Native Capillary Nanogel Electrophoresis Assay of Inhibitors of Neuraminidases Derived from H1N1 and H5N1 Influenza A Pandemics

Tetrameric neuraminidases cleave the end-capping sialylated monomer from oligosaccharide ligands at the surface of a host cell infected by the influenza A virus. This cleavage releases the replicated virions from the host cell, making drugs that inhibit neuraminidase function effective to treat influenza A infections. A capillary electrophoresis separation-based assay is reported that maintains the native structure of tetrameric viral neuraminidases derived from H1N1 or H5N1 influenza A pandemics which convert, in-real time, a substrate that mimics 6'-sialyllated threonine-linked glycans on human cells. The assay integrates the enzyme reaction with the separation and is operated using a background electrolyte containing 100 mM NaCl with a thermally reversible nanogel in a 10 μm inner diameter fused silica capillary. In addition to defining the 0.4 nL reaction zone maintained at 37 °C, the nanogel medium resolves the substrate from contaminants as well as the substrate from the product before and after the enzymatic conversion. The enzyme activity is quantifiable based on the percent conversion observed in the presence of a range of inhibitor concentrations. For 1918 H1N1 (A/Brevig Mission/1/18) neuraminidase, the inhibition constant of the transition state analog 2,3-dehydro-2-deoxy-<i>N</i>-acetylneuraminic acid (DANA) is 3.5 ± 0.8 μM (<i>n</i> = 5). The inhibition constants for oseltamivir acid (inhibiting compound of Tamiflu) and peramivir (Rapivab) are 18.2 ± 0.5 nM (<i>n</i> = 3) and 67 ± 8 nM (<i>n</i> = 3), respectively. For 2004 H5N1 (A/Vietnam/1203/2004) neuraminidase, which contained a foreign tetramerization domain to maintain the structure, the inhibition constant for peramivir is 5.4 nM.