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J Enzyme Inhib Med Chem
. 2025 Dec;40(1):2501743.
doi: 10.1080/14756366.2025.2501743. Epub 2025 May 15. Natural product sennoside B disrupts liquid-liquid phase separation of SARS-CoV-2 nucleocapsid protein by inhibiting its RNA-binding activity
Da-Wei Zhang 1 , Xiao-Shuang Xu 1 , Liangxu Xie 1 , Lei Xu 1 , Zhiguo Fu 2 , Yimin Li 3 , Xiaojun Xu 1
Affiliations
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.
Keywords: HTRF; SARS-CoV-2; liquid-liquid phase separation; nucleocapsid protein; sennoside B.
. 2025 Dec;40(1):2501743.
doi: 10.1080/14756366.2025.2501743. Epub 2025 May 15. Natural product sennoside B disrupts liquid-liquid phase separation of SARS-CoV-2 nucleocapsid protein by inhibiting its RNA-binding activity
Da-Wei Zhang 1 , Xiao-Shuang Xu 1 , Liangxu Xie 1 , Lei Xu 1 , Zhiguo Fu 2 , Yimin Li 3 , Xiaojun Xu 1
Affiliations
- PMID: 40371698
- DOI: 10.1080/14756366.2025.2501743
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.
Keywords: HTRF; SARS-CoV-2; liquid-liquid phase separation; nucleocapsid protein; sennoside B.