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

## TL;DR

This paper introduces a new capillary electrophoresis method to measure how well drugs inhibit influenza A virus neuraminidase enzymes from H1N1 and H5N1 strains.

## Contribution

A novel native capillary nanogel electrophoresis assay is developed for real-time monitoring of neuraminidase inhibition.

## Key findings

- The assay quantifies enzyme activity and inhibitor effectiveness in real time using a thermally reversible nanogel.
- Inhibition constants for DANA, oseltamivir acid, and peramivir were determined for H1N1 and H5N1 neuraminidases.
- The method maintains the native structure of tetrameric neuraminidases during analysis.

## Abstract

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-N-acetylneuraminic acid (DANA) is 3.5 ± 0.8 μM (n = 5). The inhibition constants for oseltamivir acid (inhibiting
compound of Tamiflu) and peramivir (Rapivab) are 18.2 ± 0.5 nM
(n = 3) and 67 ± 8 nM (n =
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.