Macromolecular Antiviral Agents against Zika, Ebola, SARS, and Other Pathogenic Viruses

Abstract

Viral pathogens continue to constitute a heavy burden on healthcare and socioeconomic systems. Efforts to create antiviral drugs repeatedly lag behind the advent of pathogens and growing understanding is that broad-spectrum antiviral agents will make strongest impact in future antiviral efforts. This work performs selection of synthetic polymers as novel broadly active agents and demonstrates activity of these polymers against Zika, Ebola, Lassa, Lyssa, Rabies, Marburg, Ebola, influenza, herpes simplex, and human immunodeficiency viruses. Results presented herein offer structure-activity relationships for these pathogens in terms of their susceptibility to inhibition by polymers, and for polymers in terms of their anionic charge and hydrophobicity that make up broad-spectrum antiviral agents. The identified leads cannot be predicted based on prior data on polymer-based antivirals and represent promising candidates for further development as preventive microbicides.

Keywords: Zika virus; broad spectrum antivirals; microbicides; polyanions; polymers.

Figure 1

Chemical formulas of the polymers used in this study. Carboxylates (in red): poly(acrylic acid), PAA; poly(methacrylic acid), PMAA; poly(ethylacrylic acid), PEAA; poly(propylacrylic acid); PPAA, poly(vinylbenzoic acid), PVBzA. Phosphates/phosphonates (in green): poly(vinylphosphonic acid), PVPA; poly((2‐methacrylamidoethyl)phosphonic acid), PMPA; poly((2‐acrylamidoethyl)phosphonic acid), PAPA; poly((2‐methacrylamidoethyl)phosphate), PMEP; poly((2‐acrylamidoethyl)phosphate), PAEP. Sulfonates (in blue): poly(vinylsulfonic acid), PVSA; poly(3‐sulfopropyl acrylate), PSPA; poly(2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid), PAMPS; poly(vinylbenzenesulfonate), PSVBS.

Figure 2

Identification of polyanionic inhibitors of ZIKV. A) Vero E6 cells were incubated with 0–100 mg L⁻¹ of the polyanions for 90 min and then infected with ZIKV MR766 (“cell treatment”). Infection rates were determined 3 d later by MTT assay. B) ZIKV MR766 was incubated for 15 min with polyanions (0–100 mg L⁻¹) and then mixtures were added to Vero E6 cells, resulting in tenfold dilutions of the potential drugs, as indicated in the legend (“virion treatment”). Results are presented as mean ± standard error derived from two independent experiments performed in triplicate for each data point.

Figure 3

Lead polymers inhibit ZIKV. A) PSVBS, PVBzA but not PEAA inhibit ZIKV MR766 infection of Vero E6 cells under “cell treatment” conditions. The experiment was performed as described in Figure 2A but infection was quantified 36 h post infection by virus immunodetection assay using the flavivirus antibody 4G2. B) PSVBS, PVBzA, and PEAA block ZIKV MR766 infection of Vero E6 cells under “virion treatment” conditions, as measured by ZIKV immunodetection assay. C) The three anti‐ZIKV polymers are not toxic. Vero E6 cells were exposed to the three polymers and cell viability was assessed by MTT 3 d later. Results are presented as mean ± standard error derived from two independent experiments performed in triplicate for each data point.

Figure 4

Fluorescence microscopy of ZIKV inhibition by polymers. A) PSVBS and B) PVBzA inhibit Vero E6 cell infection under “cell treatment” conditions (also see Figure S1A, Supporting Information). C) PEAA blocks ZIKV when pre‐exposed to virions (also see Figure S1B, Supporting Information). To visualize infection, cells were fixed, incubated with the anti‐ZIKV mouse antibody 4G2 that detects the viral E glycoprotein, and labeled with a secondary goat anti‐mouse IgG coupled to Alexa Flour 488 (green). Cell nuclei were stained with Hoechst (blue), and the cytoskeleton with labeled actin‐specific phalloidin (red). Shown are merged confocal images taken 3 d post infection. D) Rhodamine B‐coupled PEAA interacts with ZIKV. Rhodamine B‐coupled PEAA (200 mg L⁻¹) was incubated with buffer (no virus) or indicated dilutions of the ZIKV MR766 stock for 15 min and then imaged by confocal microscopy in the absence of cells (see Figure S2, Supporting Information).

Figure 5

Polymers inhibit clinically relevant ZIKV isolates. A) HeLa cells were exposed to PSVBS and PVBzA for 90 min and then infected with the FB‐GWUH‐2016 and PRVABC59 ZIKV isolates. B) Both viruses were exposed to PEAA for 15 min, and then used to infect HeLa cells. Three days later, cells were fixed, stained for ZIKV E protein (green), nuclei (blue), and actin (red), and analyzed by confocal microscopy (also see Figures S3 and S4, Supporting Information).

Figure 6

Inhibitory activity of polyanions against HIV‐1 (“virus treatment” mode). Polymers were first mixed with HIV‐1 for 10 min and then these mixtures were used to inoculate TZM‐bl cells. Infection rates were determined 3 d later by quantifying β‐galactosidase activities in cellular lysates. Results are presented as mean ± standard error derived from two independent experiments performed in triplicate for each data point.

Figure 7

Inhibitory activity of polyanions on infectivity of HSV‐2 under “virus treatment.” HSV‐2 particles were exposed to polymers, incubated for 1 h, and the mixtures were used to inoculate ELVIS cells. Infection rates were determined 36 h later through quantifying β‐galactosidase activities in a chemiluminescence‐based assay. Results are presented as mean ± standard error derived from two independent experiments performed in triplicate for each data point. For results obtained in the “cell treatment” setup, see Figure S8 (Supporting Information).

Figure 8

Antiviral activity of polyanions against lentiviral pseudotypes carrying glycoproteins derived from A) Lyssa virus, B) Rabies virus, C) Ebola virus, D) Marburg virus, E) SARS virus, F) Lassa fever virus, G) influenza virus. Respective target cells were exposed to 50 mg L⁻¹ of the polymers and then infected with the pseudoparticles. Cellular entry is mediated by the glycoproteins and infection rates were determined 3 d post infection by quantifying luciferase activities in cellular lysates. Shown are the mean % infection rates obtained from polyanion treated cells relative to the untreated control (100%). Results are presented as mean ± standard error derived from two independent experiments performed in triplicate for each data point. pp: pseudoparticle.

Figure 9

Summary of the maximum antiviral activities of tested polyanions.