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. 2023 Feb 28;26(4):106309.
doi: 10.1016/j.isci.2023.106309. eCollection 2023 Apr 21.

FDA-approved Abl/EGFR/PDGFR kinase inhibitors show potent efficacy against pandemic and seasonal influenza A virus infections of human lung explants

Robert Meineke  1 , Sonja Stelz  1 , Maximilian Busch  1 , Christopher Werlein  2 , Mark Kühnel  2   3 , Danny Jonigk  2   3 , Guus F Rimmelzwaan  1 , Husni Elbahesh  1
Affiliations

FDA-approved Abl/EGFR/PDGFR kinase inhibitors show potent efficacy against pandemic and seasonal influenza A virus infections of human lung explants

Robert Meineke et al. iScience. .

Abstract

Influenza viruses (IVs) cause substantial global morbidity and mortality. Given the limited range of licensed antiviral drugs and their reduced efficacy due to resistance mutations, repurposing FDA-approved kinase inhibitors as fast-tracked host-targeted antivirals is an attractive strategy. We identified six FDA-approved non-receptor tyrosine kinase-inhibitors (NRTKIs) as potent inhibitors of viral replication of pandemic and seasonal IVs in vitro. We validated their efficacy in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors and assessed their effect(s) on host responses. Their overlapping targets suggest crosstalk between Abl, EGFR, and PDGFR pathways during IAV infection. Our data and established safety profiles of these NRTKIs provide compelling evidence for further clinical investigations and repurposing as host-targeted influenza antivirals. Moreover, these NRTKIs have broad-spectrum antiviral potential given that their kinase/pathway targets are critical for the replication of many viruses.

Keywords: Cell biology; Virology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
Effect of NRTKI treatment on IAV replication, infectivity, and viability (A) Table 1. Main targets of FDA-approved NRTKIs used in this study. (B) A549 cells were infected with NL09 or NL11 at MOI = 1 +/− indicated NRTKIs at [0.25x, 0.5x, or 1x]max concentration for 72 h. Viral titers were quantified by TCID50/mL assay at 24, 48, and 72 hpi and visualized by Heatmap of fold-change in viral titers relative to DMSO treated (n = 4/condition). Also seeFigure S1. (C and D) A549 cells were infected with NL09 or NL11 at MOI = 1 and incubated for 48 h in the presence of SMKIs ([0.5x]max concentration). Fluorescence microscopy images were acquired from cells stained for infection by anti-IAV NP antibody (red), and nuclei by using NucBlue Live ReadyProbes (blue). Data visualized by Heatmap are % infectivity (C) and % cell viability (D) relative to untreated infected cells or mock-infected treated cells, respectively (n = 4/condition) Also seeFigure S2. Images quantified by ImageJ software. p values were determined by Mann-Whitney tests compared to untreated cells.
Figure 2
Figure 2
MOI-independent effect of NRTKIs on IAV infection A549 cells were infected with NL09 and NL11 at (MOI = 0.1 or 3) and incubated for 72 h in presence of NRTKIs at [0.25x]max (gray) and [0.5x]max (white) concentrations. At 24, 48, and 72 hpi, supernatants were collected and viral titers quantified by TCID50/mL assay (n = 4). L.o.d.: limit of detection. Means ± SD are shown. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. p values were determined by Mann-Whitney tests compared to untreated cells.
Figure 3
Figure 3
NRTKIs inhibit ex vivo IAV infection (A) hPCLS were infected with NL09 and NL11 (104, 105 or 106 TCID50/200ul). Viral titers were quantified by TCID50/mL assay at 2, 16, 24, 48, 72, 96, and 144 hpi (8 donors; n = 24/virus). (B) Heatmap visualization of NRTKI cytotoxicity in hPCLS treated with [1x]max and [10x]max concentration up to 144 h. At each time point, LDH release was measured using LDH-Glo Cell Viability Assay and normalized to DMSO control and relative to 1% Triton X-100 treated cells (positive control) (8 donors/n = 24). (C) hPCLS were infected with NL09 or NL11 (105 TCID50/200ul) and incubated for 120 h with NRTKIs (Defactinib 50uM; Acalabrutinib 5uM; Ibrutinib 5uM; Bosutinib 5uM; Nilotinib 10uM; Saracatinib 0.125uM). Virus was quantified by TCID50/mL assay at 2, 12, 24, 48, 72, and 120 hpi (3 donors; n = 6/condition). (D) NL11 infected hPCLS were fixed 120 hpi and PFA-fixed paraffin-embedded (PFPE) PCLS were cut into 2 μm thick section. H&E staining and viral anti-NP staining (brown) at 10× magnification (Scale bar: 200 um). (E) Semi-quantitative analysis of virus infection (anti-NP staining) was performed for tested NRTKIs and normalized to tissue-area in each section using FIJI image-analysis software. Means ± SEM are shown. l.o.d.: limit of detection. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. Significance (p values) was determined by Mann-Whitney tests compared to untreated cells.
Figure 4
Figure 4
NRTKI inhibition of IAV is stable (A) Stability of SMKI treatment on NL09 and NL11 replication was determined by serial passaging (5 times) using MDCK cells infected at MOI = 0.001 for 72 h in the presence of the [1xmax] NRTKI concentrations (n = 4) at each passage. At each passage, virus titers were quantified to inoculate the next passage at MOI = 0.001 again. Means ± SD are shown. (B) NL09 and NL11 virus stocks were pre-incubated with control (DMSO) or the [1xmax] concentration of respective NRTKI for 4 h at 37°C and A549 cells were then infected using a 1:1000 dilution and incubated for 72 h, after which, virus titers were determined by TCID50/mL assay (n = 3). Means ± SD are shown. p values were determined by Welch t-tests compared to untreated cells; ns, not significant.
Figure 5
Figure 5
NRTKI-specific effects on viral entry A549 cells were pretreated with NRTKIs for 2 h then infected with NL09 or NL11 strains (MOI = 10) for 0.5 h +/− NRTKIs [1xmax]. Cells were fixed and permeabilized and virions detected by anti-NP (green) antibody, F-Actin detected by ActinRed-555 (red), and nuclei detected using NucBlueLive ReadyProbes (blue). Virion localization was visualized by confocal microscopy (n = 2) (Scale bar: 25um).
Figure 6
Figure 6
NRTKIs affect IAV RNA replication (A) A549 cells were transfected with pPOLI-358-FFluc and pmaxGFP plasmids. At 24 hpt, cells were infected with NL09 or NL11 at MOI = 1 +/− indicated NRTKIs at [0.5x or 1x]max concentrations. At 48 hpt (24 hpi), luciferase activity was measured and normalized to GFP expression (MFI). (B) GFP-normalized polymerase activity of untreated NL09-or NL11-infected cells is shown. (C) A549 cells were transfected with pPOLI-358-FFluc and pmaxGFP plasmids and co-transfected with NL09 or NL03-minigenome plasmids. At 6 hpt, NRTKIs were added to the medium. At 30 hpt (24 h of treatment), luciferase activity was measured and normalized to GFP MFI. Bars indicate values relative to untreated cells normalized to GFP. (D) GFP-normalized polymerase activity of untreated NL09 or NL03 minigenome transfected cells is shown. Triplicate measurements from triplicate samples (n = 3); error bars indicate ±standard deviation (SD). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. p-values determined by Brown-Forsythe and Welsh ANOVA compared to untreated.
Figure 7
Figure 7
Effect of NRTKIs treatment on STAT3 and NFkB activation A549 cells were infected with NL09 or NL11 at MOI = 1, treated with NRTKIs at [1xmax] concentration and total proteins isolated from whole cell lysate at 18 and 48 hpi. Immunoblot assay was performed for phospho/total STAT3, IAV-NP and bActin (A), and phospho/total NFkB, IAV-NP and bActin (B). See alsoFigure S3. All measurements were taken from two Western blots from two-independent experiments (n = 2). All values are relative to untreated virus-infected cells. P = phosphor, T = total. Error bars indicate ±standard deviation (SD). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant (p > 0.05). p-values determined by Student’s t test compared to untreated virus-infected cells.
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