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

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: > 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.