Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-β (adenovirus transforming growth factor-β) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-β-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-β, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

Keywords: Src family kinase; idiopathic pulmonary fibrosis; lung fibrosis; preclinical models; tyrosine kinase.

Figure 1.

Computational drug repurposing identifies saracatinib as a potential therapeutic for idiopathic pulmonary fibrosis (IPF). (A) Schematic of the in silico approach used to identify novel disease indications for compounds. Drug signatures were obtained for 32 compounds in two different cell lines at two dosages. Each drug signature was compared with a library of disease signatures generated from publicly available data, and a connectivity score was generated for each disease–compound pair. Filtering and secondary analyses were performed to identify novel disease indications for each of the compounds. (B) Disease enrichment analysis results show enrichment of Disease Ontology identifier: 50156/IPF signatures among disease signatures that are transcriptomically connected to saracatinib. (C) Connectivity scores between an IPF disease signature and publicly available drug signatures (saracatinib, dasatinib, bosutinib, pirfenidone, nintedanib, and NM-PP1; obtained from LINCS L1000). FDR = false discovery rate; GEO = Gene Expression Omnibus; MDS/PCA = multi-dimensional scaling/principal components analysis; NM-PP1 = PP1 analog II; QC = quality control.

Figure 2.

Saracatinib inhibits TGF-β (transforming growth factor-β)-induced phenotypic changes in human lung fibroblasts (NHLFs). Cells were serum-starved overnight and then incubated with inhibitors (saracatinib 0.3 μM, nintedanib 1 μM, pirfenidone 20 μg/ml, or vehicle [DMSO]) for 60 minutes followed by stimulation with human recombinant TGF-β (2 ng/ml) or vehicle control for the indicated times. (A–C) Quantitative real-time PCR (qRT-PCR) analysis for (A) smooth muscle alpha (α)-2 actin (Acta2), (B) collagen type I alpha 1 chain (Col1a1), and (C) serpin1 or plasminogen activator inhibitor 1(PAI-1) in the indicated treatment groups of NHLF (mean + SEM); *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 6). (D) Representative western blots showing saracatinib inhibition of TGF-β–induced phosphorylation of Smad3 in human lung fibroblasts; data presented as mean + SEM; n = 6; *P < 0.05. (E) Representative images of α-SMA (α smooth muscle actin) staining (red) together with F-Actin (filamentous actin; green) and DAPI (blue) show fluorescent staining in human fibroblasts after TGF-β stimulation in the indicated treatment groups using confocal microscopy. (F and G) Quantification of α-SMA and F-Actin staining shown as integrated density; ***P < 0.001. (H) Volcano plot showing genes that are differentially expressed in cells treated with TGF-β and saracatinib compared with TGF-β alone (false discovery rate [FDR] < 0.05), negative fold change (blue), and positive fold change (red). (I) Functional enrichment of significantly differentially expressed genes (FDR < 0.05) in response to saracatinib (only the top 10 gene sets are shown). All gene sets shown are significant at FDR < 0.05 and are from Hallmark (H) or Kegg (K). AU or au = average intensity; NS = nonsignificant.

Figure 3.

Saracatinib inhibits pulmonary fibrosis in bleomycin and adenovirus TGF-β (transforming growth factor-β) mouse models. (A–H) Evaluation of bleomycin-induced lung fibrosis. (A) Quantitative analysis of hydroxyproline in lung homogenates from indicated groups of mice. Lung collagen content increased significantly in bleomycin-treated mice receiving vehicle control (fold change = 2.9); **P < 0.01 and ***P < 0.001. (B and C) Quantitative real-time PCR analysis on mouse lungs for (B) smooth muscle alpha (α)-2 actin (Acta2) and (C) collagen type I alpha 1 chain (Col1a1) in the indicated treatment groups. (D and F) Representative images and quantitative measurements of Masson’s Trichrome staining of lung sections in the indicated groups of mice. (E and G) Representative images (dorsal view of three-dimensional reconstructions and axial view) and quantifications of micro–computed tomography on mouse lung tissues in the indicated groups. Gross abnormality resulting from bleomycin-induced lung fibrosis is alleviated after treatment. Aerated lung volume measurements showed a significant decrease in bleomycin-treated mouse lungs (P value ⩽ 0.001; fold change > 2), whereas saracatinib and nintedanib significantly attenuated the bleomycin-induced radiographic alterations in the lung parenchyma (P value ⩽ 0.001). (H) Lung compliance measurements of the lungs in the indicated groups are shown as static compliance (Cst). (I–O) Evaluation of Ad-TGF-β (adenovirus transforming growth factor-β)-induced lung fibrosis. (I) Quantitative analysis of hydroxyproline in lung homogenates from indicated groups of mice. The hydroxyproline assay revealed a significant increase in lung collagen content for mice receiving Ad-TGF-β (fold change = 1.8; P value ⩽ 0.001), which is decreased significantly by saracatinib. (J and K) Quantitative real-time PCR analysis on mouse lungs for (J) Acta2 and (K) Col1a1 in the indicated treatment groups. (L and N) Representative images and quantification of Masson’s Trichrome staining of lung sections in the indicated groups of mice. (M and O) Representative images and quantification of α-SMA (α smooth muscle actin) staining of lung sections in the indicated groups of mice. All data are presented as mean + SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 6 in saline and n ⩾ 12 in bleomycin and Ad-TGF-β–treated groups). NS = nonsignificant.

Figure 4.

Saracatinib inhibits pulmonary fibrosis in ex vivo murine precision-cut lung slices (PCLSs) in bleomycin and Ad-TGF-β (adenovirus transforming growth factor-β) models. Mouse PCLSs were generated from both bleomycin (Day 14) and Ad-TGF-β (Day 21) models. Treatment with saracatinib (0.6 μM), nintedanib (1 μM), pirfenidone (1 mM), or vehicle control was administered in the first 24 hours after slicing (time 0 h). All lung slices were isolated 5 days after treatment for analysis (time 120 h). (A–F) Effect of saracatinib, nintedanib, and pirfenidone in PCLSs isolated from the bleomycin mouse model of pulmonary fibrosis. (A and B) Quantitative real-time PCR analysis on mice PCLSs in bleomycin model for smooth muscle alpha (α)-2 actin (Acta2) and collagen type I alpha 1 chain (Col1a1) in the indicated treatment groups. (C and D) Representative live images using second harmonic generation microscopy (SHG) and quantification assessments of PCLS samples at times 0 and 5 days (120 h) after indicated treatments. (E and F) Representative images and quantification assessments of Masson’s Trichrome staining of PCLS slides at times 0 and 5 days (120 h) after indicated treatments. (G–L) Effect of saracatinib, nintedanib, and pirfenidone in PCLSs isolated from Ad-TGF-β–treated mouse models of pulmonary fibrosis. (G and H) Quantitative real-time PCR analysis of mouse PCLSs in the Ad-TGF-β model for Acta2 and Col1a1 in the indicated treatment groups (I and J). Representative live images using SHG and quantification assessments of PCLS samples at times 0 and 5 days (120 h) after indicated treatment. (K and L) Representative images and quantification assessments of Masson’s Trichrome staining of PCLS samples at times 0 and 5 days (120 h) after indicated treatment. All data are presented as (mean + SEM); *P < 0.05, **P < 0.01, and ***P < 0.001 (n ⩾ 6 in all groups). au = average intensity; NS = nonsignificant.

Figure 5.

Saracatinib treatment results in the reversal of transcriptional changes observed in idiopathic pulmonary fibrosis (IPF) mouse models. (A) Comparison of number and direction of significantly differentially expressed (DE) genes (false discovery rate [FDR] < 0.05) in bleomycin and bleomycin/saracatinib treatment groups. Bleomycin administration induced significant differential expression of almost 7,000 genes (adjusted P value < 0.05) in the bleomycin treatment group when compared with the control group. Bleomycin-treated mice that also received saracatinib had significant changes in 2,940 genes compared with mice treated with bleomycin alone. A total of 2,628 differentially expressed genes are common between treatment groups. Of these, 1,689 are upregulated by bleomycin and downregulated by saracatinib, and 938 are downregulated by bleomycin and upregulated by saracatinib. (B) Heatmap of top 100 differentially expressed genes (BS vs. BV). (C) Top Hallmark gene set enrichment analysis (GSEA) in bleomycin murine experiments (adjusted P value < 0.05) ranked by saracatinib effect. Blue bars show BS versus BV and red bars show BV versus SV. The positive or negative signs indicate log fibrotic cocktail (FC) directions. (D) Top Hallmark GSEA in Ad-TGF-β (adenovirus transforming growth factor-β) experiments ranked by saracatinib effect (adjusted P value < 0.05). Blue bars show TS versus TV and red bars show TV versus EV. The positive or negative signs indicate logFC directions. (E) Cytoscape network analysis of common differentially expressed genes shared by both the bleomycin and TGF-β mouse models that are reversed by saracatinib. Node size: expression level; node color: logFC (red is up, blue is down); node border width: negative log adjusted P value. Bleomycin experimental conditions: SV = saline + vehicle; BV = bleomycin + vehicle; BS =  bleomycin + saracatinib; BV versus SV = bleomycin effect; BS versus BV = saracatinib on bleomycin. Ad-TGF-β experimental conditions: EV = control + vehicle; TV = TGF-β + vehicle; TS = TGF-β + saracatinib; TV versus EV = TGF-β effect; TS versus TV = saracatinib on TGF-β. ER = endoplasmic reticulum; GPCR = G protein coupled receptors.

Figure 6.

Saracatinib attenuates pulmonary fibrosis at the level of gene expression and collagen protein accumulation in ex vivo human precision-cut lung slice (hPCLS) models. (A) Schematic view of the experimental design. hPCLSs isolated from patients with idiopathic pulmonary fibrosis (IPF) were cultured and treated with saracatinib (0.6 μM) or vehicle for 5 days; IS (IPF + saracatinib) and IV (IPF + vehicle). In an independent experiment, the hPCLSs harvested from healthy donors were also cultured and treated with fibrotic cocktail (FC), (containing 5 μg TGF-β [transforming growth factor-β], 50 μg PDGF-AB [platelet-derived growth factor-AB], 10 ng TNF-α [tumor necrosis factor-α], and 10 mg lysophosphatidic acid), or control cocktail (CC), with saracatinib or vehicle for 5 days; CCV (control lung + CC + vehicle), CCS (control lung + CC + saracatinib), CFV (control lung + FC + vehicle), and CFS (control lung + FC + saracatinib). (B–D) Representative images and quantitation measurements of Masson’s Trichrome staining on the harvested slides from all groups at the end of the time points. (E–G) Representative images and quantitation measurements of α-SMA (α-smooth muscle actin) staining on the harvested slides from all groups at the end of the time points. (H) Venn diagram of the number of genes differentially expressed after saracatinib treatment in PCLSs from control hPCLSs with FC (CFV vs. CFS) (number of genes = 149) or IPF (IV vs. IS) (number of genes = 60) among all measured genes (number of genes = 761). (I) Heatmap of 149 differentially expressed genes (DEGs) in control hPCLSs with FC (CFV vs. CFS). Genes are ordered from highest to lowest fold change; z-scores are calculated across samples. (J) Heatmap of 60 DEGs in IPF PCLSs (IV vs. IS). Genes are ordered from highest to lowest fold change; z-scores are calculated across samples. (K) Volcano plot of the combined DEG results of both data sets; x-axis = log2 fold change of DEGs between CFS versus CFV; y-axis = log2 fold change of DEGs between IS versus IV. (L) Neutrophil extracellular trap (NET)-associated neutrophil elastase (mU/ml) measured from the phorbol myristate acetate (PMA)-induced neutrophil extracellular traps after 6 hours of incubation with saracatinib or vehicle. All data are presented as mean + SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 (n ⩾ 6 in all groups). CTR = control; NS = nonsignificant.