Antiviral Mechanism of Virucidal Sialic Acid Modified Cyclodextrin

Abstract

We have reported that CD-6'SLN [6-sialyllactosamine (6'SLN)-modified β-cyclodextrin (CD)] can be a potential anti-influenza drug because it irreversibly deactivates virions. Indeed, in vivo, CD-6'SLN improved mice survival in an H1N1 infection model even when administered 24 h post-infection. Although CD-6'SLN was designed to target the viral envelope protein hemagglutinin (HA), a natural receptor of 6'SLN, it remains unclear whether other targets exist. In this study, we confirm that CD-6'SLN inhibits the influenza virus through an extracellular mechanism by interacting with HA, but not with neuraminidase (NA), despite the latter also having a binding pocket for the sialyl group. We find that CD-6'SLN interacts with the viral envelope as it elicits the release of a fluorophore embedded in the membrane. Two similar compounds were designed to test separately the effect of 6'SLN and of the undecyl moiety that links the CD to 6'SLN. Neither showed any interaction with the membrane nor the irreversible viral inhibition (virucidal), confirming that both components are essential to membrane interaction and virucidal action. Unlike similar antiviral cyclodextrins developed against other viruses, CD-6'SLN was not able to decapsulate viral RNA. Our findings support that combining viral protein-specific epitopes with hydrophobic linkers provides a strategy for developing antiviral drugs with a virucidal mechanism.

Keywords: 6′SLN; antiviral mechanism; hemagglutinin; hydrophobic linker; influenza; membrane interaction; virucidal.

Figure 1

(a) Schematic drawing of the influenza virus. (b) Chemical structure of virucidal CD-6′SLN; schematic drawing of the influenza virus. (c) Chemical structure of CD-(Mal-PEG8)₇-6′SLN.

Figure 2

CD-6′SLN inhibition of H1N1 during various stages of viral infection. Cell pretreatment: compound was applied to the cells for 1 h, and the virus was added after washing the cell. Virus pretreatment: the virus was incubated with the compound for 1 h and added to the cells. Cotreatment: virus and compound were added simultaneously to the cells. Post-treatment: compound was added after viral attachment.

Figure 3

CD-6′SLN bind to H1N1 hemagglutinin instead of neuraminidase. (a) Binding of CD-6′SLN, 6′SLN and CD-(S-C11-COOH)₇ to immobilized his-tagged hemagglutinin (H1N1, A/California/04/2009). (b) Dose-response inhibition of neuraminidase (H1N1, A/California/04/2009) catalytic activity by CD-6′SLN and oseltamivir carboxylate.

Figure 4

RNA exposure assay (a) Schematic drawing demonstrating the mechanism of the RNA exposure assay. The compound and virus were co-incubated for 3 h and then treated either with RNAse or buffer, followed by viral lysis. The RNA quantities in the lysate were quantified by RT-qPCR. (b) Cycle threshold difference (ΔCt = Ct_RNase − Ct_control) of H1N1 samples treated with 100 μg/mL CD-6′SLN and 100 μg/mL SDS. Results are expressed as average ± standard deviation based on two independent experiments. The asterisk represents the p-value (*, <0.05) calculated by a two-tailed unpaired t-test.

Figure 5

CD-6′SLN interacts with the viral envelope. (a) Fluorescence intensity of R18-labeled H1N1 treated with 100 μg/mL CD-6′SLN, 100 μg/mL CD-(S-C11-COOH)₇, 100 μg/mL CD-(Mal-PEG8)₇-6′SLN or 1% triton X-100. (b) Infectious H1N1 titre of the virus-CD-6′SLN mixture over time. Every triangle represents the viral titre determined in a single well. (c) Chemical structure of CD-(S-C11-COOH)₇.