How the study of natural compounds can improve antiviral activity against human respiratory syncytial virus

Introduction: Human Respiratory Syncytial Virus is the main cause of hospitalization due to respiratory diseases among children under 5 years of age, and it is responsible for pneumonia and bronchiolitis. The RSV Matrix protein plays key roles in the virus life cycle. It is initially found in the nucleus early in infection, where it serves an inhibitory transcriptional role. Later, it is located in viral inclusion bodies, coordinating viral assembly and budding at the plasma membrane. The study of natural compounds and their properties can be used to design new drugs with antiviral and anti-inflammatory effects against RSV. Specifically, for respiratory viruses with therapeutic potential, we find polyphenolic phytoalexins (Resveratrol) and Coumarin. Resveratrol is a polyphenol (3,4',5-trihydroxy-trans-stilbene), a natural phytoalexin produced by plants. Coumarin is also a natural compound that shows potential as a drug candidate due to its properties of stability, solubility, low toxicity, and antiviral activity. Objectives: Evaluate the effects of the interaction between the proposed ligands and the matrix protein. Determine the thermodynamic profile of the interaction, calculating the association constants and the nature of the interaction. The experimental data will be used as a guide for conducting computational techniques to determine potential interaction sites. Methods: The M protein gene was cloned into pD441-NHT vector, the expression was proceeding in Escherichia coli and the protein was purified by affinity and molecular exclusion chromatography. To analyze the interaction parameters, ligand titrations were performed using fluorescence spectroscopy, and circular dichroism was utilized to analyze the protein's secondary structure. The determination of potential interaction sites was conducted computationally using docking (DOCK6 UCSF) and molecular dynamics techniques (Gromacs Package), refined by experimental descriptions of the interaction.Results: Using fluorescence spectroscopy with tryptophan as a probe, the results show that Coumarin and Resveratrol interact with the Matrix protein. The obtained data showed a dissociation constant of approximately 5.6x104 M-1 for both molecules and the thermodynamic profile of the interactions indicates a hydrophobic binding with a stoichiometry (n) of 1 ligand per protein. The phenomenon of quenching fluorescence showed a disturbance in the chemical environment of Try in solution in the presence of the compounds. Additionally, Circular Dichroism experiments indicate that these interactions do not affect the protein's secondary structure. Computationally, we tested multiple potential regions of the protein where the ligands could bind without steric restrictions. Guided by the experimental results, we refined the models and identified a pocket that aligns with all theoretical and experimental aspects. Finally, we performed molecular dynamics simulations to assess the stability of the interaction, obtaining further information about secondary structure, fluctuations, and deviations arising from the ligand-protein interactions. Conclusions: The present study demonstrated an interaction between Resveratrol and Coumarin with the M protein. This interaction is moderate and predominantly hydrophobic. These characteristics allowed us to identify the interface region as promising for this interaction to occur. Biologically, these results of viral protein interaction with antioxidants enable us to interfere with potential effects on viral assembly or the formation of mature viruses, both of which are mediated by the M protein.