Biophysical studies of the interaction of hRSV Non-Structural 1 protein with natural flavonoids and their acetylated derivatives by spectroscopic techniques and computational simulations.

Human respiratory syncytial virus (hRSV) infections are one of the most causes of acute lower respiratory tract infections in children and elderly. The development of effective antiviral therapies or preventive vaccines against hRSV is not available yet. Thus, it is necessary to search for protein targets to combat this viral infection, as well as potential ways to block them. Non-Structural 1 (NS1) protein is an important factor for viral replication success since reduces the immune response by interacting with proteins in the type I interferon pathway. The influence of NS1 on the cell's immune response denotes the potential of its inhibition, being a possible target of treatment against hRSV infection. Here, it was studied the interaction of hRSV NS1 with natural flavonoids chrysin, morin, kaempferol, and myricetin and their mono-acetylated chrysin and penta-acetylated morin derivatives using spectroscopic techniques and computational simulations. The fluorescence data indicate that the binding affinities are on the order of 105 M-1, which are directly related to the partition coefficient of each flavonoid with Pearson's correlation coefficients of 0.76-0.80. The thermodynamic analysis suggests that hydrophobic interactions play a key role in the formation of the NS1/flavonoid complexes, with positive values of enthalpy and entropy changes. The computational approach proposes that flavonoids bind in a region of NS1 formed between the C-terminal α3-helix and the protein core, important for its biological function, and corroborate with experimental data revealing that hydrophobic contacts are important for the binding. Therefore, the present study provides relevant molecular details for the development of a possible new strategy to fight infections caused by hRSV.

Probing cocaine-antibody interactions in buffer and human serum.

BACKGROUND: Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. RESULTS: MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. CONCLUSIONS: High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide valuable information for characterization of their interactions and thermodynamic properties. In addition MST measurements of antibody affinity in the presence of biological fluids will provide a better opportunity to make reliable decisions and facilitate the design of cocaine vaccines and immunization conditions. The methods should be more widely adopted in characterization of antibody complexes.