Sialidase Assay

To date, various assay methods for sialidase activity have been developed. In principle, the hydrolytic reaction catalyzed by sialidase can be assayed by quantitating the released sialic acid or aglycone. The thiobarbituric acid procedure described by Warren (1959) or its modification by Aminoff (1961) is used frequently for the colorimetric determination of released, free sialic acid. 

In addition to these methods, new techniques based on the principle of enzyme-linked immunosorbent assay (ELISA) have recently been introduced for sialidase assay. A glycoprotein or ganglioside substrate is coated in plastic microtiter wells and incubated with an enzyme preparation, followed by detection of the desialylated product using an appropriate ligand. Thus, the activity of Clos- 

Viral membrane sialidases have been solubilized with the aid of detergents or proteolytic enzymes such as pronase or trypsin (Drzeniek et al., 1966 Laver, 1978). The intact influenza virus sialidase (about 240 kDa) can be solubilized 

The kinetic properties and substrate specificity of viral sialidases have been investigated by many researchers (Drzeniek et aL, 1966 Drzeniek, 1972, 1973 Huang and Orlich, 1972 Kessler et aL, 1977 Thomas et al., 1978 Corfield et al., 1981b, 1982, 1983 Cabezas a/., 1989 Kleineidam a/., 1990 Kodama et al., 1991 Xu et al., 1993). The pH optima of these enzymes range between 5.5 and 6.5 with Neu5AcLac as the substrate, and the values are 1-2 mM. 

A kinetic study on the biosynthesis of influenza A virus sialidase has been reported recently (Hogue and Nayak, 1992). The enzyme protein is first synthesized as a monomer and, within 10 min, a significant fraction of the monomer is assembled into dimers or tetramers in the endoplasmic reticulum. The mature enzyme is detected at the host cell surface 30 min after its synthesis. Its concentration is increased to the maximum level after 1 hr, and decreases later, presumably because of its incorporation into the virion. 

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Table 2 Sensitivity of influenza virus sialidase variants isolated in the clinical setting to sialidase inhibitors in sialidase assays ...

As many conflicting observations have been made in this field, better characterization of human sialidases, especially those from the readily obtainable fibroblasts, is needed, as well as standardized, natural substrates and sialidase-assay conditions. 

FIGURE 1. Initial velocity-pH relationship for V. cholerae sialidase. Assay mixtures containing 0.10 /Ltg of enzyme protein, 3 x lO" m Gmi, Goia, Goib, Gti and 4 mM Ca + were prepared in 0.01 m Tris, which had been adjusted to the desired pH values with HO Ac in the presence and absence of added strong electrolyte (NaCl). Curve a, 0.01 m Tris-acetate alone curve b, 0.01 m Tris-acetate containing 0.10 m NaCl. 

Neu5Ac2en 4, a micromolar inhibitor of influenza virus sialidase 4 x 10 M (A/N2)] (Holzer et al. 1993), was first identified as a very good inhibitor in the late 1960s (Meindl and Tuppy 1969). A series of C-5 modified Neu5Ac2en derivatives provided the first improved in vitro inhibitors compared with the parent compound 4. The replacement of the C-5 A-acetyl moiety with a A-trifluoroacetyl group resulted in the most potent inhibitor of this series, 2-deoxy-2,3-didehydro-A-trifluoroacetylneuraminic acid 10 [A] 8 x 10 M (A/Nl)] (Meindl et al. 1974). While these C-5 modified compounds were also very effective in cell culture assays (Palese et al. 1974a Palese and Compans 1976), none, including the parent... 

Oseltamivir carboxylate 18 is used in sialidase inhibition assays Numbering used is that of A/N2 Abed et al. (2008)... 

Figure 2. Competitive Binding Assay for CT The mixture of sample solution and P-CT-B (l,(XX)-fold diluted. List Biological Laboratories) was preincubated at room temperature for 30 min, and then added to a GMi-coated microtiter plate (50 ng for each well). CT bound to Gmi was determined spectrophotometrically at 405 nm, after adding a solution of 2,2 -azino-bis-p-ethylbenzothioazoline-6-sulfonic acid) as a substrate. A quantitative analysis of sialic acid using TB A method (25) indicate that sialidase-treatment completely eliminated sialic acids of CMP (6). The degree of hydrolysis with pronase-treat CMP was 71 % (6), which was measured by the TNBS method according to the NOVO (NOVO Industry) manual (26).

HeLa cells were cultured for 24 hr with (open bars) and without (closed bars) 5 mM sodium butyrate, harvested, and assayed for indicated enzyme activity. A GM3-sialidase activity assayed with GM3 specifically labeled with [,iC]- N-acetylneuraminic acid as substrate ("14JJ. B Sialyltrasferase activity assayed with CMP-[, acetylneuraminic acid and lactosylceramide as substrates, and synthesis of labeled GM3 determined (17). Data... 

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

A virus in both sialidase inhibition and plaque-reduction assays. Alkyl ethers up to twelve carbon atoms in length exhibited similar inhibitory activity to Zanamivir against influenza A virus sialidase, however, showed a pronounced improvement in plaque-reduction assay compared to the parent triol 1. Alkylation of the C-7 hydroxyl with two-carbon substituents bearing terminal hydroxyl, amino, azido, and acetamido groups yielded inhibitors 61-64 and did not significantly affect the binding and had similar potency to that of ethyl or propyl ethers 65 or 66 (Fig. 9). 

Removal of the C-8 and C-9 hydroxy groups in order to produce a hydro-phobic side-chain, gave such derivatives as 67, which were evaluated against influenza A and B virus sialidases and used in a viral-replication assay.82 While these molecules displayed similar efficacy to the parent triol against influenza A virus sialidase, they were relatively ineffective against influenza B virus. 

Fig. 11. C-6 Carboxamide derivatives of 4-amino-4-deoxy-Neu5Ac2en (9) assayed as inhibitors of influenza virus sialidase. IC50 values against sialidase from influenza A are given.

Fig. 18. Thia, carba, and aza analogues of cyclohexene ethers were assayed for inhibition of influenza A virus sialidase. IC50 values are reported in parentheses.

Using the sialosides obtained, a 96-well plate based coupled enzymatic assay for high-throughput colorimetric screening of structures that can be recognized by different sialidases was developed (Scheme 7) (75). Briefly, individual sialosides are incubated with an appropriate amount of a certain sialidase and an excess amount of exogalactosidase or hexosaminidase. If the... 

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