Antiviral Candidates for Treating Hepatitis E Virus Infection

Abstract

Globally, hepatitis E virus (HEV) causes significant morbidity and mortality each year. Despite this burden, there are no specific antivirals available to treat HEV patients, and the only licensed vaccine is not available outside China. Ribavirin and alpha interferon are used to treat chronic HEV infections; however, severe side effects and treatment failure are commonly reported. Therefore, this study aimed to identify potential antivirals for further development to combat HEV infection. We selected 16 compounds from the nucleoside and nonnucleoside antiviral classes that range in developmental status from late preclinical to FDA approved and evaluated them as potential antivirals for HEV infection, using genotype 1 replicon luminescence studies and replicon RNA quantification. Two potent inhibitors of HEV replication included NITD008 (half-maximal effective concentration [EC₅₀], 0.03 μM; half-maximal cytotoxic concentration [CC₅₀], >100 μM) and GPC-N114 (EC₅₀, 1.07 μM, CC₅₀, >100 μM), and both drugs reduced replicon RNA levels in cell culture (>50% reduction with either 10 μM GPC-N114 or 2.50 μM NITD008). Furthermore, GPC-N114 and NITD008 were synergistic in combinational treatment (combination index, 0.4) against HEV replication, allowing for dose reduction indices of 20.42 and 8.82 at 50% inhibition, respectively. Sofosbuvir has previously exhibited mixed results against HEV as an antiviral, both in vitro and in a few clinical applications; however, in this study it was effective against the HEV genotype 1 replicon (EC₅₀, 1.97 μM; CC₅₀, >100 μM) and reduced replicon RNA levels (47.2% reduction at 10 μM). Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.

Keywords: broad-spectrum antivirals; direct-acting antivirals; hepatitis E virus; hepatitis therapy development.

FIG 1

Screening of broad-spectrum antivirals against HEV G1 replication. (A) A schematic representation of the HEV genome (top) is shown for comparison with the subgenomic replicon (bottom), with nonstructural proteins encoded by ORF1 and the structural proteins and phosphoproteins encoded by ORF2 and ORF3, respectively. Nucleotide positions, 5′ untranslated regions (UTR), and 3′ polyadenylated tails [poly(A)] are indicated. The HEV G1 subgenomic replicon pSK-HEV-2-Luc (bottom) (38) shows the disruption of the ORF2 capsid gene (nucleotides 5148 to 5816), with the firefly luciferase gene as described in reference . (B) An initial screen of 16 broad-spectrum antivirals (Table 1) for inhibitory activities against the human HEV G1 subgenomic replicon pSK-HEV-2-Luc through quantitation of luminescence is shown. All compounds were examined at a fixed concentration of 10 μM, and the percentages of mock-treated HEV replication (compound vehicle only, 0.5%, vol/vol, DMSO) for each compound are plotted. The black horizontal dotted line represents 100% HEV replication (0% inhibition), while the red dotted line represents 50% HEV replication (50% inhibition). The NA positive control 2CMC is used to demonstrate effective inhibition of HEV replication. Mean values ± SEM are shown.

FIG 2

Dose-response curves and cytotoxicity profiles of lead inhibitory compounds against HEV replication. The HEV inhibitory and cytotoxicity effects of four compounds identified in Fig. 1B are shown. Dose-response graphs were generated by quantification of luminescence (red bars, left y axis), and effects on cell viability were examined using a fluorescent resazurin-to-resorufin assay (blue lines, right y axis). The black dotted horizontal lines represent 50% inhibition. The EC₅₀ and CC₅₀ values are shown on the graphs and in Table 2 for compounds NITD008 (0.019 μM to 2.5 μM) (A), GPC-N114 (0.16 μM to 25.0 μM) (B), dasabuvir (0.16 μM to 25.0 μM) (C), and sofosbuvir (0.16 μM to 25.0 μM) (D). The synthesis of HEV replicon RNA was reduced by three broad-spectrum antivirals compared to the mock-treated control (0.5%, vol/vol, DMSO), as quantified by qRT-PCR, using primers to detect the HEV RdRp. Sofosbuvir and GPC-N114 were examined at 10 μM (E), and NITD008 was examined at concentrations of 0.04 to 2.50 μM (F). The NA 2CMC (10 μM) was used as a positive control, and RNA levels were normalized to the housekeeping gene β-actin, while the relative fold expression was calculated using the ΔΔC_T method. P < 0.001 (***); P < 0.01 (**); P < 0.05 (*). Mean values ± SEM are shown.

FIG 3

GPC-N114 and NITD008 exhibited synergistic inhibition of HEV replication. GPC-N114 and NITD008 were examined in combination against HEV replication in vitro as quantified by relative luminescence. GPC-N114 (0.16 to 5.00 μM) and NITD008 (0.005 to 0.16 μM) were evaluated in a 32:1 concentration ratio. (A) Isobologram of GPC-N114 and NITD008 in combination. Data were analyzed using the Chou-Talalay method (Compusyn software) with an average combination index of 0.4 over 50% (blue dots), 75% (red squares), and 90% inhibition (green triangles), indicating synergism. (B) Cytotoxicity effects of the two drugs in combination over the same concentration range as that described for panel A were assessed using a fluorescent resazurin-to-resorufin assay. Mean values ± SEM are shown.