The glutamate receptor antagonist ifenprodil inhibits hepatitis E virus infection

Abstract

Infection with hepatitis E virus (HEV) represents a global problem, with over 20 million people infected annually. No specific antiviral drugs are available for treating HEV infection, necessitating the development of novel targeted therapeutics. Here, we report that the N-methyl-D-aspartate receptor (NMDAR) antagonist ifenprodil, a clinically approved drug used to treat idiopathic pulmonary fibrosis (IPF), is an HEV inhibitor in liver-derived cells. In vitro investigation demonstrates that ifenprodil suppresses viral protein expression in a dose-dependent manner in human hepatoma cells by inhibiting early stages of viral infection. We also found that ifenprodil modulates host cell intrinsic biological processes distinct from virus-induced innate immunity, inhibiting HEV RNA accumulation in primary human hepatocytes. Finally, the inhibitory effect of ifenprodil in vivo was also tested in rabbits challenged with the HEV-3ra CHN-BJ-R14 strain. Fecal virus shedding was below the limit of detection in two animals for both ribavirin-treated and ifenprodil-treated rabbits compared to vehicle-treated control animals. Our data demonstrate that ifenprodil is an effective anti-HEV compound with potential as a therapeutic candidate for the treatment of HEV infection.

Keywords: FDA-approved drugs; NMDA-receptor antagonist; hepatitis E virus; ifenprodil.

FIG 1

Antiviral activity of ifenprodil against HEV in human hepatoma cells. (A) Dose-dependent non-enveloped HEV (neHEV; Kernow-C1/p6) inhibition by ifenprodil on intracellular HEV ORF2 expression in HepG2 cells (n = 3). Normalized infections in percent (%) were fitted by four-parameter log-logistic model to determine half-maximum EC₅₀ and half-maximum CC₅₀. (B) Representative whole well images of neHEV infected cells treated with either vehicle control (DMSO) or 50 µM ifenprodil. (C) Western blot analysis (n = 2) of HEV-3 Kernow-C1 p6-FL infected HepG2/C3As treated with 25 µM ifenprodil. Treatment with 25 µM RBV and DMSO served as positive and negative control, respectively. (D) Antiviral effect and (E) cytotoxic effects of 20 µM ifenprodil on wild boar neHEV 83-2-27 virus. Statistical significance was determined by an ordinary one-way ANOVA and corrected for multiple comparison by Dunnett. P-values < 0.05 were considered significant. (F) Representative immunofluorescence images illustrate the impact of ifenprodil on the infectivity of neHEV.

FIG 2

Combination treatment with RBV and time-of-drug addition analysis. (A) In vitro effect of antiviral combinations on HEV infection in HepG2/C3A cells. HEV inhibition during treatment with 0.39 µM (light green), 25 µM (dark green) or 50 µM (purple) ifenprodil and simultaneous titration of RBV (from 0 to 25 µM). (B) A two-dimensional map of synergy scores shown for the combination of ifenprodil with RBV. Synergy scores were based on ZIP synergy analysis determined with SynergyFinder (19). Determined ZIP score was −0.37. The ZIP score stands for the response beyond expectation in percentage. In the range of −10 < ZIP < 10, the compounds are likely to act in an additive manner, while a score ≥10 indicates synergism, and <−10 shows antagonism. (C, top) Experimental setup of the time-of-drug-addition assay. Hepatoma HepG2/C3A cells were inoculated with neHEV at 0 h and treated with DMSO, 0.1 µM K11777, or 25 µM RBV for 1 h (−1) before, during (0), and 2, 4, 8, 12, 24, 48, and 72 h after infection until fixation at 96 h p.i. At 8 h after infection, inoculum was removed and replenished with fresh medium-containing drugs after several washes with PBS. (C, bottom) The inhibitory effect of 20 µM ifenprodil on neHEV infection when added at different time points pre- or post-infection is depicted by the green curve. The broad-spectrum RNA virus inhibitor RBV (25 µM) served as positive control (dark green curve). Data presented represent mean  ±  SD from three independent experiments. (D–F) Hepatoma cells were transfected with HEV-3 Kernow-C1 p6-Gluc subgenomic replicon and treated with RBV (100–12.5 µM; 1:2 serial dilution) or ifenprodil (100–12.5 µM; 1:2 serial dilution). Depicted are normalized relative light units (RLUs) measured 24 (D), 48 (E) and 72 h (F) post-electroporation. DMSO was employed as vehicle control. The depicted values represent means ± SD from two independent experiments.

FIG 3

Antiviral activity of ifenprodil against HEV in PHH. (A) The effects of ifenprodil on intracellular ORF2 expression in PHH (n = 3). PHH were infected with Kernow-C1/p6 virus for 3 days and treated with 12.5, 25, and 50 µM ifenprodil. Treatment with DMSO and 25 µM RBV served as negative and positive controls, respectively. (B) Cell viability was determined by LDH release assay. Data depict means + SD of three independent experiments. To test the significance of mean differences, one-way ANOVA, followed by Dunnett multiple comparison test, was used. P-values > 0.05 were considered to be not significant (ns). (C) Representative immunofluorescence images of PHH. ORF2 = green; DAPI = blue.

FIG 4

Distinct host transcriptional responses upon HEV infection and ifenprodil treatment in PHH. (A) PHH from three different donors were either inoculated with HEV-3 Kernow-C1 p6 virus or with DMEM and/or treated with 25 µM ifenprodil. Total RNA was extracted 48 h p.i., and Illumina sequencing was performed before mapping to human (Hg38) or HEV (Kernow-C1 p6) genome scaffolds. Volcano plots visualize DEGs induced in infected versus uninfected cells (condition 1; C1), for ifenprodil versus vehicle control (DMSO) treated cells (condition 2; C2), and in infected + ifenprodil treated versus infected + vehicle treated cells (condition 3; C3). (B) Percentage of HEV p6 mapped reads of total mapped reads, under DMSO and ifenprodil treatment. (C) Numbers of upregulated and downregulated DEGs (reads per kilobase million [RPKM] ≥ 0.5 and fold change ≥ 2) under the indicated conditions. Purple bars indicate upregulated genes, and green bars represent downregulated genes. (D) Number of significantly enriched GO-terms activated in condition 1, 2, and 3 (false discovery rate [FDR] ≤ 0.05). (E) Dot plot visualizes representative enriched GO categories under the indicated conditions. Circle size corresponds to the size ratio of enriched gene relative to total genes in that category, color to the z-score, and border to significance (black border = FDR ≤ 0.05; no border = ns). (F–H) Heat maps visualizing dysregulation of genes (mean log₂ FC) associated with the GO category “response to virus” (GO:0009615; panel F), “regulation of cell cycle” (GO0051726; panel G) “lipid biosynthesis process” (GO0008610; panel H) under the indicated conditions. Color corresponds to log₂-fold change (LFC). (I) Comparison of the mean log₂ FC of genes clustering in the GO category “response to virus” upon infection of PHH under the indicated conditions. Statistical significance was calculated using two-tailed, paired t-test, ****P < 0.0001. Data presented derived from infection n = 3 biological replicates. FC, fold change.

FIG 5

In vivo efficacy of ifenprodil against HEV. (A) Schematic representation of the in vivo study setup. Twelve-week-old male rabbits were inoculated with 5 × 10⁶ copies HEV RNA by intravenous injection (n  =  5 in the vehicle-treated group, n = 5 in the compound-treated group). Ifenprodil treatment was given daily by intraperitoneal injection, beginning 14 days p.i. until 31 days p.i. (B) The suppressive effect of ifenprodil on the viral load in the feces on day 11, 18, 25, 42, 49, and 56 after infection. Rabbits received the treatment once daily with RBV 33.3  mg/kg (dark green dots), vehicle (grey dots), or ifenprodil at 10 mg/kg (bright green dots). Individual data points are represented by dots.