STAT3 inhibitor and application thereof

Abstract

The invention provides an STAT3 inhibitor and application of the STAT3 inhibitor. The STAT3 inhibitor is prepared from psoralen, isopsoralen, salidroside and quercetin. Wherein the psoralen, the isopsoralen, the salidroside, the quercetin and other small molecule ligand compounds can inhibit A549 cell proliferation, are concentration-dependent, and have statistical significance; the psoralen, the isopsoralen, the salidroside and the quercetin are extracted from natural traditional Chinese medicinal materials, so that the psoralen, the isopsoralen, the salidroside and the quercetin are good in drug tolerance and very small in side effect.

Description

Technical Field

[0001] The present invention belongs to the field of medicines, and in particular relates to a STAT3 inhibitor and application thereof. Background Art

[0002] Lung cancer is the leading cause of cancer death worldwide, with approximately 1.8 million deaths from lung cancer in 2020. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Compared with small cell lung cancer, NSCLC is relatively insensitive to chemotherapy. About two-thirds of NSCLC cases are diagnosed at an advanced stage, with limited surgical options. Research surveys show that the 5-year net survival rate of lung cancer patients in the United States is only 15%. The discovery of anti-tumor drugs targeting epidermal growth factor receptor (EGFR) and third-generation EGFR tyrosine kinase inhibitors (EGFR-tkis) is an important milestone in lung cancer treatment, but the adaptability, toxicity, side effects and drug resistance of these drugs affect their long-term efficacy. Therefore, it is necessary to develop potential new drugs with higher sensitivity and fewer side effects to improve clinical efficacy. Non-small cell lung cancer has a high incidence and high malignancy. Although targeted therapy provides ideas for lung cancer treatment, chemotherapy remains the standard treatment for most patients with NSCLC without gene mutations. Finding new anti-tumor drugs that are safe, efficient and non-resistant has become the focus of lung cancer research.

[0003] STAT3 (signal transducer and activator of transcription 3) is the most important member of the STAT family and is involved in regulating genes related to cell physiological and pathological processes. STAT3 can be phosphorylated and activated by the Janus kinase family. In IL-6 signaling, IL-6ST promotes JAK2 phosphorylation and STAT3 phosphorylation, STAT3 homodimerizes, transports to the nucleus to bind to DNA and activates the transcription of STAT3 target genes, thereby participating in a variety of biological functions, including tumor cell proliferation, survival, self-renewal, tumor invasion, angiogenesis and immunogenicity. In addition to IL-6, studies have shown that STAT3 can also be activated by a variety of cytokines and chemokines, including IL-21, IL-22, IL-27, IL-5, IL-9, IL-10, IL-11, IL-12, IFN-γ, and tumor necrosis factor-α. STAT3 has been reported to promote tumor proliferation, survival, and angiogenesis by upregulating the expression of anti-apoptotic proteins (such as survivin, Bcl-xL, and Mcl-1), cell cycle regulators (such as cyclin D1 and c-Myc), and vascular endothelial growth factor (VEGF). In most NSCLC patients, the AKT, ERK, and STAT3 signaling pathways are constitutively activated. It has been reported that in NSCLC, STAT3 is activated at a high level in more than 50% of patients. STAT3 is considered to be a key target for the progression of various malignancies, and abnormally active STAT3 can promote tumorigenesis, drug resistance, and metastasis in NSCLC. Downstream signaling of the STAT3 pathway can lead to lung cancer, and STAT3 can promote the expansion of myeloid-derived suppressor cells (MDSCs), thereby inducing immunosuppression in lung cancer. High activation of STAT3 in the tumor microenvironment can promote the expression of epithelial to mesenchymal transition (EMT)-related transcription factors, such as Snail, Twist, and ZEB1, and promote the expression of genes involved in metastasis, thereby participating in cancer progression. Cisplatin is still the main chemotherapy drug for advanced non-small cell lung cancer. STAT3 expression is associated with the chemosensitivity of tumor cells, and its inhibition can enhance the chemosensitivity of cisplatin. Therefore, finding new drugs with STAT3 inhibitory activity is crucial to improving the treatment of non-small cell lung cancer. Some compounds targeting STAT3 have been evaluated in preclinical and clinical studies, such as flubendazole, metformin, dihydroartemisinin (DHA), cucurbitacin B, etc. Chemically synthesized drugs usually have adverse side effects and poor patient tolerance. There is increasing attention to the development of new anticancer therapeutic drugs from natural sources, which are well tolerated and have few side effects. Summary of the invention

[0004] The purpose of the present invention is to provide a STAT3 inhibitor and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A STAT3 inhibitor comprises psoralen, isopsoralen, salidroside and quercetin.

[0006] Furthermore, the STAT3 inhibitor is used in the preparation of anti-non-small cell lung cancer drugs.

[0007] The structural formula of the psoralen is as follows: The structural formula of isopsoralen is as follows: The structural formula of the salidroside is as follows: The structural formula of the quercetin is as follows: The advantages of the present invention are: The present invention provides a new STAT3 inhibitor and its application in the preparation of an anti-non-small cell lung cancer drug. Small molecule ligand compounds such as psoralen, isopsoralen, salidroside and quercetin can inhibit the proliferation of A549 cells in a concentration-dependent manner with statistical significance; and these four compounds do not inhibit the proliferation of RAW264.7 cells, but can promote cell growth. Psoralen and isopsoralen are extracted from the natural Chinese medicinal material Psoralea corylifolia, and have good drug tolerance and little side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1-1 The mode of interaction between Stattic and STAT3 SH2 domain; Figure 1-2 The mode of action between psoralen and STAT3 SH2 domain; Figure 1-3 The mode of action between isopsoralen and STAT3 SH2 domain; Figure 1-4 The mode of action between salidroside and STAT3 SH2 domain; Figure 1-5 The mode of action between quercetin and STAT3 SH2 domain; Figure 2-1 The results of the SPR affinity test between psoralen and STAT3 (KD=80.92μM); Figure 2-2 The results of the SPR affinity test between isopsoralen and STAT3 (KD = 28.11 μM); Figure 2-3 The results of the SPR affinity test between salidroside and STAT3 (KD=85.93μM); Figure 2-4 The SPR affinity test results of quercetin and STAT3 (KD=45.57μM); Figure 3-1a IC for different drugs 50 result; Figure 3-1b Results of MTT assay of A549 cell proliferation ability treated with different drugs; Note: Compared with the 0 μmol / L concentration control group **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; Figure 3-2a Comparison of cell migration at 0, 24, and 48 h after scratching; Figure 3-2b is the migration rate of cells after scratching for 24 h and 48 h; Figure 3-2c The effect of 500 μmol / L psoralen, salidroside, quercetin and isopsoralen on the survival rate of RAW264.7 cells; Note: Compared with the blank concentration control group, ** P <0.01;* P <0.05. DETAILED DESCRIPTION

[0009] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0010] Through molecular docking technology, the CDOCKER binding energy of four small molecule ligands, psoralen, isopsoralen, salidroside, and quercetin, with the SH2 domain of STAT3 protein was calculated, and the docking results were visualized to observe whether the docking site was suitable for targeted binding. Secondly, the SPR technology was used to detect the interaction between four small molecule compounds, psoralen, isopsoralen, salidroside, and quercetin, and STAT3 protein to detect whether it was specific binding. Then, the effects of the four compounds on the proliferation ability of A549 cells were detected.

[0011] 1 Materials and methods 1.1 Experimental Methods 1.1.1 Molecular docking The crystal structure of STAT3 protein was downloaded from the Protein Data Bank (PDB), and the PDB ID is 1BG1. Before docking, the Discovery Studio 2019 Client (DS) software was used to optimize and modify the protein, delete the DNA chain and water molecules, and select the STAT3-SH2 domain as the docking site (amino acid position is 586 to 685 of STAT3), and the radius of the site is 10 Å. The mol structures of four candidate compounds, psoralen, isopsoralen, salidroside, and quercetin, were obtained in the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), and the CDOCKER module under the DS software was used for protein-receptor docking. Stattic was selected as a positive control and docked together with the candidate compounds. The optimal conformation was evaluated according to the CDOCKER binding energy.

[0012] 1.2 Experimental Results 1.2.1 Molecular docking results Molecular docking is to calculate the force and affinity of the connection between the known small molecule ligand and the target protein crystal structure, and obtain the optimal parameters for the binding between the small molecule and the target protein. Therefore, molecular docking can accurately determine the specific location of the ligand binding to the protein and determine whether it can specifically bind to the protein target.

[0013] In order to evaluate whether the candidate small molecule ligands can specifically bind to the active site of STAT3 protein, molecular docking technology was used to calculate the interaction between four candidate ligands and the SH2 domain in STAT3 protein. The SH2 domain is the binding site of many non-peptide STAT3 inhibitor molecules. The surface of SH2 domain protein has three subpockets: phosphorylated Tyr705 (p-Tyr705) binding pocket (pY pocket, residues 591, 609-620), Leu706 subsite (pY+1 pocket, residues 626-639), and hydrophobic side pocket (pY-X pocket, residues 592-605). However, the affinity of the three pockets is not equal, and the pY pocket has the highest affinity. As an adaptor protein, it participates in the dimerization process of STAT3 protein, connects activated receptors and downstream signaling molecules, and mediates the upregulation of signaling pathways, which is closely related to the occurrence and development of non-small cell lung cancer. Therefore, the STAT3-SH2 domain protein (amino acid position 586 to 685 of STAT3) was selected for docking with the candidate ligand and the positive small molecule Stattic.

[0014] The results of Stattic docking of positive small molecules are as follows Figure 1-1As shown in the figure, the Stattic positive small molecule inhibitor can form hydrogen bonds with Lys-591, Arg-609, and Ser-636 amino acid residues, and form an alkyl hydrophobic interaction with Pro-639, and the CDOCKER binding energy is -23.494 kJ. Figure 1-2 As shown in Figure 2, psoralen can form a cation-π interaction with Lys-591, a hydrogen bond with the Arg-609 amino acid residue, a π hydrophobic interaction with Val-637, and an alkyl hydrophobic interaction with Pro-639, with a CDOCKER binding energy of -18.6669 kJ. Figure 1-3 As shown in Figure 2, isopsoralen can form a cation-π interaction with Lys-591, a hydrogen bond with the amino acid residue Arg-609, and an alkyl hydrophobic interaction with Pro-639, with a CDOCKER binding energy of -20.9622 kJ. Figure 1-4 As shown in Figure 2, salidroside can form strong hydrogen bonds with Ser-636, Arg-609, Glu-612, Ser-613, and Ser-611 amino acid residues, and the CDOCKER binding energy is -36.1928 KJ. Figure 1-5 As shown in the figure, quercetin can form salt bridges and cation-π interactions with Lys-591, hydrogen bonds with Arg-609 and Gln-635, and alkyl hydrophobic interactions with Pro-639, with a CDOCKER binding energy of -54.9104 KJ. The CDOCKER binding energies of Stattic and candidate small molecule compounds are shown in Table 1. According to the molecular docking results, the four compounds can form typical strong hydrogen bonds with the amino acid residue fragments at the pY pocket, indicating that they have a strong affinity with the STAT3 SH2 domain. In addition, by comparing the CDOCKER binding energy with the positive control Stattic, the binding energy is negative, indicating that the binding of the ligand to the protein is favorable; thus, it can be judged that psoralen, isopsoralen, salidroside, and quercetin are potential small molecule ligands for STAT3.

[0015] Table 1 Binding energy of Stattic and candidate small molecule compounds CDOCKER 2 Materials and methods 2.1 Experimental Methods 2.1.1 Preparation of Assay Buffer PBST Take 0.5 mL of Tween20 and add it to 2 mL of DDH2O water to make 4 mL of 20% Tween20; take 50 mL of 10×PBS and add it to a 500 mL volumetric flask, then add 1.25 mL of the 20% Tween20 prepared in the previous step, and finally make up to 500 mL with DDH2O and mix thoroughly.

[0016] 2.1.2 Protein fixation Manual mode was used for protein fixation, which was divided into the following three steps: chip activation, 100 μL NHS and 100 μL EDC were mixed and injected at a speed of 10 μL / min to start activation, and the activation time was 600 s; protein fixation, protein dilution was performed using Acetate 4.0, the flow rate was set to 10 μL / min, the concentration was 50 μg / ml, and the fixation time was 600 s; chip blocking, Ethamolamine was used to start blocking at a speed of 10 μL / min, and the blocking time was 600 s.

[0017] 2.1.3 Analyte injection Use running buffer to dilute 4 compounds including psoralen, isopsoralen, salidroside and quercetin. Set the experimental program: Method - Multi-cycle kinetics / affinity, change the channel selection to 2-1, the concentration unit is μM; set the Starup step to 3 times; set the injection parameters to 60 s contact (Contact) 120 s dissociation (Dissociation Time); flow rate 30μL / min; 25℃. All steps are performed using the system's automated sampling, and the change in the response signal is expressed in units (RU).

[0018] 2.1.4 Data Processing Data were analyzed using Biacore T200 Evaluation Software 3.0 and Origin. Affinity constants (KD) were obtained by global fitting using a steady-state affinity model (1:1).

[0019] 2.2 Results and Discussion The Biacore system is based on surface plasmon resonance (SPR) technology, which records the changes in the concentration of molecules on the sensor chip surface during the process of molecular binding and dissociation in real time, thereby monitoring the interaction between molecules in real time. In order to study the interaction between two molecules, one molecule is fixed to the chip surface, while the other molecule flows continuously over the chip surface in the form of a solution. The change in mass concentration near the chip surface is proportional to the SPR response value, and the detector can detect the binding and dissociation process of molecules in the solution and molecules on the chip surface in real time.

[0020] In order to verify the binding effect of psoralen, isopsoralen, salidroside and quercetin on STAT3 protein, the SPR technology was used to detect the affinity between the above four compounds and STAT3. The results showed that these four compounds all had stable binding signals with STAT3 protein and were concentration-dependent. The sensor signal diagram and fitting curve diagram of psoralen and STAT3 protein are shown in Figure 2. Figure 2-1 As shown in Figure 2, the KD value is 80.92 μM. The sensor signal diagram and fitting curve diagram of isopsoralen and STAT3 protein are shown in Figure 2. Figure 2-2 As shown in Figure 2, the KD value is 28.11 μM. The sensor signal diagram and fitting curve diagram of salidroside and STAT3 protein are shown in Figure 2. Figure 2-3 As shown in Figure 2, the KD value is 85.93 μM. The sensor signal diagram and fitting curve diagram of quercetin and STAT3 protein are shown in Figure 2. Figure 2-4 As shown, the KD value is 45.57 μM. The above results show that psoralen, isopsoralen, salidroside and quercetin can specifically bind to STAT3 protein respectively.

[0021] 3 Materials and methods 3.1 Experimental methods 3.1.1 Cell culture After A549 cells were purchased and received, they were placed in an incubator for 2 h before being passaged. They were cultured with 1640 medium (plus 10% serum and 1% penicillin-streptomycin) at 37°C and 5% CO2. When the cells grew to a density of 90-100%, they were passaged. After passage, they were continued to be expanded for subsequent experiments, or frozen at -80°C and in liquid nitrogen for later use.

[0022] 3.1.2 Cell proliferation assay A549 cells in the logarithmic growth phase were plated in a 96-well plate so that the plate density could grow to 70-80% on the second day. Psoralen and isopsoralen at concentrations of 0, 10, 25, 50, 100, and 200 μmol / L, quercetin at concentrations of 0, 25, 50, 100, 200, and 300 μmol / L, and salidroside at concentrations of 0, 50, 200, 350, 500, and 650 μmol / L were used to act on A549 cells, respectively. Five replicate wells were set up, and 10 μL of the corresponding concentration of drug solution was added to each well for incubation for 24 h. The culture medium was discarded, and 90 μL of culture medium and 10 μL of 5 mg / ml MTT solution were added and cultured for 4 h. The culture medium was discarded, 100 μL of DMSO was added, and the cells were shaken on a shaker for 15 min to dissolve the crystal violet, and then the absorbance value was detected at 570 nm.

[0023] 3.1.3 Cell scratch assay A549 cells in the logarithmic growth phase were plated on a 6-well plate. The plate density could reach 90-100% on the second day. Marking was done with a marker pen and ruler, and then scratched with a 200 μL pipette tip to create the same wound. After washing twice with PBS solution, the IC values ​​obtained from the respective MTT experiments were used. 50 A549 cells were treated with STAT3 small molecule compounds at similar concentrations, and photographs were taken and samples were collected at 0 h, 24 h, and 48 h of culture, and the migration rate was finally calculated.

[0024] 3.2 Results and Discussion 3.2.1 Inhibition of A549 cell proliferation by STAT3 small molecule compounds The MTT experiment was used to detect the inhibitory effects of four STAT3 small molecule compounds on the proliferation of A549 cells. Different concentrations of drug reagents were used to act on A549 cells, and the absorbance values ​​were detected. The cell survival rate and IC 50 , respectively, to obtain the IC of psoralen 50 is 94.50 μmol / L, and the IC 50 is 260.2 μmol / L, and the IC 50 is 604.3 μmol / L, and the IC 50 463.8 μmol / L (see Figure 3-1a ), the results of different drug concentrations acting on A549 cells (see Figure 3-1b ), it can be seen that psoralen has the best effect, and different concentration groups have significant differences from the blank group, and the cell survival rate decreases in a concentration-dependent manner. The subsequent experiments will use a drug concentration of 100 μmol / L for experiments; the maximum experimental group concentrations of isopsoralen, salidroside and quercetin are significantly different from those of the blank group, and they all have inhibitory effects on the proliferation of A549 cells, so the subsequent experiments will use the maximum concentrations of these three drugs for experiments, which are 200 μmol / L of isopsoralen, 650 μmol / L of salidroside and 300 μmol / L of quercetin.

[0025] 3.2.2 Inhibition of A549 cell migration by STAT3 small molecule compounds According to the results of the MTT experiment, a drug concentration was taken to act on A549 cells, and the scratch test was used to detect whether the STAT3 small molecule compound had an effect on the migration ability of A549 cells. The migration area generated by the blank group and the drug group at different time periods was compared to detect whether the STAT3 small molecule compound had an effect on the migration of A549 cells. The photographic results of the scratch test at 0 h, 24 h, and 48 h were taken (see Figure 3-2a), after calculating the migration area of ​​cells at different time periods using ImageJ, the significant differences in the migration rates of cells were made. The results showed that these four STAT3 small molecule compounds all had an inhibitory effect on the migration of A549 cells. Among them, isopsoralen had the most obvious inhibitory effect and the lowest scratch healing rate. It could significantly inhibit the migration rate of A549 cells at 24 h and 48 h ( P <0.001), followed by quercetin and psoralen, and finally salidroside (see Figure 3-2b ).

[0026] 3.2.3 Inhibition of RAW264.7 cell proliferation by STAT3 small molecule compounds Mouse monocyte / macrophage-like cell line RAW264.7 is an important experimental model for simulating human immune cells. Macrophages can phagocytize and kill pathogens, intracellular parasites, tumors and other target cells that enter the body, and play an irreplaceable role in immune regulation. The present invention selected 500 μmol / L of psoralen, salidroside, quercetin, and isopsoralen to act on RAW264.7 cells, and explored the effects of the four compounds on the proliferation of RAW264.7 cells. The results are as follows Figure 3-2c As shown in the figure, compared with the blank control group, 500 μmol / L psoralen, salidroside, and isopsoralen significantly increased the survival rate of RAW264.7 cells (** P <0.01), 500 μmol / L quercetin can also improve the survival rate of RAW264.7 cells (* P <0.05), indicating that these four compounds would not inhibit the proliferation of RAW264.7 cells, but could promote their growth.

[0027] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A STAT3 inhibitor, characterized in that Including psoralen, isopsoralen, salidroside, and quercetin.

2. The STAT3 inhibitor according to claim 1, characterized in that The structural formula of psoralen STAT3 inhibitor is as follows: 。 3. The STAT3 inhibitor according to claim 1, characterized in that The structural formula of isopsoralen STAT3 inhibitor is as follows: 。 4. The STAT3 inhibitor according to claim 1, characterized in that The structural formula of the salidroside is as follows: 。 5. The STAT3 inhibitor according to claim 1, characterized in that The structural formula of the quercetin is as follows: 。 6. Use of the STAT3 inhibitor according to claim 1 in the preparation of an anti-non-small cell lung cancer drug.

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