A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system
Jarinrat Kongkamnerd1, Adelaide Milani, Giovanni Cattoli
1Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
Context:
Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors.
Objective:
Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay.
Materials And Methods:
Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor.
Results:
The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments.
Discussion And Conclusions:
The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.
