Application of compound T4015 as inhibitor in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention provides application of a compound T4015 as an inhibitor in preparation of a medicine for treating pulmonary fibrosis, and belongs to the technical field of compound medicine. According to the invention, a bioactive compound T4015 capable of effectively inhibiting JAK / STAT and NF-kappa B signal channels is identified on the basis of a high-throughput and high-throughput screening system of STAT and NF-kappa B. Experiments prove that T4015 can effectively inhibit activation of JAK / STAT and NF-kappa B signal channels so as to cope with various immunogenicity stimuli and cell factors. Subsequently, transcriptome analysis comprehensively reveals that T4015 can significantly inhibit LPS-induced inflammation and activation of immune-related signal pathways. IPA comparative analysis further shows that the anti-inflammatory mechanism of the T4015 is related to JAK / STAT and NF-kappa B signal pathways, and the T4015 has the potential of treating pulmonary fibrosis. Besides, in a bleomycin-induced pulmonary fibrosis model, the T4015 significantly reduces mRNA expression of Il6 and Ccl2 in lung tissues and expression of fibrosis-related protein COl1, can relieve symptoms of BLM-induced pulmonary fibrosis, and is a promising candidate for treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical compounds, and in particular relates to the use of a compound T4015 as an inhibitor in the preparation of a drug for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a chronic interstitial lung disease characterized by scarring and decreased lung function. Chronic inflammatory diseases are particularly prone to progression to fibrosis, which is characterized by excessive inflammatory responses and cytokine release. Idiopathic pulmonary fibrosis (IPF) is the most common type of idiopathic interstitial pneumonia, characterized by inflammation and extensive lung remodeling leading to excessive deposition of extracellular matrix. In addition, its pathological features include alveolar epithelial cell damage, inflammatory cell infiltration, fibroblast proliferation, and extracellular matrix deposition. In the early stages, most patients are not diagnosed until the middle and late stages, resulting in high mortality. Currently, there are limited available options for modern drugs, and there is no effective treatment for IPF. The only drugs that have been clinically approved for the treatment of IPF are pirfenidone and nintedanib. Pirfenidone is a potent cytokine inhibitor that can regulate and inhibit the production of transforming growth factor β (TGF-β) and other inflammatory mediators. Nintedanib is considered a tyrosine kinase inhibitor that can slow the progression of pulmonary fibrosis. However, both drugs have varying degrees of side effects related to liver damage and cannot prolong patient survival. Therefore, finding safe and effective drugs to treat pulmonary fibrosis is a pressing issue in clinical management.

[0003] The JAK family of tyrosine kinases consists of four members: JAK1, JAK2, JAK3, and TYK2. In the JAK family, JAK1 is primarily responsible for mediating signal transduction of γc receptor cytokines and proinflammatory cytokines. JAK2 primarily affects the production of blood and bone marrow cells, while JAK3 significantly affects lymphocyte differentiation. TYK2 is important for both innate and adaptive immune responses. When cytokines, growth factors, hormones, and other signaling molecules bind to dimeric receptors associated with the JAK family, this triggers the phosphorylation of the JAK complex. In turn, phosphorylated JAK proteins activate the phosphorylation and dimerization of downstream STAT proteins, which play a crucial role in regulating the transcriptional activation of target genes. Currently, JAK inhibitors are frequently used to treat inflammatory and autoimmune diseases such as rheumatoid arthritis (RA), ulcerative colitis (UC), and psoriatic arthritis. In recent years, researchers have discovered pathological and physiological links between JAK / STAT signaling and various fibrotic diseases affecting the liver, kidneys, heart, bone marrow, and lungs. JAK inhibitors have been approved for the treatment of myelofibrosis, suggesting that the JAK / STAT pathway may also be a promising target for addressing fibrosis in other organs. It has been reported that the JAK / STAT signaling pathway is involved in the process of pulmonary fibrosis. For example, under the stimulation of IL-6, STAT3 regulates the differentiation of lung fibroblasts into myofibroblasts. In addition, in IPF patients, p-STAT3 is highly expressed in lung tissue, mainly in alveolar macrophages, alveolar epithelial cells, and fibroblasts.

[0004] Likewise, NF-κB transcription factors help induce the expression of pro-inflammatory genes and promote the synthesis of cytokines, adhesion molecules, chemokines, growth factors, and enzymes. In the NF-κB signaling pathway, cytokine stimulation triggers the phosphorylation of IKK kinase, which in turn phosphorylates downstream IκB proteins that bind to NF-κB. Phosphorylated IκB is subsequently degraded by the proteasome, releasing NF-κB to translocate into the nucleus and regulate gene expression. NF-κB has been extensively studied in various lung diseases characterized by inflammation, including severe sepsis, acute lung injury, acute respiratory distress syndrome, and cystic fibrosis. It is highly expressed during the onset of pulmonary fibrosis and is closely related to the pathogenesis of various lung diseases such as occupational lung disease and acute lung injury. Current studies have shown that inhibition of the NF-κB signaling pathway can alleviate the progression of pulmonary fibrosis.

[0005] Inflammation is an important defense mechanism of the body that can prevent microbial invasion and repair damaged tissues, and maintain the normal function of tissues and organs by resisting various injuries and infections. However, excessive inflammatory responses can lead to a variety of inflammatory-related diseases, posing a major threat to human health. Both the JAK / STAT and NF-κB signaling pathways are key intracellular pathways that can mediate the secretion of inflammatory cytokines and aggravate inflammatory responses. The JAK / STAT and NF-κB signaling pathways are indeed involved in complex and frequent cross-communication. They share a variety of common upstream regulators and downstream target genes. The target genes of JAK / STAT and NF-κB are involved in the activation of each other's signaling pathways as well as their own positive feedback loops. In addition, in the inflammatory and fibrotic processes of pulmonary fibrosis, the mutual interference between the JAK / STAT and NF-κB pathways still exists. They can jointly regulate the expression of inflammatory cytokines and chemokines, the recruitment of proinflammatory cells, and the activation of fibroblasts, thereby aggravating pulmonary fibrosis. According to current studies, abnormal activation of these two pathways can be observed in the process of pulmonary fibrosis, and blocking these two pathways can inhibit the progression of fibrosis. Therefore, dual targeting of JAK / STAT and NF-κB may be a promising therapeutic strategy for the treatment of inflammatory diseases, especially pulmonary fibrosis. Summary of the invention

[0006] In order to achieve the above object, the present invention provides a use of a compound T4015 as an inhibitor in the preparation of a medicament for treating inflammatory diseases and / or immune diseases.

[0007] Preferably, the CAS number of the compound T4015 is 1356962-34-9.

[0008] Preferably, compound T4015 exerts its inhibitory effect by inhibiting the activation of JAK / STAT and / or NF-κB signaling pathways.

[0009] Preferably, compound T4015 inhibits IL6-induced STAT3 phosphorylation, IKK phosphorylation and IκBα degradation in a concentration-dependent manner.

[0010] Preferably, compound T4015 exerts anti-inflammatory effects by inhibiting the expression of LPS-induced inflammatory cytokines and chemokines.

[0011] Preferably, the inflammatory cytokines and chemokines are at least one selected from TNFα, IFNβ, IL-17α, IL-6 and CXCL2.

[0012] Preferably, compound T4015 alleviates the symptoms of BLM-induced pulmonary fibrosis by reducing the mRNA expression of Il6 and Ccl2 and the expression of fibrosis-related protein COl1 in lung tissue.

[0013] Preferably, the inflammatory disease and / or immune disease is at least one selected from pulmonary fibrosis, pulmonary inflammation, interstitial lung disease and fibrosis, and sepsis.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are:

[0015] The present invention is based on a high-throughput high-throughput screening system for STAT and NF-κB, and identifies a bioactive compound T4015 that effectively inhibits the JAK / STAT and NF-κB signaling pathways. Based on experimental evidence, T4015 can effectively inhibit the activation of the JAK / STAT and NF-κB signaling pathways in response to various immunogenic stimuli and cytokines, including IL-6-induced STAT3 phosphorylation, LPS-induced p-IKK activation, and IFNβ-induced JAK1 and TYK2 phosphorylation. Subsequently, transcriptome analysis comprehensively revealed that T4015 can significantly inhibit LPS-induced inflammation and activation of immune-related signaling pathways. IPA comparative analysis further showed that the anti-inflammatory mechanism of T4015 is related to the JAK / STAT and NF-κB signaling pathways, and has the potential to treat pulmonary fibrosis. In addition, in the bleomycin-induced pulmonary fibrosis model, T4015 significantly reduced the mRNA expression of Il6 and Ccl2 in lung tissue and the expression of fibrosis-related protein COl1, and can alleviate the symptoms of BLM-induced pulmonary fibrosis. In addition, target capture and virtual docking analysis confirmed that the JAK / STAT and NF-κB pathways were the main targets of T4015. Taken together, these data suggest that T4015, as a dual-target inhibitor of JAK / STAT and NF-κB, is a promising candidate for the treatment of pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The workflow of high-throughput screening provided in the embodiment of the present invention is as follows: A549 cells are transfected with plasmids carrying STAT and NF-κB binding elements to establish the SKA-II system; then, the cells are seeded in a 96-well plate and incubated overnight, and then treated with compounds to start the screening process;

[0017] Figure 2 T4015 provided in the embodiments of the present invention was identified as a JAK / STAT and NF-κB signaling pathway inhibitor, wherein (A) the preliminary screening results of 7200 compounds (20 μM) using the JAK / STAT and NF-κB dual-target luciferase reporter system, the preliminary screening results are shown in the Log2FC of the treatment / vector luciferase activity; (B) the compound structure, molecular weight and canonical smiles of T4015;

[0018] (C) Drug properties of T4015 were obtained by SwissADME platform analysis. mol_MW, molecular weight; DonorHB, number of hydrogen bond donors; AccptHB, number of hydrogen bond acceptors; QPlogPo / w, Log of octanol / water partition coefficient; QPlogS, logarithm of water solubility; QPPCaco, Caco-2 permeability; QPlogBB, blood-brain barrier penetration; (DI) Peritoneal macrophages were exposed to 2.5, 5, 10, and 15 μM DMSO or T4015 for 2 h, and then stimulated with 5 ng / ml IL-6 (D), 50 ng / ml IFN-β (E) and IFN-γ (F, I) for 10 min, 20 ng / ml TNF-α for 10 min (G), or 100 ng / ml LPS for 0.5 h (h). Whole cell lysates were subjected to Western blot analysis and quantitative analysis using primary antibodies including anti-pTyr705-STAT3 and anti-STAT3 (D), anti-pTyr690-STAT2 and anti-STAT2 (E), anti-pTyr701-STAT1 and anti-STAT1 (F), anti-pSer176 / 180-IKK-α / β, anti-IκBα and anti-IKK-α (G, H), anti-pTyr1022 / 102-JAK1, anti-JAK1, anti-pTyr1054 / 1055-TYK2 and anti-TYK2 (I); (JO) Peritoneal macrophages were pretreated with DMSO or T4015 (10 μM) for 0.5 h and then stimulated with 150 ng / ml LPS for 0, 0.5, 3 and 6 h. The relative mRNA levels of TNF-α (J), IFN-γ (K), IL17α (L), CCL5 (M), CXCL10 (N), and CCL2 (O) were determined by RT-PCR;

[0019] Figure 3 T4015 provided in the embodiments of the present invention inhibits the relative mRNA expression of proinflammatory cytokines in mouse primary peritoneal macrophages induced by LPS (AE) Peritoneal macrophages were pretreated with DMSO or T4015 (10 μM) for 30 minutes and then stimulated with 150 ng / ml LPS for 0, 0.5, 3 and 6 hours; the relative mRNA levels of IL1β (A), CCL4 (B), IFN-β (L), IL1α (D) and IL6 (E) were determined by RT-PCR;

[0020] Figure 4The anti-inflammatory effect of T4015 provided in the embodiment of the present invention, wherein (A) transcriptome sequencing flow chart; (B) statistical graph of DEGs between NC, LPS, T4015 and LPS_T4015 groups; (C) heat map of typical pro-inflammatory factors in each group; (D) KEGG analysis of the top 20 category terms of up-regulated DEGs between LPS group and NC group; (E) KEGG analysis of the top 20 category terms of down-regulated DEGs between LPS_T4015 and LPS group; (FG) GSEA enrichment analysis of gene clusters down-regulated in LPS_T4015 group compared with LPS group; (H) Veen graph of DEGs up-regulated in LPS vs NC and down-regulated in LPS_T4015 vs LPS; (I) PPI network of 214 genes shared in (H); the size of the circle indicates the degree of correlation, blue indicates down-regulation, and the depth of the color indicates the Log2FC size;

[0021] Figure 5 The transcriptome analysis provided in the embodiment of the present invention illustrates the anti-inflammatory mechanism of T4015, including (A) PCA analysis of samples in the NC, LPS, T4015 and LPS_T4015 groups; (B) Gene hierarchical clustering heat map of each sample; (C) In the IPA analysis, the upstream regulatory factors IFNγ and IRF3 related network downregulated by LPS_T4015 vs LPS; (D) IPA analysis shows that the upstream regulatory factors IκBκB and NF-κB related network downregulated by LPS_T4015 vs LPS; (E) Trend analysis of genes in the NC group, LPS group and LPS_T4015 group. Profile 5 represents the gene clusters upregulated in the LPS group and downregulated in the LPS_T4015 group; (F) The top 20 category terms of the KEGG analysis of Profile 5 genes; (G) The top 20 category terms of the GO enrichment analysis of Profile 5 genes;

[0022] Figure 6 Comparative analysis of the anti-inflammatory effect of T4015 using IPA provided in the embodiment of the present invention, wherein by comparing the significant differences between LPS_T4015 and LPS and LPS and NC, typical pathway comparison analysis (A), upstream regulatory factor comparison analysis (B), biological function comparison analysis (C) and disease comparison analysis (D) were performed; Positive z scores indicate predicted activation, while negative z scores indicate predicted inhibition; (EF) Disease and functional networks with reduced disease terms in LPS_T4015 compared with LPS, including lung inflammation (E), interstitial lung disease and fibrosis (F); Red indicates upregulated genes, green indicates downregulated genes, and the depth of color indicates the Log2FC size;

[0023] Figure 7The therapeutic effect of T4015 provided in the embodiment of the present invention on BLM-induced pulmonary fibrosis in mice, wherein (A) the survival rate of NC, BLM and BLM+T4015 groups after administration. (BC) The survival rate (B) and lung index (C) of mice in NC, BLM and BLM+T4015 groups; (D) Daily body weight statistics. (EF) HE staining and Masson staining of lung tissues in NC, BLM and BLM+T4015 groups. The boxed area in 40x HE staining shows the tracheal structure, the arrow indicates the thickening of the lung septum, and the boxed area in 200x HE staining indicates the alveolar structure (E); the boxed area in 40x Masson staining circles the collagen deposition (F); (GH) The relative mRNA expression of Il6 (G) and Ccl2 (H) in lung tissue was determined by RT-PCR; (I) Western blot was performed on the lung tissue lysate of each group;

[0024] Figure 8 The target fishing and molecular docking of T4015 and its structural analogs provided in the embodiments of the present invention, wherein (A) a Venn diagram of the reverse target fishing results of T4015 using PharmMapper and GalaxySagittarius; (B) a Venn diagram of T4015 and protein JAK1 using AutoDock Vina 1.1.2 software

[0025] (C), TYK2 (D), EGFR (E), RIPK1 (F), IRAK1 (G) and TAB1 (H) were molecularly docked. The molecular docking binding energy statistics are shown in (B); (I) Heat map of the inhibitory activity of T4015 and its analogs on the JAK / STAT and NF-κB dual-target luciferase reporter system at 5μM and 10μM concentrations. Hydrogen bonds, hydrophobic interactions, salt bridges, π-πstacking interactions and halogen bonds are represented by yellow, blue, red, green and purple, respectively;

[0026] Fig. 9 The signal pathway of the reverse targeting result provided in the embodiment of the present invention and the molecular docking result of T4015, wherein (A) JAK / STAT signal pathway diagram; (B) NF-κB signal pathway diagram; (CF) molecular docking results of T4015 with proteins JAK2 (C), JAK3 (D), ZAP70 (E) and IRAK4 (F) using AutoDock Vina 1.1.2 software;

[0027] Fig.10The compound structures of T4015 analogs provided in the embodiments of the present invention, wherein (A) the compound structure of T4015 analogs with EGFR inhibitory activity; (B) the compound structure of T4015 analogs with no reported EGFR inhibitory activity. The color of the compound number background represents the inhibitory activity of the compound on the reporter system, red indicates activation, blue indicates inhibition, and the compounds in the black frame do not show any inhibitory activity. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Materials and methods used in the following examples:

[0030] Antibodies and reagents

[0031] The primary antibodies used in this study are listed in Table 1. Cell lysis buffer (Cat.9803) was from Cell Signaling Technology. Recombinant mouse IFN-β protein (Cat.12400-1), IL6 protein (Cat.216-16), and IFN-γ protein (Cat.315-05) were purchased from PeproTech, and LPS (Cat.916374) was purchased from Sigma. All compounds and libraries were purchased from TargetMol.

[0032] Table 1 Antibody list

[0033]

[0034]

[0035] Luciferase reporter assay

[0036] SKA-II cells (8000 / well) cultured in RPMI 1640 medium were seeded into a white 96-well plate (Corning, Cat. CLS3610-48EA) and incubated in an incubator containing 5% CO. 2 The cells were incubated overnight at 37°C in an incubator. The cells were then treated with the indicated concentrations of solvent or compound for 24 hours. Luciferase activity was measured using a Promega luciferase kit (Cat. E2510) and stained with Spectra L microplate reader (Molecular Devices) was used for detection.

[0037] Western Blot

[0038] Primary peritoneal macrophages were exposed to T4015 at a concentration of 0, 2.5, 5, 10 and 15 μM for 2 hours, and then stimulated with 20 ng / ml IL-6, 50 ng / ml IFN-β / INF-γ or 20 ng / ml TNF-α for 10 minutes, or stimulated with 100 ng / ml LPS for 0.5 hours. The cells were then washed with pre-cooled PBS and collected in a cell lysis buffer containing 1% protease and phosphatase inhibitors A / B. The lysate supernatant containing 20 ug of protein was separated by SDS-PAGE electrophoresis and transferred to a nitrocellulose (NC) membrane (GE Healthcare, Cat. 10600034). After blocking with 5% skim milk solution (10 mMTris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) in TBST for 1.5 hours, the membrane was incubated with a primary antibody at 4 ° C overnight. After washing three times with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours at room temperature. The immune complexes were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Cat. WBKLS0500) and captured using a Tanon 5200 imaging system.

[0039] Real-time fluorescent quantitative polymerase chain reaction

[0040] Total RNA was extracted from cultured cells or mouse tissues using RNAiso Plus (TaKaRa, Cat.9109). Genomic DNA was removed and reverse transcribed using the PrimeScript RT Kit (Roche, Cat.RR037A). The StepOne Plus Real-Time PCR System (Applied Biosystems) and FastStart Universal Green Master (Roche, Cat. 04913914001) was used to amplify cDNA samples. The primer sequences for RT-PCR are shown in Table 2.

[0041] Table 2 PCR primer sequences

[0042]

[0043]

[0044] animal

[0045] Eight-week-old female C57BL / 6J mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.), weighing approximately 24-28 g, were housed under SPF animal research conditions (50 ± 10% relative humidity, 24 ± 2 °C, 12 h light / dark cycle). Mice were adapted to laboratory conditions for at least 1 week and received free access to food and water. All animal experiments, including euthanasia, were performed in accordance with protocols approved by the Experimental Animal Committee of Qingdao Institute of Marine Biomedical Sciences.

[0046] Isolation and culture of primary mouse peritoneal macrophages

[0047] C57BL / 6J mice were intraperitoneally injected with 1 ml of BBL Thioglycollate Medium Brewer Modified buffer. After 72 hours, peritoneal macrophages were extracted with pre-cooled PBS. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco), 100 IU / ml penicillin and 100 mg / ml streptomycin.

[0048] Bleomycin-induced pulmonary fibrosis mouse model

[0049] The animals were randomly divided into three groups: control group, bleomycin-induced pulmonary fibrosis group, and T4015-treated group. Pulmonary fibrosis was induced by a single oropharyngeal instillation of 50 μL BLM (T6116, Topscience) at a concentration of 5 mg / kg on day 0 under anesthesia induced by 2,2,2-tribromoethanol (T48402, Sigma-Aldrich). Mice in the control group received an equal volume of PBS solution. Mice in the T4015 group received T4015 orally every other day. The body weight and mortality of mice were monitored daily throughout the experiment. After 25 days, the mice were sacrificed and lung tissues were collected for further analysis and measurement.

[0050] Morphological and histological analysis

[0051] On day 25, the lungs of mice were collected for histological examination. The lungs were fixed in 4% paraformaldehyde (P1110, Solarbio) at room temperature for more than 48 hours. After paraffin embedding and sectioning, hematoxylin and eosin (H&E) and Masson staining were performed according to routine histopathological evaluation methods. Images at 40X and 200X were analyzed to assess the extent of lung inflammation and fibrosis. Masson trichrome staining was quantitatively analyzed using image processing software Image J.

[0052] Molecular docking

[0053] AutoDock Vina 1.1.2 software was used for molecular docking of T4015 with proteins JAK1 (Uniprot ID: P23458), TYK2 (Uniprot ID: P29597), EGFR (Uniprot ID: Q01279), JAK2 (Uniprot ID: Q62120), JAK3 (Uniprot ID: Q62137), IRAK1 (Uniprot ID: P51617), IRAK4 (Uniprot ID: Q9NWZ3), RIPK1 (Uniprot ID: Q13546), TAB1 (Uniprot ID: Q15750), and ZAP70 (Uniprot ID: P43404). Protein preparation was performed using PyMOL 2.4, including the removal of water molecules and irrelevant ligands, and the addition of hydrogen atoms. AutoDock Tools 1.5.6 was used to generate PDBQT files for docking simulations. The docking box was set to wrap the entire protein structure. Other parameters were kept at default values. The docking results were set to output 9 docking positions. The docking conformation with the lowest binding energy and the highest clustering frequency was considered to be the most potential binding mode between the ligand and the protein. Finally, we used PLIP and Pymol 2.4 software to visualize the docking results.

[0054] Transcriptome sequencing

[0055] Mouse peritoneal macrophages were divided into four groups: a control group (NC) treated with an equal volume of DMSO, a T4015-treated group (T4015) treated with 10 μM T4015 for 2 hours, an LPS-treated group (LPS) stimulated with 150 ng / ml LPS for 0.5 hours, and a T4015 pre-treated group (LPS_T4015) treated with a combination of LPS and T4015. Total RNA of each sample was extracted from the cells using RNAiso Plus. RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using ND-2000 (NanoDrop Technologies). Sequencing libraries were performed on the NovaSeq X Plus platform (PE150). The reference genome sequence version was Mus_musculus, GRCm39. The differential analysis software was DESeq2, and the screening threshold was |log2FC|>=2, padjust<0.05.

[0056] Statistical analysis

[0057] All histograms and line graphs were prepared using GraphPad Prism 9.5. Results are expressed as mean ± SD. Significant differences between data groups were analyzed using unpaired Student's t-test or one-way ANOVA. Significant differences are indicated by *p < 0.05.

[0058] Example 1 T4015 inhibits JAK / STAT and NF-kB signaling activation in primary mouse peritoneal macrophages

[0059] In previous studies, we constructed the JAK / STAT and NF-κB dual-target luciferase reporter system SKA-II based on STAT and NF-κB elements, and established a systematic high-throughput screening system ( Figure 1 ). In this study, we screened 7200 compounds from the TargetMol compound library, including novel, clinical and marketed compounds. Through inhibitory potency (|log2FC|>1) and literature search, we obtained a 4-(3-indolyl)-2-phenylaminopyrimidine structure derivative T4015 with JAK / STAT / NF-κB inhibitory activity, CAS: 1356962-34-9 ( Figure 2 AB). Then, we used the SwissADME platform to calculate the physicochemical properties and pharmacokinetics of T4015. The parameters of Lipinski's five laws are as follows: MW = 532.087; LogP = 4.816; the number of hydrogen bond acceptors is 8.25; the number of hydrogen bond donors is 2 ( Figure 2 C), indicating that T4015 has drug-like potential.

[0060] The JAK / STAT and NF-κB pathways are key hubs for activating and maintaining inflammatory and immune responses. To determine the inhibitory effects of T4015 on the JAK / STAT and NF-κB signaling pathways, we investigated the effects of T4015 on the activation of the JAK / STAT and NF-κB signaling pathways in primary mouse peritoneal macrophages induced by various immune stimulants and cytokines.

[0061] The results showed that T4015 could inhibit the phosphorylation of STAT3 under IL6 stimulation in a dose-dependent manner (0, 2.5, 5, 10 and 15 μM) and more significantly after 5 μM, and showed a significant inhibitory effect at a concentration of 5 μM ( Figure 2 D). In addition, T4015 can effectively inhibit IFNβ-induced STAT2 activation and IFNγ-induced STAT1 activation ( Figure 2EF). In addition, for the NF-κB signaling pathway activated by LPS and TNFα, T4015 also showed concentration (0, 2.5, 5, 10, and 15 μM)-dependent inhibition of IKK phosphorylation and IκBα degradation, with more obvious inhibition after 5 μM ( Figure 2 GH).

[0062] To further explore the effect of T4015 on upstream target proteins of the JAK / STAT pathway, we examined the effect of T4015 on IFNβ-stimulated JAK1 and TYK2 phosphorylation. We found that T4015 had an inhibitory effect on the phosphorylation of both JAK1 and TYK2 ( Figure 2 I). JAK / STAT and NF-κB signaling pathways can regulate inflammation and immune responses individually or synergistically.

[0063] To further verify the anti-inflammatory properties of T4015, we also studied the effect of T4015 on the expression of inflammatory cytokines in LPS-stimulated peritoneal macrophages. The results showed that T4015 inhibited the expression of multiple inflammatory cytokines and chemokines induced by LPS, including TNFα, IFNβ, IL-17α, IL-6, and CXCL2 ( Figure 2 JO, Figure 3 AE). These findings suggest that T4015 may exert anti-inflammatory effects by inhibiting the JAK / STAT and NF-κB pathways, including the expression of their upstream key kinases and downstream target genes, in response to the body's immune system response.

[0064] Example 2 Transcriptome sequencing comprehensively demonstrates the anti-inflammatory effect of T4015 on peritoneal macrophages

[0065] To fully understand the anti-inflammatory activity of T4015, we performed transcriptome sequencing on peritoneal macrophages treated with DMSO, LPS, T4015, and LPS+T015 (LPS_T4015). Figure 4 A). The PCA diagram visualizes the clustering of each group of samples ( Figure 5 A), heat map showing the expression profile of sample genes ( Figure 5 B). These data indicate that T4015 treatment modulates LPS-induced gene expression in peritoneal macrophages. LPS induced upregulation of 547 differentially expressed genes (DEGs) and downregulation of 316 DEGs. Compared with the NC group, the LPS_T4015 group upregulated 358 DEGs and downregulated 545 DEGs ( Figure 4 B). T4015 treatment reduced the expression of many DEGs associated with LPS-induced inflammatory pathways, such as JAK / STAT-related IFIT3, IFIT1, and inflammatory cytokines such as IL1β and IL6 ( Figure 4 C) Figure 4As shown in D, the top 20 KEGG pathways enriched in the up-regulated DEGs in the LPS group and the NC group included many pro-inflammatory and immune activation pathways, such as NF-kappa B signaling pathway, IL-17signaling pathway, JAK-STAT signaling pathway, TNF signaling pathway, etc. Secondly, the top 20 KEGG pathways in the KEGG enrichment analysis of the down-regulated DEGs in the LPS_T4015 group and the LPS group included JAK-STAT signaling pathway, TNF signalingpathway, IL-17signaling pathway, Toll-like receptor signaling pathway and other inflammation and immune-related pathways ( Figure 4 E). In order to more comprehensively elucidate the regulatory effect of T4015 on gene function, we used GSEA enrichment to analyze the gene sets of LPS_T4015 and LPS groups and found that the JAK / STAT-related IL6 pathway and NF-κB-mediated TNFα signaling showed a down-regulation trend ( Figure 4 FG). In addition, IPA software analysis obtained the network diagram of the upstream regulatory factors IRF3 and IFN-γ downregulated in the LPS_T4015 group and the LPS group ( Figure 5 C), and the NF-κB and IkB-α / β network diagram ( Figure 5 D).

[0066] LPS can stimulate innate immune system cells through pathogen-associated molecular patterns (PAMPs), thereby activating immune responses and releasing inflammatory mediators. To investigate the effect of T4015 on LPS-upregulated DEGs, we compared the downregulated DEGs in the LPS_T4015 group with the upregulated DEGs in the LPS group. The results showed that T4015 pretreatment downregulated 214 LPS-induced DEGs ( Figure 4 H). Proteins related to the JAK / STAT pathway, including STAT1, IFIT2, and IFIT3, as well as proinflammatory cytokines such as IL6 and IFNβ, were located at the center of the network of these 214 DEGs ( Figure 4 I). Subsequently, we performed trend analysis on the NC group, LPS group, and LPS_T4015 group ( Figure 5E), and a profile 5 with 903 genes upregulated in the LPS group and downregulated in the LPS_T4014 group was obtained. The top 20 KEGG pathways enriched in Profile 5 genes included inflammation and immune-related pathways such as JAK / STAT, NF-κB, and Toll-like receptor signaling pathways ( Figure 5 F). In addition, GO enrichment analysis of profile 5 showed that the down-regulated trend genes were significantly enriched in many signaling pathways related to immune response and stimulation response ( Figure 5 G). These results indicate that T4015 can inhibit LPS-induced pro-inflammatory responses and immune-related signaling pathways in peritoneal macrophages.

[0067] Example 3 Comparative analysis of the anti-inflammatory effect of T4015 using IPA software

[0068] In order to further systematically elucidate the anti-inflammatory effect and biological mechanism of T4015, we used IPA software to conduct comparative analysis of LPS vs NC and LPS_T4015 vs LPS. Figure 6 A), T4015 pretreatment significantly inhibited the activation of various inflammation-related and oxidative stress-related signaling pathways induced by LPS, such as NF-κBActivation by Viruses, iNOS Signaling, IL-6Signaling, and IL-17Signaling. Comparative analysis of upstream regulatory factors showed that T4015 pretreatment downregulated many upstream regulatory factors related to inflammation, including upstream factors of the JAK / STAT signaling pathway, such as STAT1, IRF3, and IRF9, and downstream factors related to the Toll-like receptor signaling pathway, such as TLR3 and TLR4 ( Figure 6 B). In addition, in the comparative analysis of biological functions ( Figure 6 C), T4015 is predicted to significantly downregulate immune-related processes, including T lymphocyte differentiation, NK cell proliferation, and myeloid cell immunity. In addition, natural killer (NK) cells and B cells are also involved in the inflammatory process. NK cells detect infection through their activating receptors NKG2D and NKp46, release lytic granules (perforin, granzymes) or participate in TNF-related apoptosis. Macrophages and B cells are also known to activate T cells through antigen cross-presentation. In comparative disease analyses ( Figure 6 D), T4015 pretreatment reduced the possibility of inflammatory and immune diseases, such as pulmonary fibrosis, lung inflammation, sepsis, inflammatory response, etc. Figure 6 The IPA network in E shows lung inflammation, interstitial lung disease and fibrosis ( Figure 6F) Disease-related biological interactions. In these networks, T4015 significantly downregulated most genes, such as STAT1, IFNγ, IL6, and IL22 ( Figure 6 EF). IPA data analysis further demonstrated that T4015 could exert anti-inflammatory effects through JAK / STAT and NF-κB signaling pathways, indicating that it may be applied to treat inflammation-related diseases, especially pulmonary fibrosis and sepsis.

[0069] Example 4 T4015 alleviates BLM-induced pulmonary fibrosis in mice

[0070] Bleomycin is an anti-tumor drug used to treat various cancers and lymphomas. However, it has certain toxicity, the most serious side effect is pulmonary toxicity, which leads to lung structural remodeling and loss of lung function, and causes adverse reactions such as pulmonary fibrosis. Due to its simplicity and reproducibility in inducing pulmonary fibrosis, similarity to the pathological process of human pulmonary fibrosis, and validation of the in vivo anti-inflammatory effect of T4015, we used the BLM-induced pulmonary fibrosis mouse model to evaluate its therapeutic potential for inflammatory diseases.

[0071] In vivo experimental results showed that T4015 treatment increased the survival rate of mice with pulmonary fibrosis and significantly reduced lung coefficient ( Figure 7 AC), lung coefficient is an indicator of the severity of lung pathology. Compared with the BLM group, T4015 had no significant effect on the body weight of mice. However, on day 17, several very weak mice in the BLM group died, resulting in a continuous increase in the average body weight of the group ( Figure 7 D). Pathological features of pulmonary fibrosis include destruction of alveolar structure, extensive deposition of collagen, infiltration of inflammatory cells, and formation of fibrotic honeycomb lungs. Compared with the control group, mice with BLM-induced pulmonary fibrosis showed thickening of lung septa (arrows) and destruction of tracheal and alveolar structures (boxes). T4015 treatment effectively alleviated the pathological symptoms of pulmonary fibrosis and blocked the progression of pulmonary fibrosis ( Figure 7 E). In addition, after T4015 treatment, the deposition of collagen in lung tissue was significantly reduced (indicated by the box) ( Figure 7 F). In the progression of pulmonary fibrosis, inflammatory cytokines and chemokines can promote the differentiation of lung fibroblasts into myofibroblasts, thereby promoting the development of pulmonary fibrosis. As expected, T4015 administration led to a significant decrease in the expression of Il6 and Ccl2 in the lungs of mice with pulmonary fibrosis ( Figure 7 GH). Subsequently, we studied the effect of T4015 on pulmonary fibrosis-related proteins and found that T4015 reduced the expression of COl1, which is related to lung collagen formation ( Figure 7I). Collectively, these results suggest that T4015 treatment alleviates the symptoms of BLM-induced pulmonary fibrosis and may be a promising candidate for the treatment of this disease.

[0072] Example 5 Reverse targeting and molecular docking of T4015 and its structural analogs

[0073] To further investigate the molecular targets of T4015, we performed virtual target capture and molecular docking. Using the PharmMapper and GalaxySagittarius databases, we predicted potential targets of T4015 and found 76 predicted targets shared by the two databases, in addition to 110 targets predicted by PharmMappers and 424 other targets predicted by GalaxySagittarius ( Figure 8 A). Among them, JAK1, JAK2, JAK3 and TYK2 are key kinases for activating STAT signals downstream of the JAK / STAT signaling pathway ( Fig. 9 A). It is reported that EGFR activates STAT3 in a JAK1 / JAK2-dependent manner. T4015 has an inhibitory effect on TNF- and LPS-induced NF-κB activation. RIPK1, IRAK1 / 4, and TAB1 in the NF-κB signaling pathway are predicted to be drug targets of T4015 ( Fig. 9 B). In addition, ZAP70 can sense extracellular antigens, activate downstream IKK / IκB / NF-κB signaling through T cell receptors, and was predicted as a potential target of T4015 in two databases ( Figure 8 A). Subsequently, we used AutoDock Vina 1.1.2 software to perform molecular docking between these ten targets and T4015. The results showed that they could all stably bind to T4015 ( Figure 8 BH, 9C-F). Based on molecular docking, T4015 can bind to the JAK receptor binding region and phosphorylation activation domain ( Figure 8 CE, 9C-D), which is crucial for its activity. The pyrimidine and indole groups of T4015 can generate π-π stacking interactions with key kinases including JAK2, TYK2, IRAK4, and JAK3. In addition, the methylpiperazine and piperidine moieties in the T4015 structure mainly promote hydrophobic interactions and the formation of salt bridges ( Figure 8 CH, 9C-F), further stabilizing the formation of the complex.

[0074] Through target capture and molecular docking studies, we found that T4015 may inhibit JAK / STAT and NF-κB signaling pathways by targeting upstream kinase proteins such as JAK1, JAK2, TYK2, JAK3, EGFR, ZAP70, RIPK1, IRAK1 / 4 and TAB1 ( Figure 8 AH). Molecular docking results showed that the binding sites of T4015 and TYK2 include residues GLU-979, THR-668, LYS963, GLU925, TYR916, ALA242, PRO918 and GLU-287 ( Figure 8 D). The indole group of T4015 participates in the formation of hydrogen bonds and π-πstacking interactions, while the piperidine group at the tail contributes significantly to the hydrophobic interaction. The amino acid residues involved in the binding site of T4015 with ZAP70 are PHE-348, ASN-347, LYS-499, TRP-500, and ALA-494. Both the indole and piperidine parts of T4015 participate in the formation of π-πstacking interactions ( Fig. 9 E). Activity testing and structural analysis of T4015 and its analogs in JAK / STAT / NF-κB may indicate the inhibitory capacity of the macrocyclic 4-(3-indolyl)-2-aminopyridine framework and tail structure on JAK / STAT and NF-κB signaling ( Figure 8 I, Fig.10 AB). This observation may indicate a feasible direction for the structural modification of anti-inflammatory and anti-fibrotic drug candidates based on this scaffold targeting JAK / STAT and NF-κB pathways.

[0075] EGFR is predicted to be one of the drug targets targeted by T4015 ( Figure 8 AB), T4015 is structurally derived from the structural modification of the EGFR inhibitor Osimertinib. Therefore, we further collected 15 structural analogs including Osimertinib to study the relationship between the compound structure and the dual-target inhibitory effect of JAK / STAT and NF-κB ( Figure 8 I). Among these 15 analogs, 7 compounds were identified as EGFR inhibitors (Table 3), 4 were CDK inhibitors, and the others were DYRK, FLT, ALK or P-GP inhibitors. Figure 8As shown in Figure 1, compounds 2 (DYRKi), 8 (EGFRi) and 4 (EGFRi, Osimertinib) showed no inhibitory ability at 5μM and 10μM, while compound 3 (EGFRi) showed inhibitory activity only at a higher dose (10μM). At the same time, compound 1 (ALK / IGFRi) showed an inhibitory potency comparable to that of T4015, while other compounds including EGFRs, CDKis and other targeted drugs also showed stronger or weaker inhibitory activity compared to T4015. These data indicate that EGFR targeting is not closely related to the inhibitory activity of the compound on the dual JAK / STAT and NF-κB luciferase systems.

[0076] Table 3 List of T4015 analogs

[0077]

[0078]

[0079] Next, we compared the structural similarities and dissimilarities of these analogs and correlated them with their ability to inhibit a dual-target reporter system of JAK / STAT and NF-κB ( Figure 8 I, 9A-B). It was observed that the analogs with EGFR inhibitory activity all reported a common tail structure feature similar to that of Osimertinib, and their inhibitory effects on the JAK / STAT and NF-κB pathways were related to the substituents on the indole part (highlighted by green circles). Among these reported analogs with EGFR inhibitory activity, compounds 3, 4, and 8 lacking substituents or only methyl substitutions had no inhibitory activity ( Figure 8 I, 9A). Analogs without EGFR inhibitory activity all have tails different from Osimertinib (as shown in blue circles), and most of them show significant inhibitory effects on the reporter system. This suggests that the substitution of the indole group and the difference in the tail structure may affect the inhibitory ability of the JAK / STAT and NF-κB dual luciferase systems. Compounds 5, 12 and 14, which contain α, β-unsaturated amide bonds and two six-membered ring structures connected by amide bonds in the tail structure, have excellent inhibitory activity. Compounds that modify or change the 4-(3-indolyl)-2-aminopyridine skeleton will reduce or lose their inhibitory activity. For example, compound 15 has a weaker inhibitory ability without an amino group on 2-aminopyridine ( Figure 8 I, 9B). Compared with the structure of T4015 ( Figure 2 B), compound 2 with a modified indole structure had no inhibitory ability ( Figure 8I, 9B). These data suggest that the 4-(3-indolyl)-2-aminopyridine skeleton may be responsible for the inhibitory activity of T4015 and its analogs, and that the substituents on the indolyl nitrogen atom and the tail substituents of the analogs may be critical for their ability to resist JAK / STAT / NF-κB signaling.

[0080] In summary, we identified compound T4015, which exhibits inhibitory activity against JAK / STAT and NF-κB signaling pathways, and based on its anti-inflammatory properties, T4015 also has the potential to be a therapeutic agent for the treatment of inflammatory diseases, including pulmonary fibrosis.

Claims

1. Use of compound T4015 as an inhibitor in the preparation of a medicament for treating inflammatory diseases and / or immune diseases.

2. Use of compound T4015 in the preparation of a drug for alleviating BLM-induced pulmonary fibrosis.

3. The use according to claim 1 or 2, characterized in that: The CAS number of the compound T4015 is 1356962-34-9.

4. The use according to claim 3, characterized in that: Compound T4015 exerts its inhibitory effect by inhibiting the activation of JAK / STAT and / or NF-κB signaling pathways.

5. The use according to claim 4, characterized in that: Compound T4015 inhibited IL6-induced STAT3 phosphorylation, IKK phosphorylation and IκBα degradation in a concentration-dependent manner.

6. The use according to claim 3, characterized in that: Compound T4015 exerts anti-inflammatory effects by inhibiting the expression of LPS-induced inflammatory cytokines and chemokines.

7. The use according to claim 6, characterized in that: The inflammatory cytokines and chemokines are selected from at least one of TNFα, IFNβ, IL-17α, IL-6 and CXCL2.

8. The use according to claim 2, characterized in that: Compound T4015 alleviated the symptoms of BLM-induced pulmonary fibrosis by reducing the mRNA expression of Il6 and Ccl2 and the expression of fibrosis-related protein COl1 in lung tissue.

9. The use according to any one of claims 1 to 8, characterized in that: The inflammatory disease and / or immune disease is selected from at least one of pulmonary fibrosis, pulmonary inflammation, interstitial lung disease and fibrosis, and sepsis.

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