Insights into Interactions of Flavanones with Target Human Respiratory Syncytial Virus M2-1 Protein from STD-NMR, Fluorescence Spectroscopy, and Computational Simulations.

Hêmily M R Piva,Fátima P Souza,Ícaro P Caruso,Jéssica M Sá,Marcelo A Fossey,Artemiza S Miranda,Ljubica Tasic

doi:10.3390/ijms21062241

Abstract

The human Respiratory Syncytial Virus (hRSV) is the most frequent agent of respiratory infections in infants and children with no currently approved vaccine. The M2-1 protein is an important transcriptional antitermination factor and a potential target for viral replication inhibitor development. Hesperetin (HST) and hesperidin (HSD) are flavonoids from the flavanone group, naturally found in citrus and have, as one of their properties, antiviral activity. The present study reports on the interactions between hRSV M2-1 and these flavanones using experimental techniques in association with computational tools. STD-NMR results showed that HST and HSD bind to M2-1 by positioning their aromatic rings into the target protein binding site. Fluorescence quenching measurements revealed that HST had an interaction affinity greater than HSD towards M2-1. The thermodynamic analysis suggested that hydrogen bonds and van der Waals interactions are important for the molecular stabilization of the complexes. Computational simulations corroborated with the experimental results and indicated that the possible interaction region for the flavonoids is the AMP-binding site in M2-1. Therefore, these results point that HST and HSD bind stably to a critical region in M2-1, which is vital for its biological function, and thus might play a possible role antiviral against hRSV.

Highlights

The human respiratory syncytial virus is the leading cause of lower respiratory tract infections in infants and children worldwide [1,2] and is responsible for the morbidity and mortality of the elderly and immunocompromised patients [3,4]
The Saturation Transfer Difference (STD)-Nuclear Magnetic Resonance (NMR) experiments unambiguously showed that HST and HSD interacted with the protein, and the binding epitopes mapping revealed that the A and B aromatic rings of these flavanones occupied the binding site in M2-1, which was especially true for the HST A-ring, while the HSD rutinose group was exposed
The analysis of the thermodynamic parameters for the binding interaction suggests that this process is spontaneous (∆G < 0) and exothermic (∆H < 0) for both flavanones, being the M2-1/flavonoid complexes stabilized by hydrogen bonds and van der Waals interactions

Summary

Introduction

The human respiratory syncytial virus (hRSV) is the leading cause of lower respiratory tract infections in infants and children worldwide [1,2] and is responsible for the morbidity and mortality of the elderly and immunocompromised patients [3,4]. It belongs to the genus Orthopneumovirus, family. Evidence suggests that M2-1 is located between the ribonucleoprotein complex and the M protein [10] and that its association with the replisome increases the replication activity of viral RNA [11]. The M2-1 structure is predicted to comprise four functionally distinct regions: the N-terminal region linked to a zinc-finger domain [13]; the α-helix region responsible to mediate the oligomerization [12]; the globular domain capable of binding to the phosphoprotein protein and RNA; and non-structured C-terminal region [14]

Methods

Results

Conclusion