Application of histone acetyltransferase inhibitor in preparation of medicine for improving liver fatty degeneration

Abstract

The invention discloses application of a histone acetyltransferase inhibitor in preparation of a medicine for improving liver fatty degeneration. The invention discloses application of a histone acetyltransferase inhibitor for selectively inhibiting Gcn5 acetylation of histone H3 in preparation of drugs for treating and / or preventing diseases caused by liver fatty degeneration. And the histone acetyltransferase inhibitor for selectively inhibiting acetylation of Gcn5 on histone H3 is CPTH2. The compound CPTH2 reduces the protein abundance of a liver lipid synthesis transcription factor SREBP1c by inhibiting the acetylation activity of H3K9 and H3K14, and obviously improves the liver fatty degeneration of C57BL / 6J mice fed with high fat diet.

Description

Technical Field

[0001] The invention belongs to the field of small molecule chemical drugs and relates to the application of a histone acetyltransferase inhibitor in the preparation of a drug for improving liver fatty degeneration. Background Art

[0002] Hepatic steatosis refers to the excessive accumulation of fat under conditions of overnutrition, which leads to insulin resistance and thus causes metabolic disorders of fatty acids. When the accumulation of these fatty acids exceeds the liver's ability to oxidize fatty acids and secrete triglycerides in the form of very low-density lipoproteins, hepatic steatosis occurs. This causes a series of diseases, including non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, etc. In addition, the imbalance of lipid metabolism can also lead to the formation of lipotoxicity, such as cellular stress (i.e., oxidative stress and endoplasmic reticulum stress), inflammasome activation and apoptosis, and subsequently stimulate inflammation, tissue regeneration and fibrosis formation, thereby causing non-alcoholic steatohepatitis (NASH) characterized by liver inflammation and fibrosis. Ultimately, NASH will deteriorate into irreversible liver damage, including cirrhosis and hepatocellular carcinoma (HCC). Currently, several drugs that can be used for other indications have been studied in clinical trials for hepatic steatosis, such as ursodeoxycholic acid, omega-3 fatty acids, and metformin, which have not shown histological benefits; vitamin E increases the risk of bleeding and causes adverse cardiovascular consequences when used in high doses; and pioglitazone causes side effects such as weight gain, osteoporosis, fluid retention, and bladder cancer, which limit their use. Therefore, there is still a need to develop safer and more effective drugs.

[0003] Histone acetylation promotes gene expression by changing the spatial structure of nucleosomes, inducing chromatin loosening, and thus increasing gene transcription and replication. There are four main core histones (H2A, H2B, H3, and H4) in nucleosomes. Histone acetylation is regulated by histone acetyltransferases (HATs). HATs include multiple families such as the MYST family, the GCN5-related N-acetyltransferase (GNATs) family, and the p300 / CREB binding protein family. Different types of HATs may acetylate different sites of different histones, such as H3K9, H3K14, H4K8, H4K16, etc., thereby regulating the expression of different genes and playing different roles in different diseases. For example, GCN5, a member of the GNATs family, can significantly promote the acetylation of H3K9 in the promoter region of vascular endothelial growth factor, restore the function of endogenous bone marrow stem cells, and alleviate the defect of angiogenesis in the bone of osteoporotic mice; Tip60, a member of the MYST family, regulates the acetylation levels of H3K9 and H3K27 in the promoter region of SETDB1, inhibiting the invasion and migration ability of lung adenocarcinoma cells; p300 / CBP, a member of the p300 / CREB binding protein family, mediates the acetylation levels of H3K9 and H3K14 in the promoter histones of PAI-1 and p21, increasing the expression of PAI-1 and p21 and aggravating renal fibrosis.

[0004] GCN5, also known as KAT2A, is a member of the GNATs family. It has a HAT catalytic domain and its catalytic activity is highly conserved throughout eukaryotes. It can play a regulatory role by promoting histone H3 lysine 9 acetylation (H3K9ac) and H3 lysine 14 acetylation (H3K14ac). The role of GCN5 in the pathogenesis of different types of diseases has been widely studied, such as cancer, diabetes, multidrug resistance, and neurological diseases. However, the role of GCN5 in liver lipid metabolism is still unclear, and there are no reports on studies using GCN5 as a target for intervention to improve liver steatosis.

[0005] CPTH2, cyclopentylmethylene-[4-(4′-chlorophenyl)thiazol-2-yl)hydrazone, with a chemical structure as shown in formula (I), is a potent histone acetyltransferase (HAT) inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5. It has been used to inhibit histone acetyltransferase in cortical cultures, bud cells, and natural killer T cells (iNKT).

[0006] Summary of the invention

[0007] The object of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 for use in the preparation of a drug for treating and / or preventing diseases caused by hepatic fatty degeneration.

[0008] Another object of the present invention is to provide a pharmaceutical composition for treating and / or preventing diseases caused by fatty degeneration of the liver.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] Use of a histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 in the preparation of a drug for treating and / or preventing diseases caused by liver fatty degeneration.

[0011] As a preferred embodiment of the present invention, the histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 is CPTH2.

[0012] As a preferred embodiment of the present invention, CPTH2 specifically inhibits the activity of histone acetyltransferase protein GCN5, hindering its acetylation at H3K9 and H3K14 sites, thereby inhibiting the transcription of cholesterol regulatory element binding protein SREBP, a transcription factor that mainly regulates liver lipid synthesis, and then downregulating its downstream lipid synthesis-related genes, reducing lipid synthesis, and improving abnormal liver lipid metabolism.

[0013] As a preferred embodiment of the present invention, the lipid synthesis-related genes are the coding genes of acetyl-CoA carboxylase ACC, fatty acid synthase FASN, fatty acid desaturase 2, and stearoyl-CoA desaturase SCD1. The use according to any one of claims 1 to 3 is characterized in that CPTH2 can significantly reduce the liver weight and liver weight to body weight ratio of patients with hepatic steatosis, reduce the content of TG and TC in the liver and serum, reduce the content of AST and ALT, indicators of serum liver damage, increase the level of serum high-density lipoprotein HDL-c, reduce the level of serum low-density lipoprotein LDL-c, and reduce lipid accumulation in patients with hepatic steatosis.

[0014] As a preferred embodiment of the present invention, the disease caused by fatty degeneration of the liver is selected from non-alcoholic fatty liver, hyperlipidemia and atherosclerosis.

[0015] A pharmaceutical composition for treating and / or preventing diseases caused by liver fatty degeneration, comprising a histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 as an effective ingredient.

[0016] As a preferred embodiment of the present invention, the histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 is CPTH2.

[0017] As a preferred embodiment of the present invention, the pharmaceutical composition further comprises conventional pharmaceutical excipients.

[0018] Beneficial effects:

[0019] The main phenotype of hepatic steatosis is that the total cholesterol (TC) and total triglyceride (TG) content in serum and liver increase. This comes from inappropriate lipolysis in the circulation to transport fatty acids and liver lipid de novo synthesis (DNL) to the liver. At the same time, the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of liver damage in serum will also increase, and the corresponding changes in lipoprotein levels in serum will occur. The present invention shows through experiments that CPTH2 can inhibit the acetylation of H3K9 and H3K14 sites by specifically inhibiting the activity of histone acetyltransferase protein GCN5, thereby inhibiting the transcription of the transcription factor cholesterol regulatory element binding protein (SREBP) that mainly regulates liver lipid synthesis, and then downregulating its downstream lipid synthesis-related genes, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), stearoyl-CoA desaturase (SCD1), etc., reduce lipid synthesis, and improve liver lipid metabolism abnormalities. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The experimental results of H3K9ac and H3K14ac protein levels in mouse liver tissue;

[0021] Figure 2 The experimental results of SREBP1c protein level in mouse liver tissue;

[0022] Figure 3 The experimental results of the transcription levels of SREBP1c and its downstream target genes in mouse liver tissue;

[0023] Figure 4 The experimental results are for the weight of mice;

[0024] Figure 5 The experimental results of food intake of mice;

[0025] Figure 6 The experimental results are for mouse liver weight;

[0026] Figure 7 This is the experimental result of the ratio of liver weight to body weight of mice;

[0027] Figure 8 The experimental results of triglyceride (TG) content in mouse serum;

[0028] Fig. 9 The experimental results of total cholesterol (TC) content in mouse serum;

[0029] Fig.10 This is the experimental result of triglyceride (TG) content in mouse liver;

[0030] Fig.11 The experimental results of total cholesterol (TC) content in mouse liver;

[0031] Fig.12 The experimental results of aspartate aminotransferase (AST) content in mouse serum;

[0032] Fig.13 The experimental results of alanine aminotransferase (ALT) content in mouse serum;

[0033] Fig.14 The experimental results are for the content of high-density lipoprotein (HDL-c) in mouse serum;

[0034] Fig.15 The experimental results of low-density lipoprotein (LDL-c) content in mouse serum;

[0035] Fig.16 The experimental results of hematoxylin-eosin staining (H&E staining) and Oil Red O staining of mouse liver tissue;

[0036] Fig.17 These are the results of the quantitative Oil Red O staining of mouse liver. Specific embodiments

[0037] Experimental methods:

[0038] After 7-week-old male C57BL / 6J mice were adapted to the environment for one week, they were fed HFD (US Research Diets, item number D12492, fat content 60%) from 8 weeks of age to induce fatty degeneration of the liver. After 4 weeks of modeling, the mice were divided into two groups according to their body weight, namely the model group and the drug-treated group. The model group mice were orally given the solvent as a control, and the drug-treated group mice were orally given CPTH2 (20 mg / kg / d). The body weight and food intake of each group of mice were recorded for later data statistics, while continuing to feed HFD. After 6 weeks, the mice were fasted overnight, and then the mice were anesthetized for blood collection, and the liver tissue was dissected after being killed.

[0039] Example 1: Effects of CPTH2 on its targets

[0040] Experimental method: Preparation of protein samples: Weigh 20-25 mg of liver tissue and homogenize it in 0.5 ml of 1×RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors. After the homogenization is completed, let it stand for 30 minutes, then centrifuge it under low temperature conditions, transfer the supernatant and perform protein content determination (BCA determination), and dilute it according to the protein concentration to make the concentration of each group of samples consistent. Then add 5× loading buffer, heat denaturation at 100℃ for 10 minutes, cool on ice and store at -80℃. Detection: Perform western blot experiment on the obtained protein samples to detect the level of target protein in mouse liver tissue.

[0041] Experimental results: Figure 1 As shown, the levels of histone H3 lysine 9 acetylation (H3K9ac) and H3K14ac proteins in liver tissues of mice administered CPTH2 were significantly reduced compared with those fed HFD, indicating that CPTH2 inhibits histone acetylation activity.

[0042] Example 2: Effect of CPTH2 on the protein level of SREBP1c, a key transcription factor in lipid synthesis

[0043] Experimental method: The preparation and detection of protein samples are the same as above. Preparation of transcription level cDNA samples: First, weigh 15-25 mg of liver tissue and homogenize it in 0.5 ml of RNA extraction reagent. After centrifugation and discarding the precipitate, extract RNA according to the RNA extraction kit and determine its concentration. After that, reverse the RNA into cDNA according to the reagent instructions. Detection: Prepare a 10 μl qPCR reaction system according to the ratio of cDNA 1 μl, enzyme-free water 3 μl, forward primer 0.5 μl, reverse primer 0.5 μl, 2×ChamQ Blue Universal SYBR qPCR Master Mix 5 μl, and detect the relative content of the target gene in mouse liver tissue.

[0044] Experimental results: SREBP belongs to the basic helix-loop-helix leucine zipper transcription factor family, which can regulate the synthesis of cholesterol and fatty acids. Its subtype SREBP-1c is mainly involved in fatty acid and glucose / insulin metabolism, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), etc. Figure 2-3 As shown, the transcription and protein levels of SREBP1c in the liver tissue of mice given CPTH2 were significantly reduced, thereby inhibiting downstream lipid synthesis-related genes, including ACC, FASN, FADS2, and SCD1, and reducing excessive lipid synthesis in the liver.

[0045] Example 3: Effect of CPTH2 on hepatic steatosis

[0046] Experimental method: The body weight and average food intake of mice were recorded at the same time every week after grouping (marking the tail with a marker). After the experiment, all graphs and statistics were performed by GraphPad Prism8.0, and the data were expressed as mean standard error (SE). Statistical analysis was performed by t-test, and differences with p<0.05 were considered statistically significant (*, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant).

[0047] Experimental results: Figure 4-5 As shown in Figure 2, the weight of mice gradually increased with HFD feeding, but there was no significant difference in body weight and food intake between the model group and the CPTH2 administration group. Figure 6-7 As shown, compared with the mice in the model group, the liver weight and liver weight to body weight ratio (LW / BW) of the mice in the CPTH2 administration group were statistically significantly decreased, with the liver weight down by 10% and the liver weight to body weight ratio down by 13%.

[0048] Example 4: Changes in the levels of total cholesterol (TC) and total triglycerides (TG) in serum and liver

[0049] Experimental method: Separation of serum: Blood was collected from the orbits of anesthetized mice, and the blood was centrifuged after standing at low temperature for 4 hours. The upper serum was transferred to a new centrifuge tube and stored at -80 degrees Celsius. The total cholesterol (TC) and total triglyceride (TG) content in serum were detected according to the instructions of the conventional kit. Extraction of lipid components in liver tissue: 25-30 mg of liver tissue was weighed and homogenized in 0.5 ml PBS. After standing for 30 minutes, 60 ul of supernatant was centrifuged and used for BCA quantitative detection. 1 ml of organic extraction reagent (chloroform: methanol = 2:1 volume fraction) was added to the remaining sample, vortexed evenly, and shaken for 3 hours. Then 0.5 ml of 0.9% saline was added, vortexed evenly, and centrifuged. The lower organic phase was transferred to a new centrifuge tube, evaporated in a fume hood, and stored overnight. 80 ul of 0.1% Triton X-100 (prepared with anhydrous ethanol) was added to dissolve the lipids at the bottom of the tube and stored at 4 °C. The contents of total cholesterol (TC) and total triglyceride (TG) in liver tissue were detected according to the instructions of routine kits.

[0050] Experimental results: Figure 8-9 As shown in Figure 2, compared with the model group, the serum triglyceride (TG) level in the CPTH2 administration group decreased by about 50%, and the serum total cholesterol (TC) level decreased to 33% of the model group. These indicators were more obvious in the liver, such as Figure 10-11As shown, the liver triglyceride level (TG) of the CPTH2 administration group decreased to 11.9% of the model group, and the liver total cholesterol (TC) level decreased to 24.7% of the model group. These results indicate that CPTH2 can significantly improve HFD diet-induced liver fat accumulation.

[0051] Example 5: Changes in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels

[0052] Experimental method: Serum was obtained according to the operation described in Example 4. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were detected according to the instructions of the conventional kit.

[0053] Experimental results: By detecting liver damage indicators in mouse serum, it was found that Figure 12-13 As shown in the results, CPTH2 can reduce the levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), suggesting that CPTH2 can alleviate liver damage and improve liver function damaged by lipid accumulation.

[0054] Example 6: Changes in the levels of high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) in serum

[0055] Experimental method: Serum was obtained according to the operation described in Example 4. The content of high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) in serum was detected according to the instructions of the conventional kit.

[0056] Experimental results: Figure 14-15 As shown, the level of high-density lipoprotein (HDL-c) in the serum of mice in the CPTH2 administration group increased significantly by 1.5 times, while the level of low-density lipoprotein (LDL-c) decreased to 37.8% of the model group. This indicates that CPTH2 inhibits the transport of cholesterol to the periphery and promotes the transport of cholesterol to the liver for catabolism, indicating that CPTH2 can regulate HFD-induced dyslipidemia.

[0057] Example 7: HE and Oil Red O pathological sections of liver tissue

[0058] Experimental methods: The liver was fixed in 4% paraformaldehyde at 4 °C overnight, embedded in paraffin, and cut into 5 μm thick sections. Liver sections were stained with hematoxylin and eosin (H&E) to evaluate morphology. Liver sections were stained with Oil Red O and counterstained with hematoxylin to evaluate lipid accumulation. Sections were observed and captured using an upright fluorescence microscope (Olympus, BX53, Tokyo, Japan). Lipid droplets were quantified using ImageJ (Version 1.5a). GraphPad Prism 8.0 was used for graphing and statistics, and data are expressed as mean standard error (SE). Statistical analysis was performed by t-test, and differences with p < 0.05 were considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

[0059] Experimental results: Fig.16 The liver histopathological sections shown in the figure show that the HFD diet induced obvious vacuoles and red-stained lipid droplets in the liver, while the liver of mice in the CPTH2 administration group was reversed. Fig.17 As shown, the relative quantification results of the red area representing neutral lipids in the Oil Red O staining images showed that the CPTH2 administration group significantly reduced lipid accumulation in the liver.

[0060] It can be seen from the above experimental results that compared with the model group, on the one hand, the gene and protein levels of H3K9ac, H3K14ac and SREBP1c in the liver tissue of mice in the CPTH2 administration group were reduced. Correspondingly, the levels of its downstream lipid synthesis target genes were also inhibited. On the other hand, the mice in the CPTH2 administration group did not show differences in body weight and food intake, but there was a significant decrease in liver weight and liver weight to body weight ratio. At the same time, the TG and TC contents in the liver and serum were reduced, the AST and ALT contents of serum liver injury indicators were reduced, the serum high-density lipoprotein (HDL-c) level was increased, and the serum low-density lipoprotein (LDL-c) level was significantly reduced. H&E and Oil Red O stained tissue sections showed that the CPTH2 administration group significantly reduced lipid accumulation in HFD-fed mice.

[0061] In summary, compound CPTH2 significantly improved hepatic steatosis in C57BL / 6J mice fed a high-fat diet by inhibiting the acetylation activity of H3K9 and H3K14 and downregulating the protein abundance of the hepatic lipid synthesis transcription factor SREBP1c.

Claims

1. Use of a histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 in the preparation of a drug for treating and / or preventing diseases caused by hepatic fatty degeneration.

2. The use according to claim 1, characterized in that: The histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 is CPTH2.

3. The use according to claim 2, characterized in that: CPTH2 specifically inhibits the activity of histone acetyltransferase protein GCN5, hindering its acetylation at H3K9 and H3K14 sites, thereby inhibiting the transcription of cholesterol regulatory element binding protein SREBP, a transcription factor that mainly regulates liver lipid synthesis, and then downregulating its downstream lipid synthesis-related genes, reducing lipid synthesis and improving abnormal liver lipid metabolism.

4. The use according to claim 3, characterized in that: The lipid synthesis-related genes are the coding genes of acetyl-CoA carboxylase ACC, fatty acid synthase FASN, fatty acid desaturase 2, and stearoyl-CoA desaturase SCD1.

5. The use according to any one of claims 1 to 3, characterized in that: CPTH2 can significantly reduce the liver weight and liver-weight-to-body weight ratio of patients with hepatic fatty degeneration, reduce the liver and serum TG and TC contents, reduce the serum liver injury indicators AST and ALT contents, increase the serum high-density lipoprotein HDL-c level, reduce the serum low-density lipoprotein LDL-c level, and reduce lipid accumulation in patients with hepatic fatty degeneration.

6. The use according to claim 5, characterized in that: The disease caused by liver fatty degeneration is selected from non-alcoholic fatty liver, hyperlipidemia and atherosclerosis.

7. A pharmaceutical composition for treating and / or preventing diseases caused by fatty degeneration of the liver, characterized in that: The active ingredient is a histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5.

8. The pharmaceutical composition according to claim 6, characterized in that The histone acetyltransferase inhibitor that selectively inhibits the acetylation of histone H3 by Gcn5 is CPTH2.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also contains conventional pharmaceutical excipients.

Images