2Department of Biochemistry, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka-shi 4228526, 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.
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Abstract |
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Key words: hemagglutinin/influenza virus/inhibitors/resistance/sialidase
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Introduction |
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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 receptorligand 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., 1988; Wiley and Skehel, 1987
). 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., 1991; Roy et al., 1993
; Spevak et al., 1993
; Choi et al., 1997
; Guo et al., 1998
; Kamitakahara et al., 1998
; Tsuchida et al., 1998
) and sialidase (Suzuki et al., 1990
; Von Itzstein et al., 1993
; Woods et al., 1993
; Sabesan et al., 1995
; Jedrzejas et al., 1995
; Choi et al., 1996
; Lew et al., 1998
; Ikeda et al., 1998
; Atigadda et al., 1999
). 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., 1993
; Itoh et al., 1995
). 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., 1981
; Wiley et al., 1981
; Watowich et al., 1994
) and sialidase (Varghese et al., 1983
; Colman et al., 1983
; Crennell et al., 1993
). This has been confirmed by chemical modifications of sialic acid (Suzuki, 1994
; Sato et al., 1998
), 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., 1988
, 1990; Wilson et al., 1981
; Sauter et al., 1992a
,b) and sialidase (Varghese and Colman, 1991
; Varghese et al., 1992
) 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., 2000
). 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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Results |
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Considered together, the results indicate that Neu5Ac3F-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.
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Discussion |
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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, Neu5Ac3F-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., 1992
). Sialic acid derivatives without aglycone are also considered to mimic the transition state (Hagiwara et al., 1994
). 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., 1982
). 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., 1990
) 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., 2000
). 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 Neu5Ac3F-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
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, Neu5Ac3F-DSPE (4), a C-3-modified
-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.
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Materials and methods |
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C-3-Modified sialyl DEPE derivatives.
Four sialyl DSPE derivatives (14) were prepared as described elsewhere (Sun et al., 2000).
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 (1.71.8 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 0.11.0 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., 1992). Viral HA units were determined in microtiter plates using 0.5% chicken erythrocytes as described previously (Suzuki et al., 1983
).
Inhibition assay for low pHinduced homolysis
The inhibitory activities of synthetic C-3-modified sialyl DSPE derivatives on the low pHinduced hemolysis were determined as described previously (Suzuki et al., 1983; MacDonald et al., 1984
; Portner et al., 1987
; Guo et al., 1998
). 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., 1986
).
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 Dittmers method (Dittmer, 1965). 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 Dittmers 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)--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)
. 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., 1983). Briefly, MDCK cell monolayers were maintained in Eagles 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 (1500 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., 1995
). The LDH activities in the medium were determined according to the manufacturers 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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