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

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.

4-Methylesculetin, a natural coumarin with intestinal anti-inflammatory activity, elicits a glutathione antioxidant response by different mechanisms.

4-methylesculetin (4 ME) is a natural antioxidant coumarin with protective effects on the intestinal inflammation, in which oxidative stress plays a key role in its aetiology and pathophysiology. Based on this, we examined the antioxidant molecular mechanisms involved in the intestinal anti-inflammatory activity of the 4 ME. For this purpose, we investigated the effects of the 4 ME on the modulation of gene expression and antioxidant-related enzyme activities in TNBS model of intestinal inflammation as well as the molecular interaction between 4 ME and glutathione reductase. Our results showed that 4 ME modulated glutathione-related enzymes, mainly increasing glutathione reductase activity. These effects were related to upregulation of glutathione reductase and Nrf2 gene expression. Fluorescence and nuclear magnetic resonance data showed that interaction between 4 ME and glutathione reductase is collisional, hydrophobic and spontaneous, in which C4 methyl group is the second epitope most buried into glutathione reductase. Molecular modelling calculation showed Lys70-B, Arg81-A, Glu381-B, Asp443-A, Ser444-A, Glu447-B and Ser475-A participated in electrostatic interaction, Lys70-B, Glu381-B and Arg81-A acted in the hydrophobic interactions and Trp73, Phe377 and Ala446 are responsible for the hydrogen bonds. Based on this, our results showed 4 ME acted by different mechanisms to control oxidative stress induced by intestinal damage, controlling the imbalance between myeloperoxidase activity and glutathione production, upregulating the glutathione S-transferase and glutathione reductase activities, preventing the Nrf2 and glutathione gene expression downregulation with consequent glutathione maintenance. Finally, 4 ME interacted at molecular level with glutathione reductase, stabilizing its enzymatic activity and reducing oxidative stress to take place in intestinal inflammatory process.