Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris

Neuraminidase (NA) is one of the two glycoproteins on the surface of influenza virus, which cleaves terminal sialic acid residues and facilitates the release of virions from infected cells. The recombinant NA from H5N1 influenza virus strain A/Vietnam/1203/04 was expressed in Pichia pastoris X33 as a 45 kDa protein that displayed a K _m of 9.96 ± 1.26 μM with fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid. Partially purified NA was used for the inhibition and kinetic assays with eight flavonoid compounds and gallic acid. Among them, gallocatechin gallate (GCG) showed the best inhibition against NA with the IC₅₀ of 8.98 ± 0.46 μM and showed a competitive inhibition pattern with K _i value of 8.34 ± 0.25 μM. In molecular docking experiments, GCG displayed a binding energy of ?13.71 kcal/mol to the active site of NA and the galloyl moiety was required for NA inhibition activity.