Verdinexor, a novel selective inhibitor of nuclear export, reduces influenza a virus replication in vitro and in vivo

Abstract

Influenza is a global health concern, causing death, morbidity, and economic losses. Chemotherapeutics that target influenza virus are available; however, rapid emergence of drug-resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza virus ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by exportin 1 (XPO1) interaction with viral nuclear export protein tethered to vRNP. RNA interference screening has identified XPO1 as a host proinfluenza factor where XPO1 silencing results in reduced influenza virus replication. The Streptomyces metabolite XPO1 inhibitor leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo, which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza virus activity of a new class of orally available small-molecule selective inhibitors of nuclear export, specifically, the XPO1 antagonist KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export and effectively inhibited the replication of various influenza virus A and B strains in vitro, including pandemic H1N1 virus, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following a challenge with influenza virus A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral loads and proinflammatory cytokine expression, while having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza virus therapeutic agent.

Importance: Antiviral drugs represent important means of influenza virus control. However, substantial resistance to currently approved influenza therapeutic drugs has developed. New antiviral approaches are required to address drug resistance and reduce the burden of influenza virus-related disease. This study addressed critical preclinical studies for the development of verdinexor (KPT-335) as a novel antiviral drug. Verdinexor blocks progeny influenza virus genome nuclear export, thus effectively inhibiting virus replication. Verdinexor was found to limit the replication of various strains of influenza A and B viruses, including a pandemic H1N1 influenza virus strain, a highly pathogenic H5N1 avian influenza virus strain, and a recently emerging H7N9 influenza virus strain. Importantly, oral verdinexor treatments, given prophylactically or therapeutically, were efficacious in limiting lung virus burdens in influenza virus-infected mice, in addition to limiting lung proinflammatory cytokine expression, pathology, and death. Thus, this study demonstrated that verdinexor is efficacious against influenza virus infection in vitro and in vivo.

FIG 1

Silencing of XPO1 gene expression results in reduction of influenza virus replication. A549 cells were transfected with nontargeting siNEG as a negative control, siMEK as a positive control, or siXPO1. (A) Cells were harvested at 96 h posttransfection to evaluate XPO1 protein knockdown efficiency by immunoblot analysis. Densitometry analysis was performed to determine the XPO1 protein level normalized to GAPDH. Percent XPO1 expression in siXPO1-transfected cells was calculated relative to that in siNEG-transfected cells. (B) At 48 h posttransfection, cells were infected at an MOI of 0.001 with influenza virus A/WSN/33 (H1N1) (B, C), A/California/04/09 (pH1N1) (D), A/Philippines/2/82-X79 (H3N2) (E), A/mute swan/MI/451072-2/06 (H5N1) (F), A/red knot/NJ/1523470/06 (H7N3) (G), A/Anhui/1/2013 (H7N9) (H), or B/Ohio/01/05 (Flu B) (I) or at an MOI of 3 with A/WSN/33 (H1N1) (J). At 48 (A) or 8 (J) hpi, cells were fixed, stained for viral NP (green) and nuclei (DAPI; blue), and visualized by fluorescence microscopy. Bars, 200 μm. (B to H) At 48 hpi, culture supernatants were collected for virus titer determination in MDCK cells and TCID₅₀s were calculated with the Spearman-Kärber formula. Data are expressed as the mean ± SE (n = 6). ****, P ≤ 0.0001; **, P < 0.01; *, P < 0.05 (relative to siXPO-transfected cells).

FIG 2

Verdinexor, a novel SINE, is efficacious against influenza A and B viruses. (A) Structure of verdinexor (KPT-335). (B to H) A549 cells were mock treated (M) or treated with increasing doses of verdinexor for 2 h at 37°C. (B) Cytotoxicity was assessed with the ToxiLight bioassay kit at 24 h posttreatment. The percent cytotoxicity in verdinexor-treated cells was determined relative to that in nontreated (0% cytotoxicity) and lysed (100% cytotoxicity) cells and graphed to evaluate the median cytotoxic concentration. (C to H) Cells were infected with influenza virus A/WSN/33 (H1N1) (C), A/California/04/09 (pH1N1) (D), A/Philippines/2/82-X79 (H3N2) (E), A/mute swan/MI/451072-2/06 (H5N1) (F), A/Anhui/1/2013 (H7N9) (G), or B/Ohio/01/05 (Flu B) (H). Culture supernatants were collected at 24 hpi for virus titer determination in MDCK cells, and experiments were repeated at least twice.

FIG 3

Verdinexor treatment resulted in nuclear accumulation of vRNP. A549 cells were pretreated for 2 h with DMSO, verdinexor (1 μM), or LMB (10 nM). Cells were infected with influenza virus A/WSN/33 at an MOI of 3 for 8 h. (A) Cells were fixed and stained for viral NP (green) and nuclei (DAPI; blue) and visualized under a fluorescence microscope. (B, C) Cells were subjected to cytoplasmic-nuclear fractionation, followed by RNA isolation from each fraction. To determine the relative nuclear-cytoplasmic distribution of influenza virus vRNA (B) or mRNA (C), RNA from both fractions was used for RT-qPCR analysis with strand-specific primers for A/WSN/33 segment 5. Viral RNA abundance was normalized to GAPDH, and the abundance relative to that in mock-infected cells was graphed. Data are expressed as the mean ± SE (n = 3). n.s., not significant; ***, P < 0.001; ****, P < 0.0001 (relative to mock-treated infected cells).

FIG 4

Verdinexor blocks influenza virus vRNP nuclear export by disrupting XPO1-NEP binding. (A, B) A549 cells were pretreated for 2 h with DMSO, verdinexor (1 μM), or LMB (10 nM). Cells were infected with influenza virus A/WSN/33 (H1N1) (A) or A/Anhui/1/2013 (H7N9) (B) at an MOI of 3. Cells were fixed and stained for viral NP (green), M2 (red), and nuclei (DAPI; blue) and visualized under a fluorescence microscope. Bars, 200 μm. (C) 293T cells were transfected with a NEP expression plasmid derived from influenza virus A/WSN/33 (H1N1), A/California/04/09 (pH1N1), or A/Anhui/1/2013 (H7N9). At 48 h posttransfection, cells were mock treated or treated with verdinexor (1 μM) for 6 h. Cells were cotransfected with a FLAG-XPO1 plasmid. Cells were harvested for coimmunoprecipitation (IP) with anti-FLAG affinity beads. Ten percent input and eluate from the beads were resolved by SDS-PAGE and subjected to immunoblot (IB) analyses of FLAG-XPO1 and NEP. Densitometry analysis of coimmunoprecipitated NEP bands was performed, and intensities of verdinexor-treated relative to mock-treated bands for each strain are indicated below the blot.

FIG 5

Verdinexor is a slowly reversible inhibitor of XPO1-mediated influenza virus replication in vitro and is bioavailable after oral administration in vivo. (A) A549 cells were mock treated (−) or treated with 1 μM verdinexor for 2 h. Following treatment, the drug-containing medium was removed and replaced with normal growth medium. Cells were incubated for different lengths of time posttreatment, prior to infection with influenza virus A/WSN/33 at an MOI of 0.01. Culture supernatants were collected at 48 hpi for virus titer determination. Data are expressed as the mean ± SE (n = 3). n.s., not significant; **, P < 0.01; ***, P < 0.001 (relative to mock-treated infected cells). (B) For a PK study of p.o. or i.v. administration of verdinexor in mice, verdinexor was given orally by gavage at 10 mg/kg (30 mg/m²) or i.v. at 5 mg/kg (15 mg/m²). Plasma verdinexor concentrations were determined with a validated ultrahigh-performance liquid chromatography–mass spectrometry method, and the mean concentration at each time point postadministration was plotted (n = 3).

FIG 6

Verdinexor reduces lung influenza A virus burdens. The efficacy of in vivo prophylactic (A to C) and therapeutic (D to F) verdinexor treatment was evaluated. (A to C) Mice were treated orally with diluent only or with 10 or 20 mg/kg verdinexor daily for 3 days (3×R_x), 2 days (2×R_x) or 1 day (1×R_x) prior to infection. (D to F) Mice were treated orally at 24, 72, or both 24 and 72 hpi with 50 mg/kg of verdinexor either as a single dose (1×R_x) or as two doses (2×R_x, 25 mg/kg every 12 h). Oral oseltamivir (10 mg/kg/day for 3 days) and diluent-only treatments were used as positive and negative controls, respectively. Mice were infected with 10 MID₅₀ of A/California/04/09 (mouse-adapted pH1N1) (B, E) or A/Philippines/2/82-X79 (H3N2) (C, F). Lungs were collected at 96 hpi and homogenized for virus titer determination in MDCK cells. Treatments were compared to the diluent-only negative-control group by Student's t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (relative to diluent-treated, virus-infected mice).

FIG 7

Verdinexor treatment reduces proinflammatory cytokine expression. (A) A549 cells were mock treated (−) or treated with 1 μM verdinexor (+) for 4 h. Cells were fixed, stained for the NF-κB p65 subunit, and visualized under a fluorescence microscope. Bars, 200 μm. (B to E) Mice were treated orally with diluent only or 20 mg/kg verdinexor daily for 3 days prior to infection with 10 MID₅₀ of mouse-adapted A/California/04/09 (MA-pH1N1). Lungs were collected at 96 hpi, and total RNA was isolated. The expression of Ifng (B), Tnf (C), Il1b (D), and Il6 (E) relative to that of Gapdh was evaluated by RT-qPCR. The data are from four mice per experimental group. *, P ≤ 0.05; **, P ≤ 0.01 (relative to diluent-treated, virus-infected mice).

FIG 8

Verdinexor treatment limits virus spread to the lower regions of the respiratory tract. (A) Experimental design. Mice were mock treated orally with diluent only (−verdinexor; C, E) or treated with 20 mg/kg verdinexor daily for 3 days (+verdinexor; B, D, F) prior to mock infection (B) or infection with 10 LD₅₀ of mouse-adapted A/California/04/09 (MA-pH1N1) (C to F). Lungs were harvested at 48 (C, D) or 96 (B, E, F) hpi, fixed, sectioned, and subjected to IHC with mouse monoclonal antibody to NP, 3,3′-diaminobenzidine chromogen, and hematoxylin counterstain. (B) Lungs from verdinexor-treated but mock-infected mice displayed an absence of viral antigen. (C) At 48 hpi in mock-treated mice infected with influenza virus, there was extensive viral antigen staining of the bronchiolar epithelium, which was sloughing into the lumen. Viral antigen was also observed in the epithelial cells of the alveolar septa. (D) At 48 hpi in verdinexor-treated mice, there was viral antigen staining of the bronchiolar epithelium; however, virus staining was minimal in the interstitium. (E) At 96 hpi in mock-treated mice, much of the bronchiolar wall was ulcerated and there was extensive and intense antigen staining of the bronchiolar epithelium, which had sloughed into the lumen. There was also viral antigen staining of epithelial cells of the alveolar septa and staining of cells in the peribronchiolar inflammation (arrow). (F) At 48 hpi in verdinexor-treated mice, the bronchiolar wall was also ulcerated and there was NP antigen staining of the bronchiolar epithelium; however, viral antigen staining in the alveolar septa was minimal. B, bronchiolar lumen. Bars, 200 μm.

FIG 9

Verdinexor treatment reduces disease pathology associated with a lethal influenza A virus challenge. Mice were orally mock treated with diluent only (−verdinexor; C, E) or treated with 20 mg/kg verdinexor daily for 3 days (+verdinexor; B, D, F) prior to mock infection (B) or infection with 10 LD₅₀ of mouse-adapted A/California/04/09 (MA-pH1N1) (C to F). Lungs were harvested, fixed, sectioned, and stained with H&E at 48 (C, D) and 96 (B, E, F) hpi for histopathology evaluation. (A) Overall lung pathology was scored on a scale of 0 to 4, where a score of 0 is unremarkable pathology; 1 is minimal changes in the bronchiolar epithelium with minimal perivascular inflammation; 2 is mild, multifocal bronchiolar epithelial changes with perivascular and peribronchiolar inflammation; 3 is moderate, multifocal bronchiolar epithelial changes with perivascular, peribronchiolar, and alveolar inflammation; and 4 is marked, diffuse bronchiolar epithelial changes with perivascular, peribronchiolar, and alveolar inflammation. Data are expressed as the mean ± SE of four or five mice per group. *, P ≤ 0.05; **, P ≤ 0.01 (relative to diluent-treated, virus-infected mice). (B to F) Representative H&E staining is shown. (B) Lungs from verdinexor-treated mice that were mock infected did not show any pathological abnormality and thus served as the baseline control group. Bronchioles had no evidence of epithelial apoptosis and had no cells in the lumen, and there were no parenchymal changes. (C) At 48 hpi in influenza virus-infected mice that received diluent-only treatment, there was extensive apoptotic cell death with sloughing of the dead cells into the bronchiolar lumen. Mild peribronchiolar inflammation was present, but the parenchyma had few changes, except for a few histiocytes in alveoli. (D) At 48 hpi in verdinexor-treated mice infected with influenza virus, the bronchiolar epithelium had areas of apoptotic cell death but there were few parenchymal changes. (E) At 96 hpi, mice that received diluent-only treatment displayed extensive cell death of the bronchiolar epithelium with narrowing of the lumen. Severe peribronchiolar and vascular inflammation was present. Extensive areas of parenchymal inflammation obliterated large areas. (F) At 96 hpi, verdinexor-treated mice displayed a level of bronchiolar epithelium cell death; however, only mild peribronchiolar and perivascular inflammation was observed. The parenchyma had few areas of inflammation that obliterate the alveoli. B, bronchioles; P, parenchyma; V, vessel; arrows, peribronchiolar inflammation. Bars, 200 μm.

FIG 10

Verdinexor treatment improves survival following a lethal influenza A virus challenge. Mice were infected with 10 LD₅₀ of mouse-adapted A/California/04/09 (MA-pH1N1). Mice were subsequently treated orally with diluent only as a control group or treated with 10 or 20 mg/kg of verdinexor every other day at 24 and 72 hpi (QOD2). A group of mice were treated with oseltamivir (10 mg/kg daily for 3 days) as a treatment control. Survival was evaluated daily for 14 days in accordance with the guidelines of the IACUC of the University of Georgia. Ten mice per treatment group were used for the survival study, and groups were compared by using the log rank (Mantel-Cox) test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 (relative to the diluent-treated, virus-infected control group); n.s., not significant (relative to the oseltamivir-treated, virus-infected group).