聚合酶（RdRp）序列高度保守，已被证实为发现抗流感新药的重要靶点。抗流感中药复方临床效果明显，是发现流感病毒RdRp抑制剂的宝库，但如何突破现有筛选方法耗时耗力、效率低下的技术瓶颈依然困难重重。本项目拟以RdRp的PA亚基为靶标，构建基于靶向亲和-高分辨活性轮廓分析的RdRp抑制剂高效筛选平台。靶向PA亚基的亲和磁性纳米材料有望快速锁定并富集中药中潜在配体群，而基于液滴微阵列的活性轮廓分析可对配体群同步进行高内涵结构和活性鉴定。二者优势互补，在缩短筛选时间、提升筛选准确性的同时，显著提高高内涵检测的灵敏度和分辨率，有利于中药潜在流感病毒RdRp抑制剂的高效发掘。本研究不仅为中药抗流感活性成分的筛选提供了强有力工具，而且对揭示其药效物质基础、推动其质量控制和谱效关系研究意义重大。

Annual influenza epidemics result in substantial mortality, heavy social and economic burden. Anti-influenza drugs are critical countermeasures for control of influenza. However, the long-term effectiveness of these drugs is a concern due to the emergence of drug-resistant strains. Thus, there is an urgent need for development of new measures to prevent and treat influenza virus infection. RNA polymerase, which consisted of three subunits, PA, PB1 and PB2, plays an essential role in the process of transcription and replication of the influenza virus. Because of its high structural conservation in influenza virus, it is a potential target for the discovery and development of new anti-influenza drugs. The effectiveness of anti-influenza Chinese herbal medicine compounds has been demonstrated over thousand years’ clinic usage. These compounds are important recourses for finding new influenza RdRp inhibitors. However, there are still bottlenecks to be addressed in order to screen out potential inhibitors in TCMs. Conventional activity-guided fractionation is a high cost and low efficient screening approach, because it involves many cycles of bioactivity test, purification and identification. Therefore, it is of great interest to develop a high efficient and high-throughput method for screening potential influenza RdRp inhibitors from TCMs. In this study, we proposed a new screening strategy integrating affinity ligand fishing and high resolution bioassay profiling technologies. PA subunit modified affinity magnetic nanomaterials will be developed for rapid locating and enriching ligands in complex matrix, while the identity and PA inhibition effect of the captured ligands will be measured on a lab-made high resolution bioassay profiling system consisted of a nano-liquid chromatography, a high-resolution mass spectrometry, a nanoliter (nL) auto-fractionation system and a microarray-based fluorescence bioassay system. After a systematical optimization and evaluation, a high resolution and rapid screening platform will finally be developed for finding influenza endonuclease inhibitors in TCMs. The proposed strategy based on affinity ligand fishing and high resolution bioassay profiling has many advantages over conventional AGF, such as low sample consumption, low cost, low false positive rate, high throughput, high sensitivity and screening efficiency etc. This research not only provides a powerful tool for screening novel influenza RdRp inhibitors, but also shows great meaning in revealing pharmacodynamic material basis and promoting comprehensive quality of anti-influenza TCMs.