CN108187049A - The application of the albumen of functional domain containing Bromo 4 and its inhibitor in treatment fatty liver and relevant disease drug is prepared - Google Patents

Abstract

The invention discloses the application of Bromo functional domain-containing protein 4 and its inhibitor in the preparation of medicines for treating fatty liver and related diseases. In the present invention, different hepatocytes (L02 cells, HepG2 cells) were used as experimental objects, treated with BRD4 inhibitors - JQ1 and OTX015 respectively, DMSO was used as a control, and then treated with (palmitate/oleic acid) PA/OA, and then treated with The cells were stained with Oil Red O, and the staining results showed that compared with the DMSO control group, the color of the inhibitor treatment group was significantly lighter. This shows that after BRD4 is inhibited, it can inhibit fat accumulation and improve the occurrence of fatty liver and related diseases Therefore, BRD4 provides a target for the development of drugs for the prevention, alleviation and/or treatment of fatty liver and related diseases.

Description

Bromo functional domain-containing protein 4 and its inhibitors are used in the preparation and treatment of fatty liver and related diseases application in disease medicine

technical field

The invention belongs to the field of gene function and application, and in particular relates to the application of BRD4 as a drug target in the screening of drugs for the treatment of fatty liver, and the use of BRD4 inhibitors in the preparation of drugs for the prevention, alleviation and/or treatment of fatty liver and related diseases Applications.

Background technique

With the continuous improvement of people's living standards, great changes have taken place in the diet structure, which gradually tends to be high in protein and high in fat. However, human metabolism is limited, which leads to the continuous accumulation of fat in the liver, which leads to fat accumulation. liver. In recent years, the incidence of fatty liver has been increasing day by day, and the patients will be accompanied by steatohepatitis, liver fibrosis and even liver cirrhosis, and will develop into hepatocellular carcinoma. At present, there is no specific drug for the prevention and treatment of fatty liver. Therefore, it is particularly important to find new targets for the preparation of drugs for preventing, alleviating and/or treating fatty liver diseases.

Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain and extra-terminal (BET) family. BRD4 is a transcriptional regulatory protein ubiquitous in mitotic cells. It plays a transcriptional regulatory role by recruiting transcriptional regulatory complexes to acetylated chromatin, and activates the transcription of downstream target genes, thereby regulating cell cycle and tumorigenesis. play an important role ^[1] . BRD4 can also interact with some important transcription factors (such as p53, etc.) to affect the transcription of downstream genes. Studies in recent years have shown that the expression and dysfunction of BRD4 can significantly affect the inflammatory response and the occurrence and development of tumors ^[2][3] . At present, the scientific research of inflammation, tumor and other diseases is carried out by targeting BET Bromodomain protein, and the search for small molecule inhibitors with good selectivity, high safety and strong biological activity is a hot spot among the hot spots. In recent years, research on BRD4 inhibitors has also achieved some results, such as the bromodomain inhibitor (+)-JQ1 ^[4] of the methyltriazole-1,4 benzodiazepine type, and OTX015, which have very high BRD4 affinity. However, the role of BRD4 and its inhibitors in fatty liver is not clear.

references:

1. Asangani I A, Dommeti V L, Wang X, et al. Therapeutic targeting of BETbromodomain proteins in castration-resistant prostate cancer [J]. Nature, 2014, 510(7504): 278-82.

2. French C A, Miyoshi I, Kubonishi I, et al. BRD4-NUT Fusion Oncogene [J]. Cancer Research, 2003, 63(2): 304-7.

3. Segura M F, Fontanalscirera B, Gazielsovran A, et al.BRD4sustainsmelanoma proliferation and represents a new target for epigenetic therapy.[J].Cancer Research, 2013,73(20):6264.

4. Filippakopoulos P, Qi J, Picaud S, et al. Selective inhibition of BET bromodomains. [J]. Nature, 2010, 468(7327): 1067.

Contents of the invention

The purpose of the present invention is to provide the application of Bromo functional domain-containing protein 4 and its inhibitor in the preparation of medicaments for preventing, alleviating and/or treating fatty liver and related diseases.

The above-mentioned purpose is achieved through the following technical solutions:

In the present invention, different hepatocytes (L02 cells, HepG2 cells) are used as experimental objects, treated with BRD4 inhibitors-JQ1 and OTX015 respectively, DMSO is used as a control, and then treated with (palmitate/oleic acid) PA/OA, and then treated with The cells were stained with Oil Red O, and the staining results showed that compared with the DMSO control group, the color of the inhibitor treatment group was significantly lighter. This indicates that the inhibition of BRD4 can inhibit fat accumulation and improve the occurrence of fatty liver and related diseases .

On this basis, the first aspect of the present invention provides the application of Bromo domain-containing protein 4 as a drug target in screening drugs for preventing, alleviating and/or treating fatty liver and related diseases.

The second aspect of the present invention provides the application of an inhibitor of Bromo functional domain protein 4 in the preparation of drugs for the prevention, alleviation and/or treatment of fatty liver and related diseases.

Preferably, the Bromo domain-containing protein 4 inhibitor is an inhibitor that inhibits BRD4 protein activity or protein level, or an inhibitor that inhibits BRD4 mRNA level, and its inhibitory activity is reversible or irreversible.

Preferably, the inhibitors that inhibit BRD4 protein activity or protein levels include antibodies to BRD4, proteins that inhibit BRD4 protein activity or protein levels, polypeptides, enzymes, natural compounds, synthetic compounds, organic substances, and inorganic substances; the inhibitors that inhibit BRD4 protein An inhibitor of activity or protein level refers to a substance that can bind BRD4 but does not produce a biological response when bound, or that the inhibitor can block, inhibit or reduce the response mediated by the agonist and can compete with the agonist for binding BRD4.

Preferably, the BRD4 inhibitor is JQ1 or OTX015 or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof.

The term "pharmaceutically acceptable salt" as used herein refers to a derivative of a pharmaceutically active compound wherein the parent compound is modified by making an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, basic or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic or quaternary ammonium salts of the parent compound formed from, for example, non-toxic inorganic or organic acids. Such conventional nontoxic salts include, for example, those derived from inorganic acids, such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and those prepared from organic acids, such as acetic, propionic, Succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, methanesulfonic acid, toluenesulfonic acid, salicylic acid, p-aminobenzenesulfonic acid, etc.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

Preferably, the BRD4 inhibitor is JQ1 or OTX015 and pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable excipients are various excipients commonly used or known in the field of pharmacy, including but not limited to: diluents, binders, antioxidants, pH regulators, preservatives, lubricants, disintegrants, etc. solution, etc.

The diluents are, for example: lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol and the like. The binder is, for example: starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, etc. The antioxidants are, for example: vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole and the like. The pH regulator is for example: hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, etc. The preservatives are, for example: methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, m-cresol, benzalkonium chloride and the like. Described lubricant is for example: magnesium stearate, micropowder silica gel, talcum powder etc. The disintegrating agent is, for example: starch, methylcellulose, xanthan gum, croscarmellose sodium and the like.

Preferably, the BRD4 antibody includes but not limited to monoclonal antibody, synthetic antibody, polyclonal antibody, multispecific antibody, human antibody, humanized antibody, chimeric antibody, single chain Fv (scFv) (including bispecific scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), and epitope-binding fragments of any of the above. In particular, antibodies for use in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecule used in the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of the immunoglobulin molecule or subclasses. Preferably, the antibodies are human or humanized monoclonal antibodies.

As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or from mice or other animals expressing antibodies from human genes.

Preferably, the inhibitor that inhibits the mRNA level of BRD4 may be its antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA, or other proteins, polypeptides, enzymes, or compounds that can inhibit the mRNA level of BRD4.

The dosage form of medicine of the present invention can be the form of oral preparation, such as tablet, capsule, pill, powder, granule, suspension, syrup etc.; It can also be the dosage form of injection administration, such as injection, powder injection etc., through Intravenous, intraperitoneal, subcutaneous or intramuscular routes. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts.

The medicament of the present invention can be administered to any animal that develops or has developed fatty liver and related diseases. These animals include humans and non-human animals such as pets or livestock.

The agents of the present invention can be administered to a subject by routes known in the art, including but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal , buccal, sublingual, intranasal, transdermal, etc.

The dose to be administered will depend on the age, health and weight of the recipient, the type of drugs to be used in combination, the frequency of treatment, the route of administration, and the like. The drug may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times daily. Doses can be administered once or more, and the administration time can range from a single day to several months or longer.

The fatty liver and related diseases include, but are not limited to: insulin resistance, metabolic syndrome, obesity, diabetes, hyperglycemia, hyperlipidemia, simple hepatic steatosis, nonalcoholic steatohepatitis, liver fibrosis, cirrhosis, Liver cancer, etc.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention discovers a new function of the BRD4 gene, that is, the BRD4 gene has the function of exacerbating fatty liver and related diseases.

(2) Based on the role of BRD4 in exacerbating fatty liver and related diseases, it provides targets for the development of drugs for the prevention, alleviation and/or treatment of fatty liver and related diseases.

(3) The inhibitor of BRD4 can be used to prepare medicines for preventing, alleviating and/or treating fatty liver and related diseases.

(4) The present invention discovers new functions of BRD4-specific inhibitors JQ1 or OTX015, which can be used to prepare drugs for preventing, alleviating and/or treating fatty liver and related diseases.

Description of drawings

Figure 1 is the oil red O staining results of L02 cells treated with inhibitors for 3 hours and treated with 0.2/0.4mM PA/OA for 12 hours; A is the control DMSO group, B is the 0.5μM JQ1 treatment group, and C is the 0.5μM OTX015 treatment group .

Figure 2 is the oil red O staining results of HepG2 cells treated with inhibitors for 3 hours and 0.1/0.2mM PA/OA for 12 hours; A is the control group DMSO group, B is the 0.5μM JQ1 treatment group, and C is the 0.5μM OTX015 treatment Group.

Detailed ways

The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way. Experimental cell lines and culture:

L02: human liver cell line, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu6.

HepG2: human hepatoma cell line,

Cells were cultured in DMEM high glucose medium (containing 10% FBS). Culture environment: 37°C, 5% CO2.

Experimental Inhibitors:

(+)-JQ1 was purchased from sigam company, item number: SML1524

OTX015 was purchased from sigam company, item number: SML1605

Oil red O staining of cells:

1. Take out the cells, suck out the medium, add PBS to rinse 3 times, and blot the PBS as dry as possible after washing.

Fix with 2.4% paraformaldehyde at 37°C for 15 minutes.

3. After the end, discard the formaldehyde, add PBS to rinse 3 times, 3 minutes each time.

4. Add 60% isopropanol to act for 30s, and wash 3 times with PBS after completion.

5. Dry the water in the ultra-clean table. After the water is completely dry, the bottom of the dish will be white.

6. Prepare the working solution of Oil Red O, Red Oil: PBS = 3:2 configuration. After the oil red is prepared, let it stand at room temperature for 10 minutes, then filter it with a 0.45 μM filter, and then use it.

7. After drying, add working solution, observe in time during the dyeing process, absorb and discard the dyeing solution after reaching the requirements.

8. Wash 3 times with PBS, add PBS to infiltrate and take pictures for observation.

[Example 1] Effect of BRD4 inhibitor treatment on lipid accumulation in L02 cells

L02 cells were divided into three groups for culture. After the cells adhered to the wall, they were treated with DMSO, 0.5 μM JQ1, and 0.5 μM OTX015 for 3 hours, and then treated with 0.2/0.4 mM PAOA for 12 hours, and stained with Oil Red O.

The staining results are shown in Figure 1. Compared with the control group DMSO group, the color of the inhibitor treatment group was significantly lighter. The results indicated that lipid accumulation was significantly inhibited in L02 cells treated with BRD4 inhibitor. [Example 2] Effect of BRD4 inhibitor treatment on lipid accumulation in HepG2 cells

HepG2 cells were divided into three groups and cultured. After the cells adhered to the wall, they were treated with DMSO, 0.5 μM JQ1, and 0.5 μMOTX015 for 3 hours, and then treated with 0.1/0.2 mM PA/OA for 12 hours before staining with Oil Red O.

The staining results are shown in Figure 2. Compared with the control group DMSO group, the color of the inhibitor treatment group was significantly lighter. The results indicated that lipid accumulation was significantly inhibited in HepG2 cells treated with BRD4 inhibitor.

The above results show that inhibiting the expression of BRD4 can significantly improve the hepatic lipid accumulation induced by PA/OA stimulation, indicating that the BRD4 gene has a significant effect on the occurrence and development of fatty liver and related diseases. BRD4 can be used to screen drugs for preventing, alleviating and/or treating fatty liver and related diseases, and BRD4 inhibitors such as JQ1 and OTX015 can be used for preparing drugs for preventing, alleviating and/or treating fatty liver and related diseases.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (8)

1. The application of Bromo functional domain-containing protein 4 as a drug target in the screening and preparation of drugs for the prevention, alleviation and/or treatment of fatty liver and related diseases. 2. The application according to claim 1, characterized in that, the drug for preventing, alleviating and/or treating fatty liver and its related diseases is a drug for inhibiting the expression of Bromo functional domain-containing protein 4. 3. The application of the inhibitor containing Bromo functional domain protein 4 in the preparation of drugs for preventing, alleviating and/or treating fatty liver and related diseases. 4. application according to claim 3, is characterized in that, the inhibitor of described Bromo functional domain protein 4 is the inhibitor that suppresses Bromo functional domain protein 4 protein activity or protein level, or inhibits the mRNA of Bromo functional domain protein 4 Levels of inhibitors whose inhibitory activity is reversible or irreversible. 5. application according to claim 4, is characterized in that, the inhibitor that described inhibition Bromo functional domain protein 4 protein activity or protein level comprises the antibody of Bromo functional domain protein 4, inhibits Bromo functional domain protein 4 protein activity or protein Proteins, polypeptides, enzymes, natural compounds, synthetic compounds, organic substances, inorganic substances at the level of proteins; the inhibitors that inhibit Bromo functional domain protein 4 protein activity or protein levels refer to the inhibitors that can bind to Bromo functional domain protein 4 but do not produce The substance of the biological response, or the inhibitor can block, inhibit or weaken the response mediated by the agonist, and can compete with the agonist for binding to Bromo domain protein 4. 6 . The application according to claim 4 , wherein the Bromo domain protein 4 inhibitor is JQ1 or OTX015 or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a metabolite thereof. 7. application according to claim 4, is characterized in that, the inhibitor of the mRNA level of described suppression Bromo functional domain protein 4 is its antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA, or other can suppress Bromo function Proteins, polypeptides, enzymes, compounds at the mRNA level of domain protein 4. 8. The application according to any one of claims 1-7, characterized in that fatty liver and related diseases include but not limited to: simple hepatic steatosis, nonalcoholic steatohepatitis, liver fibrosis, liver cirrhosis, Liver cancer, etc.