Use of a 3-oxo-benzofuran compound in the preparation of an SNX3 inhibitor and a drug for treating pulmonary fibrosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a 3-oxo benzofuran compound in preparation of an SNX3 inhibitor and a drug for treating pulmonary fibrosis. The 3-oxo benzofuran compound is a compound LC4, namely (Z)-2-(4-((6-ethoxy-3-oxo benzofuran-2(3H)-ylidene)methyl)-2,6-dimethyl phenoxy)-2-methyl propanoic acid. Researches of the application show that the compound LC4 has a binding activity with SNX3, inhibits SNX3, and influences a downstream pathway of SNX3 through regulation of the SNX3; and the compound LC4 can be used as the SNX3 inhibitor and further has a medicinal value. The compound LC4 can play a good anti-fibrosis role in a pulmonary fibrosis cell model induced by TGF-beta 1. The compound LC4 has important values in development of the SNX3 inhibitor and a drug for treating pulmonary fibrosis and other related diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine. Specifically relates to the application of a 3-oxo-benzofuran compound in the preparation of SNX3 inhibitors and lung fibrosis treatment drugs. BACKGROUND

[0002] Sorting nexins (SNXs), also known as sorting proteins, are a class of highly conserved eukaryotic proteins. In vertebrates, the main function of SNXs is to regulate the transport of intracellular molecules, including the regulation of clathrin-mediated endocytosis of proteins, the regulation of endosome maturation to mediate protein sorting and degradation. Recent studies have found that SNXs not only have important regulatory effects in some important cell signaling pathways, but also affect the occurrence and development of diseases. Sorting nexin 3 (SNX3) is the simplest subtype of the sorting nexin (SNXs) family, which only contains a PX basic domain (Phox Homology Domain). SNX3 targets the endosomal membrane through the PX basic domain to classify and regulate the recycling and degradation of cargo proteins, and has become a key regulator of protein transport involved in the pathogenesis of various diseases. In structure, SNX3 can directly or indirectly bind to the vacuolar protein sorting (VPS) family to form a retroviral protein complex. Normal life activities of cells cannot be separated from intracellular protein sorting. Endosomes, as a special organelle, are involved in the endocytosis of some important proteins and the sorting of some proteins from TGN. When SNXs go wrong, corresponding functional disorders will occur. For example, if the conserved region of the PX amino acid sequence changes, the cell cannot effectively perform endocytosis and protein sorting functions, leading to a series of related diseases. Studies have shown that SNX3 is closely related to tumors, neurological diseases such as Alzheimer's disease, and heart diseases. For example, SNX3 can cause heart damage by promoting the phosphorylation and dimerization of signal transduction and transcriptional activator 3 (STAT3) and its nuclear entry.

[0003] SNX3 inhibitors can inhibit the expression of SNX3 genes or SNX3, thereby inhibiting the action of SNXs and the expression of disease-related proteins, effectively improving various diseases such as liver fibrosis, cardiac hypertrophy, and heart failure, and providing a new means for treating diseases. Therefore, the development of SNX3 inhibitors has important clinical and pharmaceutical value.

[0004] Pulmonary fibrosis is a kind of chronic progressive tissue repair reaction, which can cause irreversible scarring and remodeling of the lung. Pulmonary fibrosis is a diffuse interstitial lung disease, which is the end stage of many interstitial lung diseases (ILD), and the vast majority of pulmonary fibrosis patients are idiopathic (idiopathic), which is called idiopathic pulmonary fibrosis (IPF). The etiology is complex, and the pathogenesis is not clear. In the context of air pollution and environmental degradation, in-depth study of the mechanism of pulmonary fibrosis and finding new intervention targets are of great significance for drug development and disease treatment.

[0005] The Chinese invention patent with publication number CN111285829A has disclosed a synthetic preparation method of compound LC4, i.e. (Z)-2-(4-((6-ethoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)-2,6-dimethylphenoxy)-2-methylpropanoic acid. The compound and the pharmaceutical composition containing the derivative can be used to improve liver fibrosis. However, it is not clear whether the compound has an effect on the function of sorting structural protein 3 (SNX3) and can be developed as an SNX3 inhibitor to play a pharmaceutical value. In addition, pulmonary fibrosis is different from liver fibrosis, and lung tissue and liver tissue are two different tissues. It is also not certain how compound LC4 affects pulmonary fibrosis. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of SNX3 inhibitors in the prior art and the limited problem of pulmonary fibrosis treatment drugs. Through research and exploration, the application of compound LC4 and its derivatives in the preparation of SNX3 inhibitors and the treatment of pulmonary fibrosis drugs is provided.

[0007] The above-mentioned purpose of the present application is realized by the following technical solutions:

[0008] The present application researches and finds that compound LC4 has binding activity with SNX3, inhibits SNX3, and affects the downstream pathway of SNX3 through the regulation of SNX3; can be used as an SNX3 inhibitor to further play a pharmaceutical value.

[0009] The present application further researches and shows that compound LC4 can play a good anti-fibrosis effect in the pulmonary fibrosis cell model induced by TGF-β1.

[0010] Therefore, compound LC4 has important value in the development of SNX3 inhibitors and the treatment of pulmonary fibrosis and other related diseases.

[0011] Therefore, the present application claims the following scheme:

[0012] Use of a 3-oxobenzofuran compound or derivative thereof in the preparation of an SNX3 inhibitor.

[0013] Use of a 3-oxobenzofuran compound or derivative thereof in the preparation of a drug for treating pulmonary fibrosis.

[0014] The 3-oxobenzofuran compound is (Z)-2-(4-((6-ethoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)-2,6-dimethylphenoxy)-2-methylpropanoic acid, referred to as compound LC4.

[0015] Further, the derivative is a pharmaceutically acceptable salt, prodrug, ester or solvate.

[0016] Still further, the drug further comprises a pharmaceutically acceptable excipient.

[0017] Still further, the pharmaceutically acceptable excipient comprises at least one of a diluent, an excipient, a disintegrant, a filler, a binder, a lubricant, a flavoring agent, a suspending agent, a surface active agent, a stabilizer.

[0018] Further, the dosage form of the drug is a tablet, a capsule, a pill, a powder, a granule or a solution.

[0019] Still further, the administration route of the drug comprises intravenous injection, intramuscular injection, subcutaneous injection or oral administration.

[0020] Based on the above application scheme, the present application provides an SNX3 inhibitor and a drug for treating and preventing pulmonary fibrosis, wherein the SNX3 inhibitor or the drug comprises (Z)-2-(4-((6-ethoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)-2,6-dimethylphenoxy)-2-methylpropanoic acid or a derivative thereof.

[0021] The pharmaceutically acceptable excipient refers to an ingredient used for converting an active compound into a dosage form suitable for administration. All pharmaceutical products or nutraceutical products including tablets, capsules, solutions, vaccines and other dosage forms contain excipients, and in most products, the excipients can account for a large proportion of the entire preparation. Some excipients can prevent the premature release of the drug to avoid damage and cause gastric irritation or gastric discomfort; some excipients can promote the disintegration of the drug into small particles to enter the blood stream faster; and other excipients can help maintain the stability of the drug so that the drug can exert the best effect when used. Therefore, the addition of one or more pharmaceutically acceptable excipients in the drug can improve the therapeutic effect of the drug on the disease.

[0022] Still further, the dosage form of the drug is a tablet, a capsule, a pill, a powder, a granule or a solution.

[0023] The present application has the following beneficial effects:

[0024] The present application researches and finds that the compound LC4 has binding activity with SNX3, inhibits SNX3, and affects the downstream pathway of SNX3 through the regulation of SNX3; can be used as an SNX3 inhibitor to further play a medicinal value. Therefore, the present application develops a new SNX3 inhibitor, which has important clinical medicinal value.

[0025] The present application further researches and shows that the compound LC4 has a good anti-fibrosis effect on pulmonary fibrosis, and has important value in the development of therapeutic drugs for pulmonary fibrosis and other related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The virtual molecular docking result shows that the SNX3 protein has significant binding with the small molecule compound LC4 (SNX3: sorting nexin 1; Affinity: affinity);

[0027] Figure 2 The immunofluorescence experiment in cells verifies that the LC4-GFP conjugate has binding with SNX3 (Vehicle: empty load; LC4-GFP: LC4 conjugated with green GFP fluorescent protein; DAPI: 4', 6-diamidino-2-phenylindole, a nuclear dye; Merge: merge; Zoom: local magnification; Quantification: quantification);

[0028] Figure 3 The immunofluorescence experiment in cells verifies that the LC4 has binding with SNX3, VPS35 and VPS26 (VPS35: vacuolar sorting protein 35; VPS26: vacuolar sorting protein 26);

[0029] Figure 4 The immunofluorescence experiment in cells verifies that the LC4 inhibits the expression of SNX3 and α-SMA protein in the fibrosis model (Vehicle: empty load group; Vehicle+TGF-β1: model group; LC4+TGF-β1: administration group);

[0030] Figure 5 The immunofluorescence experiment in cells verifies that the LC4 inhibits the expression and nuclear translocation of β-catenin protein in the fibrosis model (β-catenin: β-catenin; Vehicle: empty load group; Vehicle+TGF-β1: model group; LC4+TGF-β1: administration group);

[0031] Figure 6To verify that LC4 inhibits abnormal proliferation in the fibrosis model in cells by Edu cell proliferation experiment (Edu: 5-ethynyl-2'-deoxyuridine, cell proliferation dye; Edu positive rate (fold of Vehicle): Edu positive rate (fold of Vehicle); Vehicle: empty vehicle group; Vehicle+TGF-β1: model group; LC4+TGF-β1: drug administration group);

[0032] Figure 7 To verify that LC4 inhibits abnormal migration in the fibrosis model in cells by cell scratch experiment (wound healing rate: wound healing rate; Vehicle: empty vehicle group; Vehicle+TGF-β1: model group; LC4+TGF-β1: drug administration group). DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the examples are commercially available.

[0034] The technical solutions in the examples of the present application will be described below in a clear and complete manner. Obviously, the described examples are only a part of the examples of the present application, not all examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0035] The compound LC4 used in the following examples, i.e. (Z)-2-(4-((6-ethoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)-2,6-dimethylphenoxy)-2-methylpropanoic acid, is synthesized according to the method in CN111285829A.

[0036] Exploration of the effect of compound LC4 in Example 1 on SNX3 protein

[0037] 1. Virtual molecular docking

[0038] (1) Prepare the structure file of compound LC4, and perform structure conversion and structure optimization on it;

[0039] (2) Prepare the crystal structure file of SNX3 protein, and perform structure repair and adjustment on it;

[0040] (3) Determine the active site, which is Arg-76: 76 arginine and Lys-95: 95 lysine, and find the approximate docking pocket position;

[0041] (4) Establish docking box with predicted pocket position center as coordinate origin, shape and size are determined by project;

[0042] (5) Docking box size and other parameters are set according to ligand information;

[0043] (6) After the above steps are determined, the final docking parameters need to be set and the docking accuracy needs to be selected;

[0044] (7) Check the structure file and parameter file, call server resources according to project conditions, and start the docking program.

[0045] 2. Immunofluorescence experiment

[0046] LC4 and GFP conjugates were synthesized by a biological company for experiments, and the binding of LC4 and SNX3 was explored through GFP.

[0047] Control group:

[0048] (1) Take well-grown lung fibroblasts MRC5 and place them in a laser confocal dish, add 5 μM LC4 and treat for 1-6 h;

[0049] (2) Use 4% paraformaldehyde, fix at room temperature for 20 min, wash with PBS, then use 1% Tween-20 to permeabilize at 37°C for 20 min, then wash with PBS;

[0050] (3) Block with goat serum for 1 h, add antibodies (SNX3, 1:100) diluted according to the recommended ratio, incubate overnight, and wash with PBS;

[0051] (4) Perform operations in the dark, add fluorescent secondary antibodies corresponding to the species, incubate for 1 h, wash with PBS, then use DAPI staining solution to stain the nucleus for 10 min, and then wash with PBS.

[0052] (5) Observe the fluorescence distribution under a laser confocal microscope.

[0053] Experimental group:

[0054] (1) Take well-grown lung fibroblasts MRC5 and place them in a laser confocal dish, add 1 μL LC4 and GFP conjugate solution after 12 h, and incubate for 24 h;

[0055] (2) Use 4% paraformaldehyde, fix at room temperature for 20 min, wash with PBS, then use 1% Tween-20 to permeabilize at 37°C for 20 min, then wash with PBS;

[0056] (3) Block with goat serum for 1 h, add SNX3 antibody (SNX3, 1:1000) diluted according to the recommended proportion, respectively incubate overnight, and clean with PBS;

[0057] (4) Perform operations in the dark, add fluorescent secondary antibody corresponding to the species, incubate for 1 h, clean with PBS, and then use DAPI staining solution to stain the nucleus for 10 min, and then clean with PBS.

[0058] (5) Observe the fluorescence distribution under a laser confocal microscope.

[0059] Binding Figure 1 As shown in FIG. 2, the compound LC4 binds to the SNX3 protein through Arg-78 and Lys-95; binding Figure 2 As shown in FIG. 2, LC4 binds to SNX3 significantly.

[0060] Exploration of the effect of the compound LC4 of Example 2 on SNX3 binding

[0061] (1) Take well-grown lung fibroblasts MRC5 in a 10 cm cell culture dish, and after 12 h, add 10 μL of LC4 and GFP conjugate solution and incubate for 24 h;

[0062] (2) Extract total cell protein for quantification, take 500 μg of protein, add GFP antibody according to the recommended proportion, and at the same time, take 20 μg of protein from the total protein as an Input control sample before IP, and take about 100 μg of protein and add the same species IgG as a negative control, and the IgG group and the IP group are slowly shaken at 4°C overnight;

[0063] (3) Add 30 μl of protein A / G agarose microspheres to each group of EP tubes, mix well, and slowly shake at 4°C for 4 h. Centrifuge at 12000 g for 30 s at 4°C, and discard the supernatant.

[0064] (4) Wash the precipitate with IP Bufferl, IP Buffer2, and IP Buffer3 with a gradient decrease in NaCl concentration once each time, centrifuge at 12000 g for 30 s at 4°C after each washing, discard the supernatant, and try to aspirate the supernatant as much as possible without aspirating the precipitate.

[0065] (5) Prepare 2x loading buffer with IP Bufferl and 5x loading buffer, add 30 ul of 2x loading buffer to each group of EP tubes, mix well, and place in a 100°C metal bath for 5 min.

[0066] (6) Perform Western blot on the Input, IP, and IgG control samples, incubate SNX3 antibody (1:1000), VPS35 antibody (1:1000), and VPS25 antibody (1:1000) 4°C overnight, and then perform development and luminescence detection.

[0067] The experimental results are shown in Figure 3 Figure 6. From the results of the co-immunoprecipitation experiment, it can be seen that the compound LC4 is significantly combined with SNX3, VPS35 and VPS26.

[0068] Exploration of the effect of the compound LC4 of Example 3 on the protein level of fibrosis marker proteins

[0069] (1) Take well-grown lung fibroblasts MRC5 in a laser confocal small dish, Vehicle group: control solvent treatment for 24 h, TGF-β1+Vehicle group: use 5 ng / mL of TGF-β1 solution to induce the establishment of a cell pulmonary fibrosis model for 24 h, TGF-β1+LC4 group: give 5 ng / mL of TGF-β1+5 μM LC4 at the same time for 24 h;

[0070] (2) Use 4% paraformaldehyde to fix at room temperature for 20 min, wash with PBS, then use 1% Tween-20 to permeabilize at 37°C for 20 min, and then wash with PBS;

[0071] (3) Block with goat serum for 1 h, add SNX3 antibody, α-SMA antibody and β-catenin antibody diluted according to the recommended ratio, respectively incubate overnight, and wash with PBS;

[0072] (4) Perform operations in the dark, add corresponding species of fluorescent secondary antibody and incubate for 1 h, wash with PBS, then use DAPI staining solution to stain the nucleus for 10 min, and then wash with PBS;

[0073] (5) Observe the fluorescence distribution under a laser confocal microscope.

[0074] In combination with Figure 4 , the compound LC4 can inhibit the abnormal increase of SNX3 and α-SMA protein levels in a pulmonary fibrosis model; in combination with Figure 5 , the compound LC4 can inhibit the abnormal increase of β-catenin protein levels and also inhibit the increase of nuclear translocation in a pulmonary fibrosis model.

[0075] Exploration of the effect of the compound LC4 of Example 4 on a cell fibrosis model

[0076] 1. Cell proliferation

[0077] (1) Take well-grown lung fibroblasts MRC5 in a 6-well plate, so that the Vehicle group: control solvent treatment for 24 h, TGF-β1+Vehicle group: use 5 ng / mL of TGF-β1 solution to induce the establishment of a cell pulmonary fibrosis model for 24 h, TGF-β1+LC4 group: give 5 ng / mL of TGF-β1+5 μM LC4 at the same time for 24 h;

[0078] (2) Using BeyoClick TM EdU-488 cell proliferation detection kit according to the instructions;

[0079] (3) After treatment, observe the fluorescence distribution under the fluorescence microscope.

[0080] 2. Cell migration

[0081] (1) First, use the marker pen to draw a horizontal line evenly behind the 6-well plate, about 0.5-1 cm apart, crossing the hole;

[0082] (2) Take well-grown lung fibroblast MRC5 cells in a 24-well plate, use 5 ng / mL TGF-β1 solution for 24 h to induce the establishment of a cell pulmonary fibrosis model, and give 5 μM LC4 treatment for 24 h;

[0083] (3) The next day, use a 1 mL gun head, perpendicular to the cell plane, and draw a line on the cell layer along the line drawn on the back of the plate the day before (preferably use the same gun head between different wells);

[0084] (4) After the scratch is completed, wash the cells with sterile PBS 3 times to remove non-adherent cells, that is, the cells that were scratched, and the gap left after scratching is clearly visible, then replace fresh serum-free medium;

[0085] (5) Place the cells in a 37℃ 5% CO2 incubator for culture. Then at appropriate time points, such as 0 h, 12 h, 24 h, remove the cells, observe and measure the width of the scratch under the microscope, and take pictures.

[0086] In combination Figure 6 , compound LC4 can inhibit abnormal proliferation in the pulmonary fibrosis model; in combination Figure 7 , compound LC4 can inhibit abnormal migration in the pulmonary fibrosis model.

Claims

1. Use of a 3-oxobenzofuran compound or a salt thereof in the preparation of a drug for treating pulmonary fibrosis, wherein the 3-oxobenzofuran compound is (Z)-2-(4-((6-ethoxy-3-oxobenzofuran-2(3H)-ylidene)methyl)-2,6-dimethylphenoxy)-2-methylpropanoic acid.

2. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that The pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a disintegrant, a filler, a binder, a lubricant, a flavoring agent, a suspending agent, a surfactant, and a stabilizer.

4. The use according to claim 1, characterized in that The dosage form of the medicine is tablet, capsule, pill, powder, granule or solution.

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