A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system

Figures & data

Table 1.  Data obtained in the enzymatic characterization of recombinant neuraminidase N1 and N3.

Recombinant protein	Km ± SD (µM)^a	Mean IC50 ± SD (nM)^b	Ki (nM)^a
N1	400 ± 60	25 ± 4	23.71 ± 7.2
N3	87 ± 4	0.2 ± 0.02	0.12 ± 0.11

^aKm, IC₅₀ and Ki were obtained from three independent experiments.

^bIC₅₀ is a concentration of oseltamivir which inhibit by 50% the neuraminidase activity.

Figure 1.  Michaelis–Menten and Lineweaver–Burk plots of recombinant neuraminidase: (A, C) subtype 1, and (B, D) subtype 3. The concentration of fluorogenic substrate (MUNANA) ranged from 0 µM to 2000 µM. The kinetics were run at 37°C. The fluorescence was recorded every 30 min by using the Victor³ multi-label counter (Perkin Elmer) with excitation and emission wavelengths of 355 and 460 nM, respectively. The reported data were obtained in triplicate experiments.

Figure 2.  Dixon plots for inhibition of oseltamivir free acid on N1 (A) and N3 (B). Titration curve of oseltamivir free acid with recombinant neuraminidase subtype 1 (C) and 3 (D). The reported data are the average of a triplicate experiment.

Table 2.  Within- and between-assay determinations of IC₅₀ of oseltamivir carboxylate against neuraminidase N1 and N3.

Within-assay comparison		Between-assay comparison
IC50		IC50
N1 (µM)	N3 (nM)	N1 (µM)	N3 (nM)
27.98	0.24	26.82	0.21
29.32	0.22	24.07	0.22
27.82	0.20	24.64	0.14
26.91	0.19	24.56
22.31	0.22

Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, Blackburn GM et al. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 2006;443:45–49.

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