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

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.