Neuraminidase Inhibitors: Development and Validation of a Procedure for <i>In Vitro</i> Determination of the Inhibitory Effect

Abstract

Neuraminidase inhibitors are a class of antivirals used to  treat  influenza  infections. Screening assays for potential neuraminidase inhibitors would benefit from  the development of in vitro procedures that do not require handling viruses. The aim of the study was to  develop  and  validate  a  procedure  for  in  vitro  determination of inhibitory effects on neuraminidase (EC 3.2.1.18), using 2’-4(methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-NANA) as a fluorogenic substrate and quinonoid pigments, potential neuraminidase inhibitors, as a case study. Materials and methods: the method is based on neuraminidase cleavage of 4MU-NANA   to release fluorescent 4-methylumbelliferone, which is detected at the excitation  and emission wavelengths of 360 and 450 nm, respectively. Results: the procedure was validated for specificity, range, accuracy, and precision. It remained linear over the range of 0.31–80 μM of 4-methylumbelliferone. The accuracy for four concentration levels (including the LLOQ) was 87–114%; i.e., the relative error of accuracy evaluation was less than 15%. The intra- and inter-day precision ranged from 1.5 to 10.4% and from 2.3 to 9.6%, respectively. Inhibitory effect evaluation using zanamivir hydrate (0.6–150 nM) demonstrated the accuracy of 89–120% and the precision  of 3.1–11.0%. The IC50 values for positive controls (zanamivir hydrate and oseltamivir) were 27 ± 3 and 16 ± 2 nM, respectively. The following solvents may be used: 50% dimethyl sulfoxide, 5% Polysorbate 80, 50% ethanol, 50 and 100% methanol. If  a compound is insoluble in the solvents, it is possible to form inclusion complexes with 2-hydroxypropyl-β-cyclodextrin. For bisnaphthazarin, the natural quinonoid pigment used in the study, the IC50 amounted to 273 ± 28 nМ. Conclusion: the procedure demonstrated adequate accuracy and reproducibility and is recommended  for screening for potential neuraminidase inhibitors. In order to use the procedure for insoluble substances, the authors suggest forming inclusion complexes with cyclodextrins.