A microplate-based screening assay for neuraminidase inhibitors.

Neuraminidase (NA) represents a highly promising new target for drug development in influenza virus genes. Rapid screening of enzyme inhibitors is a key method for the identification of leading compounds. In order to speed up the screening for enzyme inhibitors of natural and synthetic origin, effective and fast assays are needed. 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA) was selected as substrate for development of a microplate-based assay. The enzymatic reaction conditions were optimized as follows: in a 100 μL reaction mixture, the final concentrations were 32.5 mM sodium acetate (pH 3.5), 20 μM 4-MUNANA, 0.005% (w/v) bovine serum albumin, and 0.42 μg/mL NA. In the study, the doseresponse relationship of oseltamivir carboxylate to NA activity was observed. In addition, an overall Z' value of 0.8 proved the systems robustness and potential for screening. The assay system developed will be a valuable tool to discover new structures for the therapeutic inhibition of NA used to treat Influenza.