Coumarins and hesperetins modified antiviral candidates for the hydrophobic pocket at the nucleoprotein/phosphoprotein interaction site of the Human Respiratory Syncytial Virus

Abstract

Human Respiratory Syncytial Virus (hRSV) is a major cause of acute respiratory disease most common in newborns, children and the elderly, responsible for complications such as Bronchiolitis and Pneumonia. Currently, there is only one treatment for pediatric patients at high risk, with Palivizumab and data on vaccine development are not satisfactory. Among the proteins encoded by the virus, nucleoprotein N stands out for its importance in protecting viral RNA forming the nucleocapsid (NC) and for its role as a template for viral replication and transcription. The efficient and specific recognition of the NC mold by RNA-dependent RNA polymerase is mediated by phosphoprotein P. This is due to the interaction of P-protein C-terminal residues in a hydrophobic pocket in the N-protein N domain (N- NTD). From the context presented, the present project proposes a detailed investigation of the interaction of coumarins: coumarin, 4-methyl-esculetin, esculetin and esculin, which showed interaction with the N-NTD through a screening via STD-NMR performed previously in the master's study. A set of techniques such as fluorescence anisotropy and nuclear magnetic resonance will make it possible to elucidate details of the N-NDT/coumarin interactions. Among other analyzed molecules, hesperetin also showed interaction with N-NTD, so this study also aims to find chemical modifications in hesperetin, promoting an increase in the affinity and specificity of this polyphenolic compound for the hydrophobic site in N-NTD. To achieve the objective of identifying chemical modifications with an antiviral potential, a combination of computational approaches to docking and molecular dynamics will be performed. Subsequently, interaction experiments such as STD-NMR and fluorescence anisotropy will be carried out, methodologies that will make it possible to characterize binding parameters of the complexes such as: dissociation constant, binding region in the protein, non-covalent interactions responsible for the stabilization of the complex and conformational dynamics of the protein in the absence and presence of these compounds. Chemical modifications to coumarins structures that have interactions (via STD-NMR and fluorescence spectroscopy) relevant to N-NTD will also be proposed. After determining the compounds modified or not that interact with the N terminal domain, cell culture assays will be carried out in order to verify the viral inhibition of these molecules against hRSV. The expected results of this proposal will enable relevant advances in generating knowledge of the interaction mechanisms of these potential inhibitors of hRSV infections and will allow the development of new strategies to combat the virus. The proposal for a drug that is a candidate to fight respiratory infections will impact the quality of life of children and the elderly, the most affected, while also promoting a reduction in public health spending. (AU)