Hesperetin targets the hydrophobic pocket of the nucleoprotein/phosphoprotein binding site of human respiratory syncytial virus

Abstract

The human Respiratory Syncytial Virus (hRSV) is one of the most common causes of acute respiratory diseases such as bronchiolitis and pneumonia in children worldwide. Among the viral proteins, the nucleoprotein (N) stands out for forming the nucleocapsid (NC) that functions as a template for replication and transcription by the viral polymerase complex. The NC/polymerase recognition is mediated by the phosphoprotein (P), which establishes an interaction of its C-terminal residues with a hydrophobic pocket in the N-terminal domain of N (N-NTD). The present study consists of biophysical characterization of N-NTD and investigation of flavonoids binding to this domain using experimental and computational approaches. Saturation transfer difference (STD)-NMR measurements showed that among the investigated flavonoids, only hesperetin (Hst) bound to N-NTD. The binding epitope mapping of Hst suggested that its fused aromatic ring is buried in the protein binding site. STD-NMR and fluorescence anisotropy experiments showed that Hst competes with P protein C-terminal dipeptides for the hRSV nucleoprotein/phosphoprotein (N/P) interaction site in N-NTD, indicating that Hst binds to the hydrophobic pocket in this domain. Computational simulations of molecular docking and dynamics corroborated with experimental results, presenting that Hst established a stable interaction with the N/P binding site. The outcomes presented herein shed light on literature reports that described a significant antireplicative activity of Hst against hRSV, revealing molecular details that can provide the development of a new strategy against this virus.

Graphical Abstract

Communicated by Ramaswamy H. Sarma

Keywords:

• Hesperetin
• hRSV nucleoprotein
• STD-NMR
• fluorescence anisotropy
• molecular docking
• molecular dynamics

Acknowledgements

The author J.M.S acknowledges the CAPES scholarship and J.V.P thanks the CNPq scientific initiation scholarship (102465/2019-3). I.P.C. acknowledges the financial support by the postdoctoral fellowship from FAPERJ (202.279/2018), PROPe UNESP, and CNPq/Universal grant (439306/2018-3). The authors thank Prof. Dr. Marcio Francisco Colombo for the access to the Äkta purifier and ISS-PC1 spectrofluorimeter and Prof. Dr. Alexandre Suma de Araújo for the access to the Calix cluster (FAPESP 2010/18169-3) for performing the molecular docking and molecular simulations. The authors recognize GridUNESP for the availability of the Gaussian 09 software and SDumont supercomputer for the calculations using the g_mmpbsa program. The authors also acknowledge for the access to the NMR laboratory of the Multiuser Center for Biomolecular Innovation (FAPESP 2009/53989-4) in IBILCE/UNESP.

Disclosure statement

The authors declare no conflicts of interest.