A new FGFR inhibitor disrupts the TGF‐β1‐induced fibrotic process

Abstract Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti‐fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3‐(2‐chloro‐6‐fluorobenzyl)‐1,6,7‐trimethyl‐1H‐imidazo[2,1‐f]purine‐2,4(3H,8H)‐dione (IM‐1918), markedly inhibited transforming growth factor (TGF)‐β‐stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α‐smooth muscle actin, on human lung fibroblasts. However, IM‐1918 neither decreased Smad‐2 and Smad‐3 nor affected p38MAPK and JNK. Instead, IM‐1918 reduced Akt and extracellular signal‐regulated kinase 1/2 phosphorylation increased by TGF‐β. Additionally, IM‐1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin‐induced murine lung fibrosis model, IM‐1918 profoundly reduced fibrotic areas and decreased collagen and α‐smooth muscle actin accumulation. These results suggest that IM‐1918 can be applied to treat lung fibrosis.

target these fibrotic growth factors and their receptors, such as TGF-β1, 4-6 fibroblast growth factor receptors (FGFR) 1-3, platelet-derived growth factor receptor α and β and vascular endothelial growth factor receptors 1-3, 7 and have received approval from the US Food and Drug Administration in 2014 for treating patients with IPF. 8 While these therapies provide a significant milestone in IPF treatment, they show some limitations and slow disease progression but do not stop or cure the disease. 9 Therefore, targeted therapies for IPF based on the cellular and molecular mechanisms of its pathogenesis are needed.
Other TGF-β-signalling target inhibitors such as fresolimumab (GC-1008) and thalidomide are currently being evaluated in clinical trials. 3,10 TGF-β is a potent pro-fibrotic cytokine for which three isoforms have been identified in mammals: TGF-β1, TGF-β2 and TGF-β3. Among these, TGF-β1 is most closely associated with IPF pathogenesis. 11 During IPF development, secreted TGF-β recruits macrophages and fibroblasts to the wound site and activates fibroblasts. It also provokes the differentiation of fibroblasts to activated myofibroblasts, affecting the production and accumulation of excessive ECM. 11 In contrast, TGF-β1 is also a well-known antiinflammatory and immunosuppressive factor, and thus approaches aimed at inhibiting TGF-β1 for IPF treatment have been attempted with caution. Recent studies demonstrated that the roles of inflammatory cells are less critical than the therapeutic effect of TGF-β1 signalling inhibition, 11,12 promoting continuous efforts to develop new TGF-β signalling inhibitors for treating patients with IPF.
In our previous study, we screened chemical libraries using a TGF-β1-responsive luciferase-reported assay system and isolated the imidazopurine compound IM-412 among several candidates.

| Luciferase reporter assay
For the 3TP-Lux reporter assay, HEK293 cells were transfected with the 3TP-Lux plasmid using Lipofectamine 2000 (Invitrogen). The cells were treated with IM-1918 2 hours prior to adding human recombinant TGF-β1 (1 ng/mL) and incubated for 24 hours. Luciferase activity was assessed using a microplate reader (Wallac Victor, Perkin-Elmer). Relative luciferase activity was normalized against cell viability.

| Western blot analysis
To analyse total proteins, cells or lung tissues were lysed with RIPA buffer (50 mM Tris-Cl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin and 1 μg/mL leupeptin) and phosphatase inhibitors (1 mM Na 3 VO 4 and 1 mM NaF). Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). After blocking non-specific antibody sites, the membranes were probed overnight at 4°C with primary antibodies. The membranes were incubated with peroxidase-conjugated secondary antibodies, and immunoreactive bands were visualized by enhanced chemiluminescence reagents (GE Healthcare). The experiments were repeated at least three times.

| Bleomycin-induced pulmonary fibrosis mouse model
Six-week-old male C57BL/6 mice were purchased from Dae-Han

| Histological analysis and immunohistochemical staining
The lung tissues were fixed in formalin for 24 hours and embedded in paraffin (Hayashi Pure Chemical Industries). Left lung tissue sections (3 µm) were cut and stained with haematoxylin and eosin or Masson's trichrome for histopathological examination and evaluation of collagen accumulation. The dewaxed sections were exposed to 3% H 2 O 2 for 10 minutes to block endogenous peroxidase activity, followed by incubation with primary antibody against α-SMA for 60 minutes at room temperature using a Cap-Plus kit (Zymed) according to the manufacturer's protocol. The sections were then incubated with the biotinylated secondary antibody for 40 minutes, and streptavidin conjugate successively for 30 minutes at room temperature. After three washes (5 minutes each) with PBS-Tween 20, the slides were exposed to diaminobenzidine solution and counterstained with Mayer's haematoxylin. The sections were mounted in Permount (Thermo Fisher Scientific), and images were obtained using a microscope.

| Data analysis
Data are represented as means ± SD. Significant differences between groups were determined by analysis of variance and Tukey's post hoc comparisons using GraphPad software version 5. Statistical significance was defined as P-values < .05.

| Imidazopurine derivative IM-1918 reduces TGF-β-induced fibrotic process
To develop a novel inhibitor of TGF-β-mediated fibrosis, we previously performed cell-based screening of chemical libraries using a reporter assay of a 3TP-Lux construct stably transfected into HEK293 cells. 13 Among the active candidate compounds, the imidazopurine derivative IM-1918 was identified to be active and inhibit the TGF-β-induced response ( Figure 1A,B). IM-1918 dose-dependently inhibited TGF-β-mediated luciferase activity (IC 50 = 4.28 μM).
To determine whether IM-1918 inhibits TGF-β1-induced cellular responses in lung fibroblast cells, the effect of IM-1918 on cell viability was measured. As shown in Figure 1C, the viability of CCD18-Lu cells was weakly decreased by IM-1918, and the concentration at which 50% inhibition of cell growth occurred (GI 50 ) was 43.58 μM.
The GI 50 values of IM-1918 for IMR-90 and WI-38 cells could not be determined. TGF-β treatment did not affect cell viability of CCD18-Lu, and the cell viability-induced by IM-1918 was not altered in the presence or absence of TGF-β ( Figure S1A,B). In addition, IM-1918 was not shown to be cytotoxic in lung fibroblasts ( Figure 1C). Next, the expression of fibrosis-associated TGF-β1 target molecules was determined. The levels of CTGF, fibronectin, pro-collagen I and α-SMA expression were remarkably increased by TGF-β1 treatment, whereas IM-1918 significantly decreased these proteins in a dose dependent manner ( Figure 1D). These data indicate that IM-1918 effectively reduced the TGF-β1-induced fibrotic process without causing cytotoxicity.
To clarify the inhibitory activity of IM-1918 towards target proteins, an in vitro kinase assay was performed (Table 1). Interestingly, most kinases tested showed no significant inhibition by IM-1918, and only seven kinases were slightly inhibited by approximately 10%.

| IM-1918 inhibits basic fibroblast growth factor (bFGF)-mediated fibrotic process
Because FGF signalling has been implicated in the pathogenesis of PF and co-operatively cross-talks with TGF-β1, 20 -1918). B, HEK293 cells were transiently transfected with 3TP-Lux reporter gene and then seeded into 96-well plates. After 24 h, IM-1918 was added 2 h prior to adding human recombinant TGF-β1 (1 ng/mL) and incubated for 24 h. Data shown are the means ± SD of three independent experiments. † † † P < .001 vs control, **P < .01 and ***P < .001 vs TGF-β1-alone treatment. C, MTT and Lactate dehydrogenase (LDH) assays were performed to determine cell viability (circles, left y-axis) and cytotoxicity (triangles, right y-axis), respectively. Cells were seeded at 8 × 10 3 cells/well into 24-well plates and treated with the indicated concentrations of IM-1918 for 24 h. Data shown are the means ± SD of three independent experiments. D, CCD18-Lu cells were treated with the indicated dose of IM-1918. Two hours later, TGF-β1 (1 ng/mL) was added, and the cells were incubated for 24 h. Total protein was isolated and analysed by Western blotting for the indicated proteins F I G U R E 2 IM-1918 inhibits TGF-β-induced fibrotic response via Erk1/2 and AKT down-regulation. CCD18-Lu cells were treated with the indicated dose of IM-1918. Two hours later, TGF-β1 (1 ng/mL) was added, and the cells were incubated for 24 h. Total protein was isolated and analysed by Western blotting for the indicated proteins. A, Smad signalling. B, Non-Smad signalling. C, FGFR

| Inhibition of FGFR3 disrupts TGF-β1-mediated fibrotic process
To verify that inhibition of FGFR3 blocks TGF-β1-induced fibrotic activity, pharmacologic inhibition of FGFR3 using AZD4547 treatment or knockdown of FGFR3 by short interfering RNA genetic inactivation was performed. AZD4547, a well-known pan-FGFR inhibitor, slightly suppressed the phosphorylation of FGFR3 at the doses tested in this study. Moreover, AZD4547 did not alter fi-

| IM-1918 attenuates BLM-induced murine pulmonary fibrosis
To investigate the efficacy of IM-1918 on fibrosis in animals, a murine BLM-induced lung fibrosis model was used. Immunohistochemical analysis revealed that BLM-induced accumulation of collagen, and α-SMA was markedly inhibited by IM-1918 ( Figure 5A). Furthermore,

| D ISCUSS I ON
TGF-β is a major causative factor of symptoms of fibrotic diseases and multifunctional cytokine that plays various roles in the body. 12 In this study, the novel compound IM-1918 effectively suppressed the expression of fibrosis-related molecules up-regulated by TGF-β1 Several studies have demonstrated that administration of TGF-β1 changes the sensitivities of FGFRs and activates the production F I G U R E 4 Knockdown of FGFR3 inhibits the expression of TGF-β1-induced fibrosis-associated molecules. CCD18-Lu cells were treated with 10 μM of AZD4547 for 2 h, following treatment with TGF-β1 (1 ng/mL) for 5 h. Total protein was isolated and analysed by Western blotting for the indicated proteins. A, Signalling molecules. C, Fibrotic markers. CCD18-Lu cells were transfected with scramble RNA or siFGFR3, and then incubated for 24 h. TGF-β1 (1 ng/mL) was added to the cells, and the cells were incubated for an additional 5 h. Total protein was isolated and analysed by Western blotting for the indicated proteins. B, Signalling molecules. D, Fibrotic markers of FGF-2 in primary human lung fibroblasts. [25][26][27] FGFs are associated with the pathogenesis of PF, and FGF2-neutralizing antibodies successfully inhibit the TGF-β1-mediated fibrotic process. 20 It has been also reported that FGF-2 and FGFR1IIIc are involved in EMT and advanced cancer progression, which may be regulated by TGF-β1 autonomously secreted from cancer cells. 25,28 Moreover, non-selective inhibition of RTKs and non-specific inhibition of FGFRs decrease BLM-induced PF in rodents. 7,29 Altered expression of FGFR1 and FGF1 proteins was observed in the lungs of patients with IPF, 30 and a specific FGFR1 inhibitor (NP603) attenuated carbon tetrachloride-induced hepatic fibrosis in rats. 31 In addition to FGF, epidermal growth factor, which acts through RTKs, can synergize with TGF-β signalling to increase collagen accumulation and interstitial fibrosis, 32,33 suggesting that a co-operative network exists between RTKs and TGF-β1. In agreement with these ob-  Band intensities corresponding to the indicated proteins were quantified by densitometry using ImageJ software, normalized to β-actin or the total form of each protein, and expressed as the fold-change compared to each control. Data were considered significant at *P < .05, **P < .01, ***P < .001 vs control; † P < .05, † † P < .01, † † † P < .001 vs BLM In summary, we demonstrated that the interplay between TGF-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. F I G U R E 6 Schematic model for anti-fibrotic activity of IM-1918. TGF-β is a well-known mediator that promotes the fibrotic process via Smad-dependent and Smad-independent pathways. Treatment with bFGF also induced activation of fibrotic molecules, in cooperation with TGF-β1. IM-1918 prevents phosphorylation and activation of FGFR3, leading to attenuation of the fibrotic process in vitro and in vivo