Inhibition of Phosphatidylinositol 3-Kinase by Pictilisib Blocks Influenza Virus Propagation in Cells and in Lungs of Infected Mice

Abstract

Influenza virus (IV) infections are considered to cause severe diseases of the respiratory tract. Beyond mild symptoms, the infection can lead to respiratory distress syndrome and multiple organ failure. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. Interestingly, the virus-supportive function of the cellular phosphatidylinositol 3-kinase (PI3K) suggests that this signaling module may be a potential target for antiviral intervention. In the sense of repurposing existing drugs for new indications, we used Pictilisib, a known PI3K inhibitor to investigate its effect on IV infection, in mono-cell-culture studies as well as in a human chip model. Our results indicate that Pictilisib is a potent inhibitor of IV propagation already at early stages of infection. In a murine model of IV pneumonia, the in vitro key findings were verified, showing reduced viral titers as well as inflammatory response in the lung after delivery of Pictilisib. Our data identified Pictilisib as a promising drug candidate for anti-IV therapies that warrant further studying. These results further led to the conclusion that the repurposing of previously approved substances represents a cost-effective and efficient way for development of novel antiviral strategies.

Keywords: Pictilisib; influenza virus; phosphatidylinositol-3 kinase; signaling.

Figure 1

Efficient inhibition of viral replication upon Pictilisib treatment. (a–d) NCl-H441 cells (a,b), A549 cells (c) or Calu-3 cells (d) were pre-incubated with the indicated concentrations of Pictilisib or solvent control (DMSO) for 30 min prior to infection. Afterwards, viral infection was performed with PR8M (H1N1) for 8 h (MOI 0.5) (a), 24 h (MOI 0.01) (b), (MOI 0.5) (d), or SC35M (H7N7; MOI 0.001) for 20 h (c) in the presence of the indicated concentrations of Pictilisib or DMSO. Data represent means + SD of three independent experiments including two (a,b,d) or four (c) biological samples. DMSO-treated cells were arbitrarily set as 100%. Statistical analysis was performed by one-way ANOVA followed by multiple comparisons test. (**** p < 0.0001, *** p < 0.001, ** p < 0.01).

Figure 2

Pictilisib efficiently inhibits IV-induced Akt activation and affects early stages of viral infection. A549 cells were left untreated or pre-incubated with the indicated concentrations of Pictilisib or solvent control (DMSO) for 30 min prior to infection with influenza PR8M (H1N1) (MOI 5) (a) and the recombinant SC35M (H7N7) (b: MOI 5; c: MOI 0.001). After 30 min of infection, viral inoculum was removed, and cells were supplemented with medium containing Pictilisib or DMSO. After the indicated times, cell-lysates were prepared and applied to protein detection by Western blot analysis. Activation of PI3K-mediated signaling was monitored by phosphorylated Akt (Ser473). Equal protein loading of the kinase was verified by detection of ERK1/2. Ongoing viral replication was demonstrated by accumulation of the viral proteins in two (NP, M1) or three (PB1) independent experiments.

Figure 3

Pictilisib treatment does not affect cell viability. NCl-H441 (a), A549 cells (b) and Calu-3 cells (c) were treated with increasing amounts of Pictilisib (1–20 µM) or DMSO in 500 µL of medium with 10% FCS. After the indicated times cells were washed with PBS and detatched with 150 µL/well Trypsin/EDTA. Subsequently, the procedure was stopped by adding 350 µL/well medium with 10% FCS. 20 µL of suspension was supplemented with 20 µL 0.1% Trypan Blue and used for counting. (d) A549 cell were transfected with a constitutive active CMV promoter luciferase plasmid. Eight hours post transfection cells were left untreated or treated with the indicated amounts of Pictilisib or cycloheximide (100 µg mL⁻¹) respectively for 18 h. (d–f) Staurosporine (1 µM) or cycloheximide (100 µg mL⁻¹)-treated cells were used as positive controls. The cleavage of PARP (113 kDa) by Pictilisib was proven by Western blot analysis (e). For this, cells were lysed on ice with RIPA buffer and transferred to SDS-PAGE and afterwards blotted onto a nitrocellulose membrane. The cleaved PARP was visualized by use of a PARP antibody (e). A549 cells (f) were left untreated or pre-incubated with Pictilisib (10 µM) or DMSO for 30 min prior to infection with influenza PR8M (H1N1) (MOI 5). After 30 min of infection, viral inoculum was removed, and cells were supplemented with medium containing Pictilisib or DMSO for 8 h. The mRNA expression of M1 and NS1 was blocked significantly by Pictilisib (f). Data represent means + SD of three independent experiments including four biological samples. DMSO-treated cells were arbitrarily set as 100%. Statistical analysis was performed by one-way ANOVA followed by multiple comparisons test. (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 4

Viral internalization is impaired upon Pictilisib treatment. A549 cells were pre-incubated for 2 h with DMSO or Pictilisib (5 µM or 10 µM) in DMEM and infected with recombinant SC35M or PR8M (MOI 200). Staining was performed with primary antibody against NP. The figure is representative for three independent experiments. White arrows show virus particles at the cell border; the yellow arrow shows a large accumulation of NP in the cytoplasm. Acquired with Axiovert200M, scale bar represents 20 µm

Figure 5

Pictilisib inhibits viral replication in the human chip model. The epithelial and endothelial sites of the human chip model were pre-incubated with 10 µM Pictilisib or solvent control (DMSO) for 30 min prior to infection. PR8M infection (MOI 0.1) was then performed for 30 min on the epithelial site of the model. The chip was incubated with Pictilisib (10 µM) or DMSO to 24 h p.i. (a,b) The cell layers of the epithelial (a) and endothelial chamber (b) were stained using primary antibodies against IV nucleoprotein (a,b), E-cadherin (a) and VE-cadherin (b), Nuclei were stained with Hoechst (a,b). (c) Viral titers were determined by standard plaque assay. The solvent control was arbitrarily set to 100%. Data represents the mean + SD of eight independent experiments. Statistical significance was determined by multiple t-tests (**** p < 0.0001).

Figure 5

Pictilisib inhibits viral replication in the human chip model. The epithelial and endothelial sites of the human chip model were pre-incubated with 10 µM Pictilisib or solvent control (DMSO) for 30 min prior to infection. PR8M infection (MOI 0.1) was then performed for 30 min on the epithelial site of the model. The chip was incubated with Pictilisib (10 µM) or DMSO to 24 h p.i. (a,b) The cell layers of the epithelial (a) and endothelial chamber (b) were stained using primary antibodies against IV nucleoprotein (a,b), E-cadherin (a) and VE-cadherin (b), Nuclei were stained with Hoechst (a,b). (c) Viral titers were determined by standard plaque assay. The solvent control was arbitrarily set to 100%. Data represents the mean + SD of eight independent experiments. Statistical significance was determined by multiple t-tests (**** p < 0.0001).

Figure 6

Pictilisib reduces virus titers and the pro-inflammatory response of IV-infected mice. (a–j) C57BL/6JRj mice were treated with 150 mg kg⁻¹ Pictilisib on day 0 and infected with IV variant HA-G222-mpJena/5258 followed by further Pictilisib treatment on day 1 and 2 after infection. Three days post infection (p.i.) mice were sacrificed to subject blood samples and lung explants. Viral titers (a), body weight of mice (b), and the secreted pro-inflammatory cytokines and chemokines (c–h; IFN-γ, CCL2, CCL5, IP-10, TNF-α, IL-6), Mx1 mRNA expression (i) and number of neutrophil granulocytes (j) are depicted for achievable interpretation of Pictilisib treatment in mice. Statistical analysis was performed by one-way ANOVA followed by multiple comparisons test. (ns = not significant, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).