Dendrimers functionalized with membrane-interacting peptides for viral inhibition

Abstract

This contribution reports the synthesis of a poly(amide)-based dendrimer functionalized at the termini with a membrane-interacting peptide derived from the herpes simplex virus (HSV) type 1 glycoprotein H, namely gH625-644. This peptide has been shown to interact with model membranes and to inhibit viral infectivity. The peptidodendrimer inhibits both HSV-1 and HSV-2 at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite of viral internalization. The 50% inhibitory concentration was 100 and 300 nM against HSV-1 and HSV-2 respectively, with no evidence of cell toxicity at these concentrations. These results show that the functionalization of a dendrimer with the peptide sequence derived from an HSV glycoprotein shows promising inhibitory activity towards viruses of the Herpesviridae family.

Keywords: antiviral activity; membranotropic peptides; peptidodendrimer.

Figure 1

(A) Structures of the poly(amide)-based azidodendrimer 1 and cartoon of the peptidodendrimer 2. The helices on the structure represent the gH625 peptide sequence. (B) Schematic representation of the CuAAC reaction to functionalize 1 with peptides. Shown is the C-terminal PrA residue. Abbreviations: CuAAC, copper catalyzed 1,3-dipolar alkyne/azide cycloaddition; PrA, propargylglycine.

Figure 2

Cell viability measured by the MTT assay for 3 and 24 hours for the dendrimer (1) and peptidodendrimer (2). Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 3

Apoptotic effects of 1 and 2 on Vero cells. FACS analysis after double labeling with propidium iodide and FITC-Annexin V of Vero cells: (A) untreated cells, (B) treated with 1, and (C) treated with 2 for 24 hours at 0.55 μM. The experiments were performed three times, and the results were always similar. Note: Insets, percentage of positive cells. Abbreviations: FACS, fluorescence activated cell sorting; FITC, fluorescein isothiocyanate.

Figure 4

Virus yield reduction assay. Vero cells were infected with either HSV-1 (blue lines) or HSV-2 (red lines) in the presence of increasing concentrations of the dendrimer 1 (triangles) or the peptidodendrimer 2 (squares). Notes: The percentage of inhibition was calculated with respect to no-compound control experiments. Data points represent an average of three experiments, and error bars represent standard deviations. Abbreviation: HSV, herpes simplex virus.

Figure 5

Posttreatment assay. HSV-1 (blue lines) and HSV-2 (red lines) infected Vero cells were treated with increasing concentrations of the dendrimer 1 (triangles) or the peptidodendrimer 2 (squares), then overlaid with CMC and incubated for 24 hours at 37°C. Notes: Plaque numbers were scored, and the percentage of inhibition was calculated with respect to no-compound control experiments. Data points represent an average of three experiments, and error bars represent standard deviations. Abbreviations: HSV, herpes simplex virus; CMC, carboxymethyl cellulose.

Figure 6

Effect of 1 and 2 on viral attachment. (A) Vero cell monolayers were infected with HSV-1 (blue lines) or HSV-2 (red lines) at an MOI of 0.1 pfu/cell for 2 hours at 4°C. After unattached virus removal, plates were treated with various concentrations of 1 (triangles), 2 (squares), or heparin (circles) for 3 hours at 37°C. Finally, cells were overlaid with CMC and incubated for 2 days at 37°C. (B) Vero cell monolayers were treated with inocula containing both the viruses and the antiviral compounds precooled at 4°C. Notes: Plaque numbers were scored, and the percentage of inhibition was calculated with respect to “no-compound” control experiments. Data are reported in triplicate, and error bars represent standard deviations. Abbreviations: HSV, herpes simplex virus; MOI, multiplicity of infection; pfu, plaque-forming unit; CMC, carboxymethyl cellulose.

Figure 7

Cell pretreatment assay. Vero cell monolayers were treated with various concentrations of 1 (triangles), 2 (squares), or heparin (circles) for 30 minutes at 4°C before being infected with precooled HSV-1 (blue lines) or HSV-2 (red lines) at 0.1 pfu/cell in the presence of the compounds for 2 hours at 4°C. After the removal of excess compounds, cells were overlaid with CMC, and incubated for 2 days at 37°C. Notes: Plaque numbers were scored, and the percentage of inhibition was calculated with respect to “no-compound” control experiments. Data points represent an average of three experiments, and error bars represent standard deviations. Abbreviations: HSV, herpes simplex virus; pfu, plaque-forming unit; CMC, carboxymethyl cellulose.

Figure 8

Virucidal assay. Different concentrations of 1 (triangles) and 2 (squares) were added to aliquots (10⁴ pfu) of HSV-1 (blue lines) or HSV-2 (red lines), and incubated at either 4°C (A) or 37°C (B) for 2 hours. After incubation, the samples were diluted to reduce the compound concentrations below the threshold for inhibition of viral replication, and the virus was titrated on Vero cells. Notes: Plaque numbers were scored and the percentage of inhibition was calculated with respect to “no-compound” control experiments. Data points represent an average of three experiments, and error bars represent standard deviations. Abbreviations: HSV, herpes simplex virus; pfu, plaque-forming unit.