Rhein inhibits respiratory syncytial virus entry by stabilizing the prefusion conformation of the fusion protein

  • J Adv Res. 2026 Apr 3:S2090-1232(26)00279-1. doi: 10.1016/j.jare.2026.03.058.
Kunhuan Yang  1 ,  Yingcai Xiong  2 ,  Junxi Song  1 ,  Zhaowei Zhang  1 ,  Keyu Tao  1 ,  Zepeng Yang  1 ,  Tao Li  3 ,  Liangyu Cai  4 ,  Jianjian Ji  5
Affiliations
  • 1. Jiangsu Key Laboratory of Children's Health and Chinese Medicine, Jiangsu Provincial Research Institute of Chinese Medicine Schools, The First Clinical College, Nanjing University of Chinese Medicine, Nanjing 210023, China.
  • 2. The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210023, China.
  • 3. Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine, Wuxi 214045, China. Electronic address: [email protected].
  • 4. Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine, Wuxi 214045, China. Electronic address: [email protected].
  • 5. Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine, Wuxi 214045, China; Jiangsu Key Laboratory of Children's Health and Chinese Medicine, Jiangsu Provincial Research Institute of Chinese Medicine Schools, The First Clinical College, Nanjing University of Chinese Medicine, Nanjing 210023, China; Jiangsu Provincial Research Institute of Chinese Medicine Schools, Nanjing University of Chinese Medicine, Nanjing, China. Electronic address: [email protected].
PMID: 41936817 DOI: 10.1016/j.jare.2026.03.058
Abstract

Introduction: Respiratory syncytial virus (RSV) remains a major cause of severe lower respiratory tract infections. The high mutability of its RNA genome necessitates novel therapeutics, with the viral fusion (F) protein representing a promising target.

Objectives: This study aimed to identify RSV F protein inhibitors via structure-based virtual screening and to systematically characterize their Antiviral efficacy and mechanism of action.

Methods: A multi-tiered virtual screening of an Antiviral compound library was performed against the prefusion RSV F protein structure. Hits were validated for binding using MST and SPR. The mechanism was elucidated through time-of-addition, membrane fusion, and lentiviral pseudotype assays. In vivo efficacy was evaluated in an RSV infected BALB/c mouse model.

Results: Screening identified 10 candidate compounds to interact with RSV F protein. Among these, Rhein bound to the prefusion F protein with micromolar affinity. Rhein exhibited potent anti-RSV efficacy with IC50 values of 498.4 nM in HEp-2 cells and 594.0 nM in A549 cells. Further experiments revealed Rhein disrupted RSV membrane fusion by enhancing the conformational stability of the RSV F protein before fusion. Rhein bound to key residues within the central cavity of the RSV F protein at VAL406 and PHE137. Moreover, Rhein demonstrated potent inhibition against the K394R and K272E mutant pseudoviruses, indicating its potential efficacy against RSV variants resistant to Palivizumab and clinical-stage fusion inhibitors. Additionally, acute toxicity and pharmacokinetic studies revealed that Rhein has an LD50 > 2000 mg/kg, along with favorable oral absorption, extensive tissue distribution, and exposure kinetics suitable for Antiviral intervention. In an in vivo study, Rhein was shown to alleviate RSV-induced pulmonary pathology by reducing viral titers, mitigating lung injury, and suppressing the inflammatory responses in the Lungs.

Conclusion: Rhein is a promising RSV fusion inhibitor, and its unique binding mode offers a valuable lead compound for overcoming drug resistance.

Keywords
Fusion protein; Respiratory syncytial virus; Rhein; Viral entry inhibitor.
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