A novel multikinase inhibitor SKLB‐YTH‐60 ameliorates inflammation and fibrosis in bleomycin‐induced lung fibrosis mouse models

Abstract Objectives Idiopathic pulmonary fibrosis (IPF) is marked by the excessive accumulation of extracellular matrix, which participates in a variety of chronic diseases or injuries and seriously threatens human health. Due to the side effects of clinical drugs, there is still a need to develop novel and less toxic drugs to treat pulmonary fibrosis. Materials and Methods SKLB‐YTH‐60 was developed through computer‐aided drug design, de novo synthesis and high‐throughput screening. We employed the bleomycin (BLM)‐induced lung fibrosis animal models and used TGF‐β1 to induce the epithelial‐mesenchymal transition (EMT) of A549 cells in vitro. Meanwhile, the protein expression of collagen I and the α‐smooth muscle actin (α‐SMA), E‐cadherin, p‐FGFR1, p‐PLCγ, p‐Smad2/3 and p‐Erk1/2 was detected by western blot. Results YTH‐60 has obvious anti‐proliferative activity on fibroblasts and A549 cells. Moreover, YTH‐60 could impair the EMT of A549 cells and suppressed fibrosis by inhibiting FGFR and TGF‐β/Smad‐dependent pathways. Intraperitoneal administration of preventive YTH‐60 could significantly reduce the degree of fibrosis in mice and regulate the imbalance of the immune microenvironment. In addition, we observed that therapeutic YTH‐60 treatment attenuated fibrotic changes in mice during the period of fibrosis. Importantly, YTH‐60 has shown an acceptable oral bioavailability (F = 17.86%) and appropriate eliminated half‐life time (T 1/2 = 8.03 hours). Conclusions Taken together, these preclinical evaluations suggested that YTH‐60 could be a promising drug candidate for treating IPF.


| INTRODUC TI ON
Idiopathic pulmonary fibrosis (IPF) is a chronic and lethal lung disease associated with fibroblast activation, myoblast proliferation and extracellular matrix (ECM) deposition. 1 Although the mechanism is unclear, most studies have shown that the increased proliferation of fibroblasts is the most direct cause of fibrosis. 2 Currently, the complex aetiology and the not accurate diagnosis of pulmonary fibrosis, leading to the treatment progress is slow. Gene therapy is a potential treatment; however, successfully applying it in clinical practice has been a formidable challenge. 3,4 Only two novel anti-fibrotic therapies approved by the FDA, a pyridinone derivative pirfenidone and a multi-target tyrosine kinase inhibitor Nintedanib. 5,6 However, due to adverse reactions and limited efficacy of current drugs, it is still far from satisfaction in protecting against pulmonary fibrosis. 7,8 Other target inhibitors of TGFβ-signalling that are currently being evaluated in clinical trials such as fresolimumab (GC-1008) and thalidomide. 9 There is still a need for developing new and less toxic drugs to treat pulmonary fibrosis. Therefore, our research group has been interested in the design, synthesis and biological evaluation of novel multi-kinase inhibitors as potential new anti-fibrosis agents.
Myofibroblasts are generally considered to be important effector cells in the development of fibrosis, 10 which are the main source of collagen I and the characteristic is expression of α-smooth muscle actin (α-SMA). Many mediators and growth factors have been involved in the process of lung fibrosis. 11,12 Among these molecules, the transforming growth factor β 1(TGFβ 1 ) is an important profibrotic factor in lung fibrosis and wound healing can markedly cause the differentiation of fibroblasts into myofibroblasts and induces EMT in alveolar epithelial cells (AECs). 13 TGFβ 1 can elevate the expression of fibrosis marker α-SMA which promoter harbours Smad binding elements, which specifically bind Smad3. 14 In addition, Studies have shown that the FGFR signalling pathway is involved in the pathogenesis of IPF and cooperatively cross-talks with TGFβ 1 . 15 After the two receptor molecules form a dimer on the membrane, the tails of the intracellular domains contact each other to activate their protein kinases to autophosphorylate the tyrosine residues in the tails, leading to the activation of downstream signals Erk and PLCγ. The FGFR signalling pathway takes an important part in tissue repair, embryogenesis and wound healing. 16 Furthermore, multiple studies have shown that TGFβ 1 could change the sensitivity of FGFR in human primary lung fibroblasts. 17,18 Much of the research indicated that FGF-2 and FGFR1IIIc are involved in EMT. 19 Antibodies that neutralize FGF2 successfully inhibited the fibrosis process mediated by TGFβ 1 . 15 In short, FGF pathway could be an ideal target for anti-fibrotic therapeutic approaches.
Our goal was to develop a new multi-kinase inhibitor that could potently block the FGF/FGFR signalling cascade with low toxicity.
Considering the anti-fibrosis potential of tyrosine kinase inhibitor, we independently developed a series of compounds. Among these compounds, YTH-60 showed outstanding kinase inhibitory activity, including FGFR1, FGFR2, FGFR3, Abl and VEGFRs. This compound has also shown anti-proliferative activities against a panel of fibroblast cells. In this study, we used the clinical drug Nintedanib as a positive control and evaluated the activity of YTH-60 in inhibiting lung fibrosis in vitro and in vivo. Moreover, further mechanistic studies revealed that YTH-60 ameliorated fibrosis by inhibiting both FGFR and TGFβ/Smad-dependent pathways. Taken together, these preclinical evaluations suggest that YTH-60 could be a promising drug candidate for the treatment of fibrotic diseases.

| Synthesis and preparation of the compound YTH-60
acetamide was synthesized by our team and the structural formula is shown in Figure 1A and Figure  as an internal standard. The purities of all final compounds were greater than 95%.

| Drugs and regents
Cell count kit-8 (CCK-8) was purchased from MedChemExpress. MTT and DMSO were purchased from Sigma. TGFβ 1 and FGF-basic were purchased from Novoprotein. PEG 400 was purchased from Sigma.
Collagenase Type IV was purchased from GIBCO. Bleomycin sulphate was purchased from Chengdu Synguider Technology Co., Ltd.

| Molecular modelling
The molecular docking studies were accomplished by using GOLD 5.0. The crystal structure of FGFR1 (PDB ID: 5UR1) was acquired from the RCSB Protein Data Bank and chosen as the structure of the reference protein. The molecular structures of compounds were constructed YTH-60 using Chemdraw software and saved as SDF format files.

| Determination of thermodynamic solubility
Weigh about 1 mg of the test compound into 1.5 mL centrifuge tube and add 0.5 mL various solvents, sonicating for 5 minutes (if the compound is completely dissolved, then added again test compound until a little solid remains). Take the supernatant after centrifugation, diluting to a suitable concentration using and measuring absorbance values by UV spectrophotometer. Weigh 10 mg compound accurately of the test compound into 1.5 mL centrifuge tube and add 1.2 mL methanol solution to prepare 8.3 mg·mL −1 mother solution. Take the above With concentration c (mg·mL −1 ) as the abscissa, absorbance A as the ordinate, mapping and fitting line to get the standard curve (The correlation coefficient should be greater than 0.998). Calculate solubility according to the standard curve of the linear equation.

| Kinase profile assay and IC 50 test
The in vitro kinase enzymatic inhibition assays were carried out by the Kinase Profiling Services provided by Eurofins (UK). The detailed protocol descriptions can be provided by the website (https://www. eurof insdi scove ryser vices.com).

| Cell lines and cell culture
The human alveolar epithelial A549 cells were purchased from (v/v) and 1% antibiotics (penicillin and streptomycin) in 5% CO 2 at 37°C.

| Cell viability assay
The cell viability of YTH-60 and Nintedanib treatment was performed by MTT and CCK8 assay. Briefly, the exponentially growing cells were seeded in 96-well (2-5 × 10 3 cells·well −1 ) and cultured for F I G U R E 1 A, Chemical structure of YTH-60. B, Docking of YTH-60 into FGFR1 kinase X-ray crystal structure in the three-dimensional structure (PDB ID:5UR1). C, A two-dimensional interaction map of YTH-60 and FGFR1

| Western blot analysis
Samples of cells and mouse lung tissues were lysed in RIPA buffer with a protease and phosphatase inhibitors (Selleckchem). The protein concentrations in the cell lysate were measured by the Lowry method.
Approximately 50 μg of total protein was separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience). After the membranes incubation with the specific primary and secondary antibodies, the reactive bands were detected with enhanced chemiluminescent substrate to horseradish peroxidase.

| Immunofluorescence assay
NIH-3T3 and HPF cells were cultured in 24-well plates, and treated with YTH-60 (5 μmol·L −1 ) and TGFβ 1 (5 ng·mL −1 ) for 24 hours. The cells were successively washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 20 minutes at room temperature, blocked for 30 minutes with 5% BSA, and then incubated with specific primary antibodies overnight at 4°C. After a wash step, the cells were incubated with specific secondary antibodies for 1.5 hours at room temperature. Then, wash the sample with PBS, drip antifluorescence quencher, and use LSM 510 laser confocal microscope to acquire images (Zeiss confocal microscope).

| Quantitative real-time PCR (qPCR)
Total RNA of lung tissues or cells was extracted by using AxyPrep Multisource Total RNA Miniprep Kit (Axygen). Reverse transcription was carried out with Evo M-MLV RT Kit with gDNA Clean (Accurate, AG11711). qRTPCR was conducted using SYBR Green Pro Taq HS (Accurate, AG11701). The PCR primers used were synthesized by Genewiz and listed in Table S1.

| BLM-induced mouse models of pulmonary fibrosis
Male C57BL/6 mice (8-10 weeks old) were purchased from HFK Bioscience Co. Ltd. All animal experiments were permitted by the Institutional Animal Care and Treatment Committee of Sichuan University in China (New Permit Number: 20190524003). Lung fibrosis was induced using BLM treatment (2 mg·kg −1 ). All C57BL/6 mice were divided into five groups (n = 10): a BLM + Nintedanib group (Nintedanib, 30 mg·kg −1 ), a BLM + YTH-60 group (YTH-60, 15 mg·kg −1 and 30 mg·kg −1 ), a control group (Sham), and a bleomycin group (Vehicle). At the beginning, the mice were anaesthetized using 10% chloral hydrate, dissolved BLM with saline and administered intratracheally. To evaluate the preventive effect, the next day after BLM challenge, YTH-60 and Nintedanib were administered intraperitoneally daily for 13 days (fibroproliferation) to the mice. All mice were sacrificed 24 hours after the last drug injection, the lungs and other organs were taken for the subsequent analysis. To test the therapeutic effect, after the 7th day of BLM treatment, mice were injected intraperitoneally with YTH-60, Nintedanib and vehicle daily for 7 consecutive days (fibroproliferation).

| Bronchoalveolar lavage fluid
After the animals were anaesthetized, rinsing the lungs with 1 mL of normal saline three times and collected bronchoalveolar lavage fluid.
The total living cell count was performed by an ADVIA 2120i.

| Histopathological staining
The left lung was detached and immediately fixed in 4% paraformaldehyde for 72 hours and then embed with paraffin wax. A microtome was used to prepare paraffin sections (3-5 μm), after deparaffinized the tissue sections were stained with H&E and Masson's trichrome.
Ashcroft scoring was used to examine the severity of pulmonary fibrosis. 20 The image analysis-based system to quantify collagen deposition was described previously. 21

| Flow cytometry
We analysed the percentage of MDSCs and macrophages by Flow cytometry (FCM). 22 Use scissors to cut approximately 30 mg of mouse lung tissues into small pieces and use 5 mL of collagenase-IV with a concentration of 1 mg·mL −1 , digested at 37°C for at least

| Pharmacokinetics study
The appropriate amount of YTH-60 was accurately weighed, added to the final volume of 5% DMSO, 40% PEG400 and 55% normal saline, and vortexed or ultrasonicated to fully mix. Then, 0.4 and 2 mg·mL −1 of clarified dosing solution was used for intravenous administration and oral gavage administration. SD rats were trans- After the blood samples were collected, they were placed on ice and centrifuged to separate plasma within 2 hours (centrifugation conditions: centrifugal force 6800 g, 6 minutes, 2-8°C). The collected plasma samples were stored at −80°C before analysis by LC-MS/ MS-18. After analysis, the remaining plasma samples were stored at −80°C for a period of 1 month.

| Statistical analysis
All in vitro studies were performed at least three times. Results are expressed as mean ± SD and P-values were determined by two-tailed Student's t test for comparison of two groups. Statistically significant P values were considered at: *P < .05, **P < .01 and ***P < .001; # P < .05, ## <0.01 and ### P < .001.

| Design, synthesis, screening, kinase inhibition profile and molecular modelling studies of YTH-60
A total of 550 novel multi-kinase compounds were designed through computer aided drug design (CADD). There are 55 lead compounds ranked in the top 10% according to Ludi Energy Estimates, and preliminary screening has been carried out through the kinase inhibition test (date not shown). Among these, (E)-N-(4-(2-(6-(2,6-dichloro-  Figure 1A and Figure S1. To clarify the inhibitory effects of YTH-60 on multiple proteins, an in vitro kinase assay was performed (Table 1). YTH-60 robustly inhib-  ALA564, LEU630 and ALA640. LYS514 of YTH-60 forms an electrostatic force. The above results speculate that YTH-60 could bind with protein stably. These results shown that YTH-60 was a potent multi-target inhibitor especially targeting FGFR1. In addition, YTH-60 has better solubility than Nintedanib (Table 2). Therefore, we chose Nintedanib as a positive control to evaluate the efficacy of YTH-60.

| YTH-60 inhibits fibroblast activation and proliferation
To

| YTH-60 exhibited anti-fibrotic activity by inhibiting FGFR and TGFβ/Smad pathways
In order to further evaluate the cellular activity of YTH-60 targeting FGFR kinase, we analysed its effects on the phosphorylation of FGFR, PLCγ and Erk. As shown in Figure 3A,B, 10 μmol·L −1 YTH-60 inhibited the phosphorylation of FGFR1 in fibroblasts. Like the inhibition of FGFR1 phosphorylation, the phosphorylation of PLCγ and Erk was also inhibited. Although the expression of nonphosphorylated FGFR1, PLCγ and Erk1/2 is also reduced, the effect on phosphorylation is more obvious. In order to further verify the FGFR pathway, we stimulated fibroblasts with FGF2 for 1 and 24 hours in vitro. As shown in Figure 3C,D, the expression of downstream protein p-ERK1/2 increased after activation of the FGFR pathway, while YTH-60 could reduce its expression. These results indicate that YTH-60 shows effective blocking of FGFRs signalling.
In order to further study the target molecule of YTH-60 in the signal pathway mediated by TGFβ 1 , we found the phosphorylation of

| YTH-60 blocks TGFβ 1 -induced EMT
Next, we want to further explore whether YTH-60 could inhibit the EMT. First, we noticed that YTH-60 suppressed the viability of A549 cells ( Figure S2C,D). Subsequently, the morphological changes of A549 cells induced by TGF-β1 were observed from ovoid epithelial cells to spindle-shaped fibroblasts cells ( Figure   4A). Compared with the control A549 cells, the cell migration ac-  Figure 4I).

| Pharmacokinetic study of YTH-60 after IV and PO administration to SD rats
To evaluate the efficacy of YTH-60 in animal models, we first examined the pharmacokinetic profile of YTH-60. Blood concentrationtime curve of the compound after oral administration and intravenous infusion are shown in Figure 5A,B. After an IV dose, the mean halflife was 4.92 hours, and the mean AUC( 0-t ) was 772.62 h·ng·mL −1 .
The mean bioavailability was 17.86% for 20 mg·kg −1 YTH-60 by oral administration. The mean half-life was 8.03 hours, and the mean AUC( 0-t ) was 1379.73 h·ng·mL −1 (Table 3 and Table S2). While, the mean bioavailability was 11.9% for 50 mg·kg −1 Nintedanib by oral administration. 23 This result suggests that YTH-60 might be a potential drug candidate with low toxicity and has better absorption than Nintedanib.

| YTH-60 prevents BLM-induced pulmonary fibrosis
To investigate the anti-fibrosis effect of YTH-60 in vivo, mice were These data initially suggest that YTH-60 has a certain protective effect on pulmonary fibrosis. Next, we further investigated the anti-fibrotic effect of YTH-60. We detected the levels of endogenous α-SMA, collagen I and TGFβ 1 mRNA in the lung tissues of BLM-treated mice. YTH-60 could reduce the expression of these indicators ( Figure 6F-H). Immunohistochemistry also showed that YTH-60 significantly decreased the expression of α-SMA, collagen I and p-FGFR1 ( Figure 6I). In addition, western blot analysis further confirmed these results ( Figure 6J).

| YTH-60 has anti-inflammatory effects in BLMinduced lung fibrosis
Because BLM could cause inflammatory cells to enter the lungs in the early stages of inflammation, 24

| YTH-60 exerts anti-fibrotic effect in BLMinduced lung fibrosis
It is not enough to administer the drug before the 7th day of the acute bleomycin model and only test the anti-fibrotic compounds, so many researchers have recommend evaluating the anti-fibrotic characteristic of any new target drugs pre-clinical investigational drugs, especially when inflammation subsides. 28 Here, we injected bleomycin (2 mg·mL −1 ) into mice, and then injected YTH-60 intraperitoneally from 7th to 14th day ( Figure 9A,B) after BLM instillation. Between the vehicle group and the YTH-60 group, the administration of YTH-60 on the 7th day of F I G U R E 4 YTH-60 blocks TGF-β1 induced EMT in A549 cells. A, The morphological changes were imaged using phase contrast microscopy. B, A549 cells were seeded in a 6-well plate for 24 h, then starved with serum-free medium for 6 h, and the medium was replaced with 5 ng·mL −1 TGF-β1 fresh medium. A scratch was made using a sterile 200 μL micropipette tip. One hour later, 5 μmol·L −1 YTH-60 was added. After 24 h, the cells were photographed by a microscope (Scale bar = 100 μm). C, Healing rate (%) = (wound area at 24 h/wound area at 0 h) × 100% (Scale bar = 100 μm). Data are presented as mean ± SD, n = 3, *P < .05; **P < .01 and ***P < .001, Scale bar = 100 μm. D, E, Quantitative real-time qPCR analysis was used to measure the mRNA levels of E-cadherin and TGF-β1 in A549 cells stimulated with TGF-β1 followed by 5 μmol·L −1 YTH-60 administration for 24 h. F, Expression changes of α-SMA, E-cadherin and vimentin using western blot in A549 cells. G, H, Analysed by densitometry. Data are presented as mean ± SD, n = 3, *P < .05; **P < 0.01 and ***P < .001. I, The levels of E-cadherin and α-SMA were evaluated by immunofluorescence experiment. Scale bar = 100 μm treatment caused significant differences in the fibrosis Ashcroft score ( Figure 9A-D). Therefore, the data show that YTH-60 can not only prevent pulmonary fibrosis in the early stage of inflammation, but also has a certain therapeutic effect in the period of fibrosis. to the lack of clear markers to assess the interstitial phenotype. 34,35 Bleomycin can induce lung damage, and subsequent fibrotic changes that similar to pathological characteristics of human pulmonary fibrosis. 28  In the field of tissue damage and inflammation, MDSCs and macrophages are essential for controlling the immune response.

| D ISCUSS I ON
Alveolar macrophages are a significant source of TGFβ 1 . 32 There is evidence suggesting that MDSCs accumulation is a result of chronic inflammation and the cytokines produced as a result of this inflammation. 33 Our findings also suggested that YTH-60 significantly reduced the number of macrophages and MDSCs in mouse lung tissue.
Our findings also suggested that YTH-60 attenuates collagen accumulation in lung tissue, which may be a follow-up result of inhibiting lung inflammation and lung injury. This may partly explain why inflammation in the lungs is reduced. Therefore, inhibiting leukocyte recruitment directly affects inflammation and tissue repair, which may partially explain the preventive effects of YTH-60 on BLM-induced pulmonary fibrosis. Therefore, we proved that YTH-60 effectively improves pulmonary fibrosis, in part because YTH-60 reduces the expression and activation of some inflammatory cytokines and fibrotic cytokines, thereby reducing multiple interactions between them. We also demonstrated that the anti-fibrotic effects of YTH-60 may be due to several mechanisms, including EMT, FGFR and TGFβ/Smad signalling pathway. In summary, in two models of pulmonary fibrosis, our findings suggest a potential treatment for pulmonary fibrosis and this compound is expected to provide a clinical strategy for idiopathic pulmonary fibrosis in the future.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.