Repurposing Kinase Inhibitors as Antiviral Agents to Control Influenza A Virus Replication

Abstract

Influenza A virus (IAV) infection causes seasonal epidemics of contagious respiratory illness that causes substantial morbidity and some mortality. Regular vaccination is the principal strategy for controlling influenza virus, although vaccine efficacy is variable. IAV antiviral drugs are available; however, substantial drug resistance has developed to two of the four currently FDA-approved antiviral drugs. Thus, new therapeutic approaches are being sought to reduce the burden of influenza-related disease. A high-throughput screen using a human kinase inhibitor library was performed targeting an emerging IAV strain (H7N9) in A549 cells. The inhibitor library contained 273 structurally diverse, active cell permeable kinase inhibitors with known bioactivity and safety profiles, many of which are at advanced stages of clinical development. The current study shows that treatment of human A549 cells with kinase inhibitors dinaciclib, flavopiridol, or PIK-75 exhibits potent antiviral activity against H7N9 IAV as well as other IAV strains. Thus, targeting host kinases can provide a broad-spectrum therapeutic approach against IAV. These findings provide a path forward for repurposing existing kinase inhibitors safely as potential antivirals, particularly those that can be tested in vivo and ultimately for clinical use.

Fig. 1.

Dose-dependent antiviral efficacy of dinaciclib, flavopiridol, and PIK-75 against influenza H7N9 virus. A549 cells were pretreated with increasing dose (¼-log₁₀ increments) of dinaciclib, flavopiridol, or PIK-75 for 2 h at 37°C. Cells were then infected with influenza A/Anhui/1/13 (H7N9) at MOI of 0.01 in the presence of TPCK-trypsin without removal of inhibitors. At 48 hpi, cells were fixed and stained for viral NP and counterstained with DAPI. (A) Representative immunofluorescence images acquired by Cellomics high-content imaging. (B–D) A percentage of infected cells were quantified using the Cellomics system. IC₅₀ values were determined using the nonlinear regression method in GraphPad Prism and summarized in Table 1. (E–G) Culture supernatants were also collected for virus titration in MDCK cells to determine inhibitors' ability to reduce infectious virus titers. Data are expressed as the mean±SE (n=3). *P<0.05; **P<0.01; ***P<0.001 compared to DMSO-treated cells.

Fig. 2.

Dose-dependent antiviral efficacy of dinaciclib, flavopiridol, and PIK-75 against pandemic H1N1 virus. A549 cells were pretreated with an increasing dose (¼-log₁₀ increments) of dinaciclib, flavopiridol, or PIK-75 for 2 h. Cells were then infected with influenza A/California/04/09 (pdmH1N1) at MOI of 0.1 in the presence of TPCK-trypsin without removal of inhibitors. At 48 hpi, cells were fixed and stained for viral NP and counterstained with DAPI. (A) Representative immunofluorescence images acquired by Cellomics high-content imaging. (B–D) A percentage of infected cells were quantified using the Cellomics system. IC₅₀ values were determined using the nonlinear regression method in GraphPad Prism and summarized in Table 1. (E–G) Culture supernatants were also collected for virus titration in MDCK cells to determine inhibitors' ability to reduce infectious virus titers. Data are expressed as the mean±SE (n=3). *P<0.05; **P<0.01; ***P<0.001 compared to DMSO-treated cells.

Fig. 3.

Dose-dependent antiviral efficacy of dinaciclib, flavopiridol, and PIK-75 against an influenza H3N2 virus. A549 cells were pretreated with an increasing dose (¼-log₁₀ increments) of dinaciclib, flavopiridol, or PIK-75 for 2 h. Cells were then infected with influenza A/Philippines/2/82-X79 (H3N2) at MOI of 0.1 in the presence of TPCK-trypsin without removal of inhibitors. At 24 hpi, cells were fixed and stained for viral NP and counterstained with DAPI. (A) Representative immunofluorescence images acquired by Cellomics high-content imaging. (B–D) A percentage of infected cells were quantified with the Cellomics system. IC₅₀ values were determined using the nonlinear regression method in GraphPad Prism and summarized in Table 1. (E–G) Culture supernatants were also collected for virus titration in MDCK cells to determine inhibitors' ability to reduce infectious virus titers. Data are expressed as the mean±SE (n=3). *P<0.05; ***P<0.001 compared to DMSO-treated cells.

Fig. 4.

Synergistic antiviral effect of kinase inhibitors. (A, B) A549 cells were treated with flavopiridol alone (A; top row), dinaciclib alone (A; first column), or a combination of the two drugs from 0.01 to 1 μM for 2 h. (C, D) Cells were treated with PIK-75 alone (C; top row), dinaciclib alone (C; first column), or a combination of the two drugs from 0.01 to 1 μM for 2 h. Cells were then infected with influenza A/California/04/09 (pdmH1N1) at MOI of 0.1 in the presence of TPCK-trypsin without removal of drugs. At 48 hpi, cells were fixed and immunostained for viral NP and counterstained with DAPI. (A, C) Representative immunofluorescence images acquired by Cellomics high-content imaging. (B, D) A percentage of infected cells were quantified with the Cellomics system from two independent experiments and the values were used to compute synergism (or antagonism) volume using the MacSynergyII algorithm. Horizontal plane (arrow; add.) represents the theoretical additive effect of the drugs. Above-the-plane volume indicates synergism, while below-the-plane volume indicates antagonism. Synergism and antagonism volumes are summarized.

Fig. 5.

Synergistic antiviral effect of kinase inhibitor with oseltamivir. A549 cells were treated with DMSO or 0.1, 1, or 10 μg/mL of oseltamivir phosphate in combination with DMSO control, dinaciclib (A, B), flavopiridol (C, D), or PIK-75 (E, F) at 0.01, 0.1, or 1 μM for 2 h. Cells were then infected with influenza A/California/04/09 (pdmH1N1) at MOI of 0.1 in the presence of TPCK-trypsin without removal of drugs. At 48 hpi, virus from the supernatant was titered in MDCK cells. Virus titers from three independent experiments were used to compute synergism (or antagonism) volume using the MacSynergyII algorithm. Virus titer results are expressed as the mean (n=3). *P<0.05; **P<0.01; ***P<0.001 compared to DMSO-treated cells.

Fig. 6.

Activity of dinaciclib, flavopiridol, and PIK-75 against oseltamivir-resistant virus. A549 cells were treated with DMSO only, oseltamivir phosphate at 10 μg/mL, dinaciclib, flavopiridol, or PIK-75 at 5 μM for 2 h. Cells were then infected with influenza A/Mississippi/3/01 (H1N1) wild type (wt) (A) or H274Y mutant (B) at MOI of 0.1 in the presence of TPCK-trypsin without removal of drugs. At 48 hpi, virus from the supernatant was titered in MDCK cells. Virus titers are expressed as the mean±SE (n=4). *P<0.05; ***P<0.001; n.s. not significant compared to DMSO-treated cells.