RESUMO
Seasonal periodic pandemics and epidemics caused by Influenza A viruses (IAVs) are associated with high morbidity and mortality worldwide. They are frequent and unpredictable in severity so there is a need for biophysical platforms that can be used to provide both mechanistic insights into influenza virulence and its potential treatment by anti-IAV agents. Host membrane viral association through the glycoprotein hemagglutinin (HA) of IAVs is one of the primary steps in infection. HA is thus a potential target for drug discovery and development against influenza. Deconvolution of the multivalent interactions of HA at the interfaces of the host cell membrane can help unravel therapeutic targets. In this contribution, we reported the effect of a multivalent HA glycoprotein association on various glycosphingolipid receptors (GD1a, GM3, GM1) doped asymmetrically into an artificial host membrane spanned across an aqueous filled microcavity array. The extent of HA association and its impact on membrane resistance, capacitance, and diffusivity was measured using highly sensitive electrochemical impedance spectroscopy (EIS) and fluorescence lifetime correlation spectroscopy (FLCS). Furthermore, we investigated the inhibition of the influenza HA glycoprotein association with the host mimetic surface by natural and synthetic sialic acid-based inhibitors (sialic acid, Siaα2,3-GalOMe, FB127, 3-sialyl lactose) using electrochemical impedance spectroscopy and observe that while all inhibit, they do not prevent host binding. Overall, the work demonstrates the platform provides a label-free screening platform for the biophysical evaluation of new inhibitors in the development of potential therapeutics for IAV infection prevention and treatment.
RESUMO
Equilibrium anomeric ratios are reported for pyranoses (hemiacetals) of glucuronic and galacturonic acid and their derivatives. These are compared to related gluco- and galactopyranoses and to deoxyfluorogluco- and deoxyfluorogalactopyranoses. An association between axial anomer stability and the sum of 1H NMR downfield chemical shifts for protons H-3 and H-5 was observed in D2O with gluco- and galactopyranoses as reference compounds. When compared to 2-hydroxytetrahydropyran in water, introduction of three OAc substituents and one carboxylic acid substituent leads to an increase in stability of the axial anomer by 0.89-1.05 kcal/mol. This is interpreted as the electron-withdrawing substituents causing a reduction in the steric (gauche) interaction and an increase in favourable Coulombic interaction between CH groups of the pyranose and the anomeric group through substituent deshielding effects. Anomer preferences for galacturonic acid and its derivatives were more sensitive to solvent polarity compared to other pyranoses, and this may be linked to the electrostatic potential and reduced stabilization of the equatorial anomeric OH group due to reduced hydrogen bonding. The latter is more notable in nonpolar chloroform. Analysis of crystal structures combined with molecular dynamics indicated there are conformational distinctions between galacturonic acid and glucuronic acid that could influence properties.
