An O-glycoside of sialic acid derivative that inhibits both hemagglutinin and sialidase activities of influenza viruses

Chao-Tan Guo2, Xue-Long Sun3, Osamu Kanie1,3,4, Kennedy Francis Shortridge5, Takashi Suzuki2, Daisei Miyamoto2, Kazuya I.-P. Jwa Hidari2, Chi-Huey Wong1,3,6 and Yasuo Suzuki1,2

2Department of Biochemistry, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka-shi 422–8526, Japan; 3Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; 4Advanced Techno-Bioscience Department, Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida-shi 194-8511, Tokyo, Japan; 5Department of Microbiology, University of Hong Kong, Queen Mary Hospital, Hong Kong; and 6Department of Chemistry, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

Received on August 21, 2001; revised on October 29, 2001; accepted on November 5, 2001.


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The compound Neu5Ac3{alpha}F-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3{alpha}F-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 µM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.

Key words: hemagglutinin/influenza virus/inhibitors/resistance/sialidase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The antigenic drift of influenza A viruses causes epidemics in the human population and results in many deaths each year. The idea of eliminating these viruses through vaccination is not very effective because antigenic changes of the virus render the old vaccine ineffective. Therefore, vaccines must be reformulated each year based on predictions that may not always closely match the new desirable vaccines required. In terms of influenza prevention, the drug amantadine and its analogue, rimantadine, are effective prophylactically but not common for the treatment of influenza A virus infection because of the side effects probably caused by inhibition of the endogenous ion channel activity (Stoof et al., 1992Go) and the rapid emergence of resistant strains (Hayden et al., 1989Go; Belshe et al., 1989Go).

Two membrane-bound glycoproteins are cooperatively associated with influenza virus replication. The glycoprotein hemagglutinin (HA) and sialidase are directly involved in the attachment and detachment of viral particles to and from the host cell, respectively. Although both recognize sialyl oligosaccharides expressed on the cell surface, HA is responsible for the attachment of viral particles to the host cell through a specific receptor–ligand interaction and the sialidase catalyzes the hydrolysis of such ligands to allow the virus to escape from the cell during the budding process (Weis et al., 1988Go; Wiley and Skehel, 1987Go). Therefore it is considered that effective inhibition of viral replication can be achieved by disturbing both processes.

Many anti-influenza compounds have been designed and synthesized against viral HA (Glick et al., 1991Go; Roy et al., 1993Go; Spevak et al., 1993Go; Choi et al., 1997Go; Guo et al., 1998Go; Kamitakahara et al., 1998Go; Tsuchida et al., 1998Go) and sialidase (Suzuki et al., 1990Go; Von Itzstein et al., 1993Go; Woods et al., 1993Go; Sabesan et al., 1995Go; Jedrzejas et al., 1995Go; Choi et al., 1996Go; Lew et al., 1998Go; Ikeda et al., 1998Go; Atigadda et al., 1999Go). The anti-influenza activities of some inhibitors targeting HA are reduced because the sialic acid residue is cleaved by the viral sialidase. If these inhibitors are resistant against sialidase, their effectiveness will be increased (Sparks et al., 1993Go; Itoh et al., 1995Go). Our idea is to develop a O-glycoside of sialic acid analog that exhibits inhibitory activities against both HA and sialidase. To achieve this goal, we synthesized a series of compounds with modification at C-3 position (see Scheme S01). The rationale was based on the following considerations. The functional groups of sialic acid residues such as carboxylic acid (C-1); 4-, 8-, 9-OHs; and the acetamido group at C-5 have been demonstrated to play an important role in the binding of the influenza A virus HA (Wilson et al., 1981Go; Wiley et al., 1981Go; Watowich et al., 1994Go) and sialidase (Varghese et al., 1983Go; Colman et al., 1983Go; Crennell et al., 1993Go). This has been confirmed by chemical modifications of sialic acid (Suzuki, 1994Go; Sato et al., 1998Go), however the physiological role of the C-3 position of Neu5Ac has not been elucidated. Also, our analysis of the crystallographic data of HA (Weis et al., 1988Go, 1990; Wilson et al., 1981Go; Sauter et al., 1992aGo,b) and sialidase (Varghese and Colman, 1991Go; Varghese et al., 1992Go) indicated that modification of this position may be acceptable (Figure 1). We have reported that a sialic acid analog with the axial hydrogen at C-3 position replaced by the fluorine atom acts as an inhibitor of both proteins (Sun et al., 2000Go). The one carrying distearoylphosphatidyl-ethanolamine (DSPE) is considered to be especially useful because of its potential to form a liposome for multivalent interaction.



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Scheme 1. C-3-modified sialoside.

 


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Fig. 1. An idea of inhibiting two classes of carbohydrate-related proteins, hemagglutinin (HA) and sialidase of influenza virus. The illustrations were created to reflect the original three-dimensional structures of these proteins based on the available resources from protein data base. HA: Wilson et al., 1981Go; Wiley et al., 1981Go; Watowich et al., 1994Go. Sialidase: Varghese et al., 1983Go; Colman et al., 1983Go; Crennell et al., 1993Go. A bound structure of sialoside and HA (H3) where X stands for the site of modification (A). A cartoon of sialidase (N2) catalytic site holding the sialoside (B), which was drawn based on a crystal structure of sialidase with sialic acid having a boat conformation. X also indicates the site of modification to be made. Note that the substituent X at C-3 position in both cases points away from the binding sites.

 
Based on this background, we describe here an {alpha}-O-glycoside of sialic acid derivative that inhibits HA and sialidase as well as replication of human influenza virus.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Inhibitory activities of C-3-modified derivatives against low pH–induced hemolysis of erythrocytes
It was shown previously that C-3-modified sialyl DSPE derivatives were recognized by hemagglutinin (H3), which was evident from the thin-layer chromatography (TLC)/virus-binding assay and HA inhibition assay. To further test the effect of these compounds, we have examined hemolysis tests. As shown in Table I, inhibitory effect against low pH–induced hemolysis caused by A/Aichi/2/68 (H3N2) strain was observed for four synthetic sialyl DSPE derivatives (14), but no inhibition was observed for A/PR/8/34 (H1N1) strain (data not shown).


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Table I. Binding activities and inhibitory activities of C-3-modified sialyl DSPE derivatives (14) on the viral hemagglutination and the low pH–induced hemolysis by human influenza virus A/Aichi/2/68 (H3N2)
 
C-3-Modified sialyl DSPE derivatives resist the hydrolysis by influenza virus sialidases
Viral sialidase is used to release progeny viruses from the surface of infected cells by cleaving O-glycosidic linkage of sialic acid, thus inhibitors containing such linkage lose anti-influenza effect. To examine the stability of synthetic C-3-modified sialyl DSPE derivatives against influenza virus sialidases, two neuraminidase subtypes of influenza viruses, N1 (A/PR/8/34 strain) and N2 (A/Aichi/2/68 strain), were incubated with each derivative. As shown in Figure 2, Neu5Ac-DSPE (1) was completely hydrolyzed after incubation at 37°C for 30 min with each influenza virus in the presence of 1% taurodeoxycholate (TDC). Compound 1 was shown to resist sialidase treatment to some extent in the absence of TDC. However, in the presence of TDC, Neu5Ac-DSPE (1) was destroyed by the enzyme. Similar activation effect has been reported previously (Sun et al., 2000Go), but the mechanism of the apparent activation was not fully understood (Sugano et al., 1978Go; Saito et al., 1979Go; Suzuki et al., 1980Go). It is possible that the presence of the liposome surface close to sialic acid residue may have interfered with the sialidase action when the experiment was carried out in the absence of TDC. Under the same conditions, marked differences in hydrolysis rate were observed between compound 1 and C-3-modified sialyl DSPE derivatives (24).



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Fig. 2. Stability of synthetic C-3-modified sialyl DSPE derivatives against influenza virus sialidase. The stability of synthetic C-3-modified sialyl DSPE derivatives (14, 10 µM) were assayed by a Dittmer’s reagent. Detection of the free DSPE released from these derivatives was performed after catalytic hydrolysis for 30 min with sialidases from human influenza A viruses (1 µg/µl virus protein) A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains. The data are expressed as mean ± SD of three independent experiments. See Materials and methods.

 
Inhibitory activities of C-3-modified sialyl DSPE derivatives against the sialidases from human influenza A viruses
To examine whether the C-3-modified sialyl DSPE derivatives (24) are inhibitory against sialidases, the enzymic hydrolysis of the p-nitrophenyl (PNP) {alpha}-glycoside of sialic acid (Neu5Ac) as a substrate was carried out. After incubation of A/PR/8/34 (H1N1) or A/Aichi/2/68 (H3N2) virus (5 µg/µl) with each derivative at a concentration of 250 µM at 37°C for 1 h, strong inhibitory activity was observed for Neu5Ac3{alpha}F-DSPE (4) but not by other derivatives examined (Figure 3). The IC50 values of 4 against A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) viruses were 63 and 31 µM, respectively.



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Fig. 3. Inhibitory activities of synthetic C-3-modified sialyl DSPE derivatives against human influenza A virus sialidase. The inhibitory activities of synthetic C-3-modified sialyl DSPE derivatives against human influenza A virus sialidase [A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains] were determined as described under Materials and methods. The data are expressed as mean ± SD of three independent experiments. Each experiment was carried out in duplicate. Neu5Ac-DSPE (1) (closed circles), Neu5Ac3ßOH-DSPE (2) (open circles), Neu5Ac3{alpha}OH-DSPE (3) (closed triangles), Neu5Ac3{alpha}F-DSPE (4) (open triangles).

 
Inhibitory activities of Neu5Ac3{alpha}F-DSPE against the sialidase from various influenza A viruses
To examine the specificity of Neu5Ac3{alpha}F-DSPE (4) against various influenza A virus sialidases, 16 strains of influenza virus isolated from humans, ducks, and pigs were tested. A broad inhibitory spectrum of Neu5Ac3{alpha}F-DSPE (4) against the sialidases of all influenza viruses tested was shown (Table II). The IC50 values of Neu5Ac3{alpha}F-DSPE (4) against these viral sialidase activities varied from 31 to 500 µM with 4-MU-Neu5Ac as the substrate and from 31 to 250 µM with Neu5Ac-DSPE (1) as the substrate.


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Table II. Inhibitory activities of Neu3{alpha}F-DSPE (4) against the sialidases from various influenza A viruses
 
Neu5Ac3{alpha}F-DSPE inhibited cell infection by influenza virus in the early and late stages
To confirm the effects of synthetic sialyl DSPE derivatives, neutralization of the infectivity of influenza virus A/Aichi/2/68 (H3N2) strain by the derivatives was determined by the abolition of cytopathic effects in Mardin Darby canine kidney (MDCK) cells while the activity of lactate dehydrogenase (LDH) released from the infected cells was used to estimate influenza virus infection. As shown in Figure 4A, synthetic sialyl DSPE derivatives inhibited the infection of A/Aichi/2/68 virus in a dose-dependent manner (IC50, 5–70 µM). Neu5Ac3{alpha}F-DSPE (4) markedly inhibited the infection compared with those of the other sialyl DSPE derivatives examined. The IC50 value of Neu5Ac3{alpha}F-DSPE (4) against A/Aichi/2/68 (H3N2) was 5.6 µM, eightfold stronger than those of compounds 2 and 3.



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Fig. 4. Neutralization of human influenza virus infection with the sialyl DSPE derivatives. After incubation of A/Aichi/2/68 (H3N2) virus with the sialyl DSPE derivatives, the mixture was added to culture media of MDCK cells and incubated (A). After incubation with the pretreated virus, the inoculum was removed and the cells maintained with Eagle’s minimum essential medium for 20 h (B). The inhibitory activities of the derivatives were determined after incubation of the synthetic derivatives with infected cells that were inoculated with virus for 20 h (C). The data are expressed as mean ± SD of three independent experiments. Each experiment was carried out in duplicate. Neu5Ac3ßOH-DSPE (2) (circles), Neu5Ac3{alpha}OH-DSPE (3) (closed triangles), Neu5Ac3{alpha}F-DSPE (4) (open triangles).

 
Because two distinct proteins, HA and sialidase, are independently involved in early and late stages of infection, the mechanism of this inhibitory effect was investigated further. For this, a set of experiment was carried out. After incubation of the virus with the derivatives, the mixture was added to the culture medium of MDCK cells. As shown in Figure 4B, the inhibitory activity of Neu5Ac3{alpha}F-DSPE (4) was dropped significantly, whereas the other derivatives showed similar inhibitory activities as in Figure 4A. On the other hand, to examine the inhibitory activities of the derivatives against the newly released virus, the LDH activities from infected cells were determined by adding the derivatives to the infected cells after incubation. Only Neu5Ac3{alpha}F-DSPE (4) prevented the cytopathic effects (Figure 4C).

Considered together, the results indicate that Neu5Ac3{alpha}F-DSPE (4) acts not only at the early stage of infection by attachment of HA to its ligands but also at later stage with release of progeny virus, whereas the other derivatives only inhibited the adhesion of virus to cellular membrane. The stronger inhibitory effect observed in Figure 4A is thus probably due to the synergetic effect of inhibition of both processes.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this study, we have demonstrated that Neu5Ac3{alpha}F-DSPE (4) inhibited the attachment of virus to the host cell surface and the release of new-virus particles from infected cells. The inhibitory mechanism of Neu5Ac3{alpha}F-DSPE (4) involves inhibition of both HA and sialidase of the influenza A virus. Furthermore, C-3-modified sialic acid derivatives generally resisted both acidic and enzymatic hydrolysis, indicating that these compounds have potential as leads for inhibitory activity optimization. Most of the functional groups of sialic acid residues have been experimentally identified to be important for the binding of viral HA (Suzuki, 1994Go; Sauter et al., 1992aGo), although substitution of 4-OH with O-Ac group does not affect HA (H3) binding as measured by 1H-nuclear magnetic resonance (Sauter et al., 1989Go). Recently it was also reported that the 7-(R)-fluoro-2,3-ene sialic acid derivative inhibited the interaction between HA (H1) and ganglioside GM3 (Sato et al., 1999Go). However, participation of the deoxy (C-3) position has not been investigated in any study. In addition, it is known that the O-glycosidic linkage is stabilized dramatically by introducing an electron withdrawing substituent to the position next to the anomeric center, C-3 in sialic acid (BeMiller, 1967Go; Varghese et al., 1992Go; Okamoto et al., 1992Go). The acid and enzyme labile glycosidic bond of sialic acid may become resistant against these conditions, and this may lead to longer availability of drugs.

Based on these considerations, we reported some activities of a series of synthetic O-glycosides of sialic acid analogs, which indicated that the modification of Neu5Ac at the C-3 position does not affect the binding affinity to the influenza HA as shown in the TLC binding assay and hemagglutination inhibition assay. The sialyl DSPE derivatives were shown not to bind to the H1 subtype of human influenza A virus A/PR/8/34 (H1N1) strain. The inhibitory activities of these compounds against influenza-induced hemolysis is consistent with the binding specificities found by the binding assay and hemagglutination inhibition assay.

Among the DSPE derivatives of C-3-modified sialic acids, Neu5Ac3{alpha}F-DSPE (4) was shown to not only resist acid- and sialidase-catalyzed hydrolysis but also inhibit the influenza sialidases (N1, N2, N3, and N5) of various origins. The loss of inhibitory activities of compound 2 and 3 is perhaps due to a steric impediment of the relatively large hydroxyl group at C-3 position. Most of the inhibitors of sialidases reported so far are derived from the 2,3-ene compound that has the half-chair conformation to mimic the transition state of the enzyme reaction (Varghese et al., 1992Go). Sialic acid derivatives without aglycone are also considered to mimic the transition state (Hagiwara et al., 1994Go). Different approaches accommodating noncleavable glycosidic linkage has been also addressed in find sialidase inhibitors, for instance, trisaccharides having Neu5Ac as thioglycoside were shown to be inhibitors (Ki value of µM range) of sialidase from Arthrobacter sialophilus (Kessler et al., 1982Go). Although no HA inhibitory effect was investigated, the ganglioside analogs having thioglycosidic linkage was synthesized and shown to have µM range Ki values against influenza sialidases. (Suzuki et al., 1990Go) In contrast to these results, it should be emphasized that our corresponding PNP-glycosides having an axial substituent (OH or F) at C-3 position of sialic acid showed very potent inhibitory activities (Ki = 1.1 and 2.2 µM, respectively) comparable to that of the 2,3-dehydro compound (Sun et al., 2000Go). Because only the difference in the structures is the aglycon, similar inhibitory mechanism is expected for compound 4 (see Figure 1B)

The inhibitory activity of Neu5Ac3{alpha}F-DSPE (4) to the cellular infection of influenza virus was eightfold stronger than that of the other two derivatives examined. The inhibitory effect of Neu5Ac3{alpha}F-DSPE to influenza virus may involve at least two mechanisms. One is the inhibition of attachment of virus to cellular surface receptors, and the other is the inhibition of viral sialidase to prevent the release of new-viruses budding from infected cells.

In conclusion, Neu5Ac3{alpha}F-DSPE (4), a C-3-modified {alpha}-sialoside, was found for the first time to inhibit two independent classes of proteins, HA and sialidase. This was also confirmed by the observed synergetic inhibitory effect on the influenza infection of cultured MDCK cells. The compound may be useful as a lead for the inhibitory activity optimization.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
IV3Neu5AcnLc4Cer and IV6Neu5AcnLc4Cer were prepared from human erythrocytes (Wherrett, 1973Go) and human meconium, respectively (Nilsson et al., 1981Go). 4-MU-NeuAc and PNP-NeuAc were purchased from Sigma.

C-3-Modified sialyl DEPE derivatives.
Four sialyl DSPE derivatives (14) were prepared as described elsewhere (Sun et al., 2000Go).

Preparation of liposome
The sialic acid-decorated liposomes were formed to contain 10 mol % of sialylphospholipids (14) by mixing with distearoylphosphatidylcholine (DSPC) and cholesterol (CH). All liposomes were prepared by mixing chloroform-methanol solution of DSPC (9.6 mg, 0.012 mmol), CH (0.9 mg, 0.0024 mmol), and sialylphospholipid (1.7–1.8 mg, 0.0016 mmol) in a round-bottom flask and concentrating by evaporation to produce a dried film, which was swelled in 2.0 ml saline on a Vortex mixer. The resulting suspension was then extruded several times (Extruder, Lipix Biomembrane) by passing through a polycarbonate membrane filter (Nuclepore; pore size 0.1–1.0 µm). The particle size of the liposome was determined by dynamic light scattering (Dynapro-801 TC), and the mean size of each liposome was 76, 80, 74, and 72 nm, respectively. The liposome thus obtained was directly used for biological assay. The concentration of the sialyl DSPE analogs used in the following experiments was based on the molarity of each derivative in the liposome.

Influenza viruses
The following influenza viruses were used in this study; human: A/PR/8/34 (H1N1), A/Singapore/1/57 (H2N2), and A/Aichi/2/68 (H3N2) strains; swine: A/swine/Hokkaido/2/81 (H1N1) and A/swine/Italy/309/83 (H3N2) strains; and avian: A/duck/HK/36/76 (H1N1), A/duck/HK/273/78 (H2N2), A/duck/HK/24/76 (H3N2), A/duck/HK/849/80 (H4N1) A/duck/HK/313/78 (H5N3) A/duck/HK/13/76 (H6N1), A/duck/HK/47/76 (H7N2), A/duck/HK/86/76 (H9N2), A/duck/ HK/33/76 (H10N1), A/duck/HK/44/76 (H11N3), and A/duck/HK/862/80 (H12N5) strains (Table II). The viruses were propagated in the allantoic cavities of 11-day-old chicken eggs for 48 h at 35°C and purified by sucrose density gradient centrifugation (Suzuki et al., 1992Go). Viral HA units were determined in microtiter plates using 0.5% chicken erythrocytes as described previously (Suzuki et al., 1983Go).

Inhibition assay for low pH–induced homolysis
The inhibitory activities of synthetic C-3-modified sialyl DSPE derivatives on the low pH–induced hemolysis were determined as described previously (Suzuki et al., 1983Go; MacDonald et al., 1984Go; Portner et al., 1987Go; Guo et al., 1998Go). Briefly, viruses [A/Aichi/2/68 (H3N2) or A/PR/8/34 (H1N1); 29 HA units] were incubated with different concentrations of derivatives at 4°C for 1 h and then added to 20 mM acetate buffered saline (pH 5.0) containing 2.5% human erythrocytes. The released-hemoglobin from erythrocytes was determined after incubation at 37°C for 30 min (Suzuki et al., 1986Go).

Sialidase assay
Method 1: Hydrolysis of synthetic sialyl DSPE derivatives by influenza virus sialidase.
Relative stabilities of synthetic DSPE derivatives against influenza virus sialidase were expressed with detecting of cleaved-DSPE by using Dittmer’s method (Dittmer, 1965Go). After incubating the influenza virus [A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains] with synthetic sialyl DSPE derivatives (14) for 1 h at 37°C, the DSPE cleaved was determined using Dittmer’s reagent in a TLC plate because C-3-modified sialic acid was not detected by a resorcinol reagent or a thiobarbituric acid assay.

Method 2: Hydrolysis of synthetic sialyl PNP derivatives by influenza virus sialidase.
Virus suspension (2 µg/µl protein) and 500 µM of synthetic sialyl PNP derivatives were mixed in the 0.1 M acetate buffer (pH 5.0) containing 1% TDC (50 µl). After incubated for 20 min at 37°C, the reaction was stopped with 0.2 N NaOH. Hydrolyses of the PNP derivatives were monitored by measuring the increase of absorbance at 415 nm using an MTP-32 microplate reader (Corona Electric, Japan).

Sialidase inhibitory assay
2'-(4-Methylumbelliferyl)-{alpha}-D-N-acetylneuraminic acid (4-MU-Neu5Ac) and Neu5Ac-DSPE were used as the substrates of influenza virus sialidases. After incubation of influenza viruses (virus protein 1 µg/µl) with serially diluted derivatives for 2 h at 4°C, the treated-virus mixture was added to 0.5 mM substrae (4-MU-Neu5Ac or Neu5Ac-DSPE), and then incubated for 1 h at 37°C. The Neu5Ac released was determined by the thiobarbituric acid method according to Aminoff (1961)Go. The relative inhibition activities of synthetic sialyl DSPE derivatives were expressed with the concentration of Neu5Ac released.

Neutralization assay
The neutralizations of synthetic sialyl DSPE derivatives on the infection of influenza virus to MDCK cells were determined as described previously (Suzuki et al., 1983Go). Briefly, MDCK cell monolayers were maintained in Eagle’s minimum essential medium containing 5% fetal calf serum. One hundred microliters of TCID50 (50% tissue-culture infectious dose) of A/Aichi/2/68 in the presence of sialy DSPE analogs (1–500 µM) was inoculated at 34.5°C for 1 h. The cells were examined using a light microscope for the progression of viral-induced cytopathic effect after incubation at 34.5°C for 20 h. The LDH that was released from MDCK cells was examined for virus neutralization by simply modified colorimetric assay (Watanabe et al., 1995Go). The LDH activities in the medium were determined according to the manufacturer’s instructions. Briefly, the medium (0.0125 ml) was diluted to 1:4 with phosphate buffered saline and mixed with 0.05 ml LDH reagent (Shinotest, Japan). The mixture was incubated at 37°C for 10 min, and the reaction was stopped by the addition of 0.1 ml of 0.5 N HCl. Absorbance was measured at 550 nm (reference at 630 nm). The assays were performed in duplicate.

To investigate the inhibitory mechanism of the derivatives, the process of virus infection was divided into early (S1) and late stages (S2) depending on the inoculum to cells by virus. As shown in Figure 4 (right), three different schedules of experimental procedure were carried out. After preincubation with the inhibitor and virus, the mixture was added to MDCK cells. In Figure 4A, the mixture was maintained throughout the process; in Figure 4B, the mixture was discarded after inoculation for 1 h and the cells were incubated with the virus. In Figure 4C, the inhibitor was added to the cells after inoculation of the cells with the virus for 1 h to investigate whether the inhibitors had any effect in the virus release from cells.


    Acknowledgments
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
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This work was supported in part by grants-in-aid 06454211, 05274101, 08457098, 07044286, 07557166 (Y.S.) for Scientific Research from the Ministry of Education, Science and Culture of Japan and Leading Research Utilizing Potential of Regional Science and Technology (Y.S.). This research was also supported in part by the Science and Technology Agency of the Japanese government.


    Abbreviations
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CH, cholesterol; DSPC, distearoylphosphatidy-lcholine; DSPE, distearoylphosphatidyl-ethanolamine; HA, hemagglutinin; LDH lactate dehydrogenase; MDCK, Madin Darby canine kidney; 4-MU-Neu5AC, 2'-(4-Methylumbelliferyl)-{alpha}-D-N-acetylneuraminic acid; PNP, p-nitrophenyl; TDC, taurodeoxycholate; TLC, thin-layer chromatography.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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