New methylene blue derivatives suggest novel anti-orthopoxviral strategies

Abstract

Decades after the eradication of smallpox and the discontinuation of routine smallpox vaccination, over half of the world's population is immunologically naïve to variola virus and other orthopoxviruses (OPXVs). Even in those previously vaccinated against smallpox, protective immunity wanes over time. As such, there is a concomitant increase in the incidence of human OPXV infections worldwide. To identify novel antiviral compounds with potent anti-OPXV potential, we characterized the inhibitory activity of PAV-866 and other methylene blue derivatives against the prototypic poxvirus, vaccinia virus (VACV). These compounds inactivated virions prior to infection and consequently inhibited viral binding, fusion and entry. The compounds exhibited strong virucidal activity at non-cytotoxic concentrations, and inhibited VACV infection when added before, during or after viral adsorption. The compounds were effective against other OPXVs including monkeypox virus, cowpox virus and the newly identified Akhmeta virus. Altogether, these findings reveal a novel mode of inhibition that has not previously been demonstrated for small molecule compounds against VACV. Additional studies are in progress to determine the in vivo efficacy of these compounds against OPXVs and further characterize the anti-viral effects of these derivatives.

Keywords: Antiviral; Methylene blue; Novel inhibitor; PAV-866; Poxviruses; Vaccinia virus.

Fig. 1

Treatment with PAV-866 and its derivatives decreases VACV yield with low cytotoxicity. HeLa cells were infected with VACV-NLS-GFP at MOI 3 and treated with different concentrations of PAV-866 and its 10 derivatives. After 24 h, cells were collected, and total virus yield was determined by plaque assay in the absence of inhibitors. Virus yields (pfu/mL) for each concentration tested are shown as filled black bars. The dotted lines in the graphs represent the total virus yield in the untreated control (blue, top) and in the presence of AraC (black, bottom). The right y-axis corresponds to percent cytotoxicity with respect to a lysed cell control, and the % cytotoxicity for each compound at the concentrations tested are shown as red circles. Values shown represent the mean of two or more replicates.

Fig. 2

Reduction in VACV spread and early gene expression by PAV-866 and derivatives(A) Percentage of GFP⁺ cells in the presence of inhibitors AraC (DNA replication) and ST-246 (EV membrane wrapping). HeLa cells were infected with VACV-NLS-GFP at MOI 0.4 for 24 h in the absence and presence of inhibitors. Samples were fixed and stained with DAPI to visualize total number of cells. The percentage of infected cells was determined as the ratio of GFP⁺ nuclei to total number of nuclei. Values represent the mean of two or more replicates±SEM. (B) Percent VACV spread in the presence of serially diluted PAV-866 and derivatives. Shading of cells corresponds to degree of viral inhibition (darkest cells indicate low viral spread or high viral inhibition). Corresponding EC₅₀ values also shown. C) HeLa cells were infected with VACV-LUC virus at MOI 3 in the presence or absence of four concentrations of 10 PAV-866 analogs. After 2 h, cells were lysed and luciferase activity was measured as a surrogate for early gene expression. The values plotted represent the percent relative luminescence units (RLU) of each treatment with respect to cells infected with virus alone. The dotted line represents the 50% RLU value relative to the virus only treatment. The mean of two or more replicates is shown±SEM.

Fig. 3

PAV-866 derivatives act directly on viral particles to decrease infectivity. A) Concentrated VACV-LUC (equivalent to 3 MOI) was incubated in the presence (Condition 1) or absence (Condition 2) of concentrated PAV-164 or PAV-174 in a small volume at 37 °C for 1h. Both groups were diluted 75-fold, and compounds were added to the virus-only tubes resulting in two treatments (final concentrations 5 μM and 0.05 μM) for each compound in each condition. Both conditions included virus-only controls. The diluted virus-compound mixtures were added to cells for 1 h at 4 °C to allow viral adsorption. After removing the inoculum and washing, cells were incubated at 37 °C until harvest. Each treatment was tested in triplicate. B) Virus entry measured by luciferase assay. Bars represent the mean±SEM percent RLU of compound treatments with respect to virus only. C) Virus yield determined by plaque assay. Values plotted represent the mean±SEM percent virus yield of compound treatments with respect to virus only. For both B and C, black and gray bars represent Conditions 1 and 2 respectively. Statistical significance determined using the Holm-Sidak method, with alpha = 5.000%. Asterisks indicate p values < 0.05.

Fig. 4

Pre-treatment of cells with PAV-866 derivative can block VACV infection. (A) Schematic representation of the three treatment conditions tested. The incubation of cells with virus for 1 h at RT represents the viral adsorption step, and compounds (2 μM) were added at different time points pre and post adsorption as illustrated. (B) Virus yield (pfu/mL) at 24 h for all three treatments measured by plaque assay. The red line represents the input virus titer determined based upon AraC treatment titers from Condition 3. Bars represent average of two or more replicates±SEM.

Fig. 5

Reduced virus binding and membrane fusion of VACV in presence of PAV-866 derivative. (A) Percent YFP⁺ cells in the presence or absence of compound PAV-164 quantified by flow cytometry. Bars colored based upon treatment group: virus only (gray) and PAV-164 (orange). Values represent the average of two replicates±SEM. Statistical significance measured using one-way ANOVA and post-hoc Dunnet's multiple comparisons test. P values indicated within graph: * = p < 0.05, ** = p < 0.005 and ns = not significant. (B) Fusion of viral and cell membranes in the presence or absence of compound PAV-164 evaluated by flow cytometry-based detection of DiO⁺ cells. Histogram plots showing overlay of DiO intensity peaks in the presence (orange) and absence (gray) of three different concentrations of PAV-164. Peaks normalized to mode (y-axis), and the x-axis represents the intensity of DiO staining.

Fig. 6

PAV-866 derivative exhibits broad OPXV antiviral activity. HeLa cells were infected with four different OPXVs: VACV, CPXV, MPXV and Akhmeta virus for 24 h in the absence and presence of compound PAV-164. Virus yields (pfu/mL) determined by plaque assay. Bars represent the mean of three replicates±SEM.