INTERACTION STUDY OF THE POLYANIONS HEPARIN AND ENOXAPARIN WITH THE CORE DOMAIN OF HRSV M2-1

As one of the leading causes of acute respiratory diseases worldwide, the human Respiratory Syncytial Virus (hRSV) is an important etiological agent of respiratory infections, such as bronchiolitis and pneumonia, especially in newborns, children and elderly. Although recent advances in hRSV vaccine development have benefited elderly individuals and pregnant women, Palivizumab - a monoclonal antibody targeting the viral fusion protein - remains the only approved therapeutic option, despite adverse reactions and high cost. The hRSV M2-1 protein functions as an antitermination factor for the viral RNA polymerase, simultaneously binding to mRNA and the phosphoprotein P through its core domain (cdM2-1). Thereby, cdM2-1 plays a vital role in viral replication, presenting itself as a potential target for the development of antivirals that inhibit this event. Moreover, due to an extensive positively charged surface, cdM2-1 is a target for the binding of negatively charged molecules, such as polyanions. In this sense, the present study aims to evaluate the interaction of the cdM2-1 with enoxaparin (4.5 kDa) and heparin (14.8 kDa) using fluorescence spectroscopy and Nuclear Magnetic Resonance (NMR). The fluorescence quenching experiments revealed hyperbolic binding isotherms, from which dissociation constants of 730 μM and 43 μM were determined for the interactions of enoxaparin and heparin with cdM2-1, respectively. Thermodynamic analysis showed that the cdM2-1/heparin complex exhibits the enthalpic term as the major contribution to the favorable binding free energy, highlighting the role of hydrogen bonding and van der Waals interactions. In contrast, the cdM2-1/enoxaparin complex is characterized by a dominant entropic contribution, indicative of electrostatic interactions driving complex formation. Competition experiments based on fluorescence anisotropy, using polyanions and a Rhodamine-labeled fragment of phosphoprotein P (residues 90-110), demonstrated that both heparin and enoxaparin bind to the M2-1 interface responsible for interactions with P and RNA. The cdM2-1 residues of this biologically relevant binding site were mapped using 15N HSQC spectra by NMR spectroscopy. Therefore, the findings of this study provide valuable molecular insights into the interaction of negatively charged ligands with the M2-1 protein, supporting the rational design of potential antiviral agents capable of disrupting the hRSV replication cycle.