Zhang Da-Wei, Xu Xiao-Shuang, Xie Liangxu, Xu Lei, Fu Zhiguo, Li Yimin, Xu Xiaojun
Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China.
Department of Orthopedics, Changzhou Hospital of Traditional Chinese Medicine, Changzhou, China.
J Enzyme Inhib Med Chem. 2025 Dec;40(1):2501743. doi: 10.1080/14756366.2025.2501743. Epub 2025 May 15.
The nucleocapsid protein (NP) of SARS-CoV-2, an RNA-binding protein, is capable of undergoing liquid-liquid phase separation (LLPS) during viral infection, which plays a crucial role in virus assembly, replication, and immune regulation. In this study, we developed a homogeneous time-resolved fluorescence (HTRF) method for identifying inhibitors of the SARS-CoV-2 NP-RNA interaction. Using this HTRF-based approach, we identified two natural products, sennoside A and sennoside B, as effective blockers of this interaction. Bio-layer interferometry assays confirmed that both sennosides directly bind to the NP, with binding sites located within the C-terminal domain. Additionally, fluorescence recovery after photobleaching (FRAP) experiments revealed that sennoside B significantly inhibited RNA-induced LLPS of the NP, while sennoside A displayed comparatively weaker activity. Thus, the developed HTRF-based assay is a valuable tool for identifying novel compounds that disrupt the RNA-binding activity and LLPS of the SARS-CoV-2 NP. Our findings may facilitate the development of antiviral drugs targeting SARS-CoV-2 NP.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的核衣壳蛋白（NP）是一种RNA结合蛋白，在病毒感染期间能够发生液-液相分离（LLPS），这在病毒组装、复制和免疫调节中起着关键作用。在本研究中，我们开发了一种用于鉴定SARS-CoV-2 NP-RNA相互作用抑制剂的均相时间分辨荧光（HTRF）方法。使用这种基于HTRF的方法，我们鉴定出两种天然产物，番泻苷A和番泻苷B，它们是这种相互作用的有效阻断剂。生物层干涉术分析证实，这两种番泻苷均直接与NP结合，结合位点位于C末端结构域内。此外，光漂白后荧光恢复（FRAP）实验表明，番泻苷B显著抑制NP的RNA诱导的LLPS，而番泻苷A的活性相对较弱。因此，所开发的基于HTRF的检测方法是鉴定破坏SARS-CoV-2 NP的RNA结合活性和LLPS的新型化合物的有价值工具。我们的研究结果可能有助于开发针对SARS-CoV-2 NP的抗病毒药物。
