Bioconjugate Chem. 1999, 10, 271−278
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Inhibition of Viral Adhesion and Infection by Sialic-Acid-Conjugated Dendritic Polymers Jon D. Reuter,†,‡ Andrzej Myc,† Michael M. Hayes,† Zhonghong Gan,§ Rene Roy,§ Dujie Qin,| Rui Yin,| Lars T. Piehler,| Roseita Esfand,| Donald A. Tomalia,| and James R. Baker, Jr.*,† Center for Biologic Nanotechnology, Department of Internal Medicine, Division of Allergy, 1150 West Medical Center Drive, Room 9220 MSRB III, University of Michigan, Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Michigan 48109, Department of Chemistry, University of Ottawa, Canada, and Michigan Molecular Institute, Midland, Michigan 48640. Received August 19, 1998; Revised Manuscript Received December 21, 1998
Multiple sialic acid (SA) residues conjugated to a linear polyacrylamide backbone are more effective than monomeric SA at inhibiting influenza-induced agglutination of red blood cells. However, “polymeric inhibitors” based on polyacrylamide backbones are cytotoxic. Dendritic polymers offer a nontoxic alternative to polyacrylamide and may provide a variety of potential synthetic inhibitors of influenza virus adhesion due to the wide range of available polymer structures. We evaluated several dendritic polymeric inhibitors, including spheroidal, linear, linear-dendron copolymers, comb-branched, and dendrigraft polymers, for the ability to inhibit virus hemagglutination (HA) and to block infection of mammalian cells in vitro. Four viruses were tested: influenza A H2N2 (selectively propagated two ways), X-31 influenza A H3N2, and sendai. The most potent of the linear and spheroidal inhibitors were 32-256-fold more effective than monomeric SA at inhibiting HA by the H2N2 influenza virus. Linear-dendron copolymers were 1025-8200-fold more effective against H2N2 influenza, X-31 influenza, and sendai viruses. The most effective were the comb-branched and dendrigraft inhibitors, which showed up to 50000-fold increased activity against these viruses. We were able to demonstrate significant (p < 0.001) dose-dependent reduction of influenza infection in mammalian cells by polymeric inhibitors, the first such demonstration for multivalent SA inhibitors. Effective dendrimer polymers were not cytotoxic to mammalian cells at therapeutic levels. Of additional interest, variation in the inhibitory effect was observed with different viruses, suggesting possible differences due to specific growth conditions of virus. SA-conjugated dendritic polymers may provide a new therapeutic modality for viruses that employ SA as their target receptor.
INTRODUCTION
Influenza virus is a highly contagious, enveloped RNA virus which is responsible for several severe pandemics (Mulder and Hers, 1972). Two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), determine the antigenic specificity of viral subtypes (Lamb and Krug, 1996). The virus is highly mutagenic, and frequent alterations in HA glycosylation leading to altered antigenicity are considered to be responsible for the episodic resurgence of influenza. The capacity to modify viral antigenic sites enables influenza virus to overcome inhibitory effects of neutralizing antibodies and elude many anti-viral compounds including neuraminidase inhibitors (Lamb and Krug, 1996; Schulze, 1997). The first step of infection by influenza virus is the binding of the viral HA to sialic acid receptors on the host cell, followed by receptor-mediated endocytosis of the virus into the cell (Wiley and Skehel, 1987). One potential approach to anti-viral therapy involves the inhibition of virus/host cell binding via blocking of the viral receptor (Kiefel et al., 1996; Weis et al., 1988). Ideal preventative agents must be nontoxic to mammalian cells, broadly * To whom correspondence should be addressed. Phone: (734) 647-2777. Fax: (734) 936-2990. E-mail:
[email protected]. † Center for Biologic Nanotechnology. ‡ Unit for Laboratory Animal Medicine. § Department of Chemistry. Michigan Molecular Institute.
specific across viral subtypes and cost effective. Previous reports have demonstrated that millimolar concentrations of solubilized monomeric sialic acid (SA) prevent influenza A agglutination of chicken erythrocytes (cRBC), presumably by binding to the HA molecule on the virus (Pritchett and Paulson, 1987; Weis et al., 1988). However, host cell viral receptor sialyl moieties have great lateral mobility on the cellular surface, allowing each influenza virion to use the estimated 300-600 envelope hemagglutinin spikes to develop strong polyvalent binding to host cell receptors (Choi et al., 1997; Weis et al., 1988). Thus, while the binding of soluble R-sialoside monomeric derivatives to hemagglutinin is very weak (KD ≈ 2 mM), cooperative interaction allows influenza virus attachment to the surface of an erythrocyte with KD (HA) over
