Limited inhibitory effects of oseltamivir and zanamivir on human sialidases

Abstract

Oseltamivir (Tamiflu) and zanamivir (Relenza), two extensively used clinically effective anti-influenza drugs, are viral sialidase (also known as neuraminidase) inhibitors that prevent the release of progeny virions and thereby limit the spread of infection. Recently mortalities and neuropsychiatric events have been reported with the use of oseltamivir, especially in pediatric cases in Japan, suggesting that these drugs might also inhibit endogenous enzymes involved in sialic acid metabolism, including sialidase, sialyltransferase, and CMP-synthase, in addition to their inhibitory effects on the viral sialidase. The possible inhibition could account for some of the rare side effects of oseltamivir. However, there has been little direct evidence in regard to the sensitivities of animal sialidases to these drugs. Here, we examined whether these inhibitors might indeed affect the activities of human sialidases, which differ in primary structures and enzyme properties but possess tertiary structures similar to those of the viral enzymes. Using recombinant enzymes corresponding to the four human sialidases identified so far, we found that oseltamivir carboxylate scarcely affected the activities of any of the sialidases, even at 1 mM, while zanamivir significantly inhibited the human sialidases NEU3 and NEU2 in the micromolar range (K(i), 3.7 +/- 0.48 and 12.9 +/- 0.07 microM, respectively), providing a contrast to the low nanomolar concentrations at which these drugs block the activity of the viral sialidases.

Figures

FIG. 1.

Comparison of the endogenous expression levels of four sialidases in the human brain and lung. Quantitative analyses of the transcripts were performed with real-time PCR using porphobilinogen deaminase as an internal standard as described in Materials and Methods. Bars indicate the standard deviations of the means for experiments performed in triplicate.

FIG. 2.

The effects of oseltamivir carboxylate and zanamivir on sialidase activities. (A) After SDS-PAGE of NEU2 (left) and NEU3 (right) enzymes purified by FLAG affinity chromatography, protein staining, Coomassie brilliant blue (CBB) for NEU2 and silver staining for NEU3, and Western blotting (WB) were performed to evaluate the degree of purification. The enzymes were assayed with increasing concentrations of 4MU-NeuAc or GM3 as a substrate with or without zanamivir (B) or oseltamivir carboxylate (C). The Km and Ki values were calculated from these results.

FIG. 3.

Effects of oseltamivir and zanamivir on desialylation of endogenous substrates of 293T cells. (A) Alterations of glycoproteins by transfection of NEU2 into 293T cells were observed by PNA and RCA lectin blotting. Intensities of several protein bands (right panel) indicated were markedly increased in the NEU2-transfected cells compared to the mock-transfected cells, but no significant changes were observed in the NEU2-transfected cells after treatment with any of the drugs at the concentration of 500 μM. (B) The glycolipids were examined by TLC in the NEU3-transfected cells. The transfected cells showed a marked decrease in GM3 amounts compared to mock-transfected cells. Neither addition of oseltamivir phosphate nor that of oseltamivir carboxylate changed the patterns, while zanamivir inhibited GM3 hydrolysis by NEU3. The relative intensities of the bands corresponding to GM3 in TLC are shown in the right panel.