Deverlopment of non-cell based assays for screening of inhibitors against avian influenza neuraminidase

Abstract

A screening for avian influenza neuraminidase inhibitors is presently limited to be operated only in the biosafety level-3 laboratory. This is due to the use of high virulence strains of avian influenza virus as a direct source of neuraminidase enzymes in the screening assay. It is, therefore, necessary to develop new screening methods with high safety and efficiency for being used in a general laboratory in order to speed up the seach for new neuraminidase inhibitors. In this research work, two recombinant neuraminidases obtained from H7N1, H7N3, and a viral neuraminidase from inactivated H5N1 were used as the safe enzyme sources for screening. This assay is based on the use of florescence method which is highly sensitive with the limit of detection at the level of as low as nanomolar scale. Various compounds, including 5 synthetic oseltamivir analogs and 35 naturally occurring flavonoids from Thai medicinal plants: Dalbergia parviflora and Belamcanda chinensis were tested for their potential inhibitory activities. The results showed that the analog PMC-35 exhibited higher inhibitory activity against H7N3 neuraminidase than oseltamivir, the analog PMC-36, on the other hand, exhibited its highest activity against that of H7N1 whereas both analogs gave similar activity to oseltamivir in inhibiting H5N1 neuraminidase. For the natural flavonoids, the inhibitory activity on the neuraminidases appeared to be weaker in micromolar level range. However, it was found, interestingly, that the structures of flavonoids themselves had a quenching effect on the observed inhibitory activity which leads to the overestimated inhibitory efficacy of some compounds. Thus, it is necessary to determine the degree of quenching of each flavonoid in order to obtain accurate inhibitory values of this group of natural products.