Application of ANKRD22 in the preparation of therapeutic drugs for metabolic-related fatty liver disease

Abstract

The present invention relates to the field of biomedicine, and discloses the use of ANKRD22 in the preparation of a drug for treating metabolic-related fatty liver disease. The present invention first discovered that there is a correlation between ANKRD22 and MAFLD, and ANKRD22 can be used as a target for treating MAFLD, and can meet the needs of early treatment of MAFLD; in addition, the present invention also provides 4 small molecule compounds that can target ANKRD22 and promote the expression of ANKRD22, which have important clinical potential application prospects for the treatment of MAFLD.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of ANKRD22 as a target in the preparation of a drug for treating metabolism-related fatty liver disease. Background Art

[0002] Metabolic-associated fatty liver disease (MAFLD) is currently the most common chronic liver disease in the world, which can eventually progress to cirrhosis, liver cancer, and liver failure. MAFLD patients have an insidious onset, slow progression, and are generally asymptomatic. Most patients have comorbidities such as obesity, type 2 diabetes, dyslipidemia, and / or hypertension. Currently, a large number of studies at home and abroad focus on the occurrence and development of MAFLD and its molecular mechanisms, but there is still no specific early treatment for MAFLD.

[0003] Domestic and international MAFLD diagnosis and treatment guidelines unanimously recommend lifestyle changes as the first-line measure and cornerstone for treating fatty liver and its related inflammatory fibrosis. Patients with simple fatty liver can completely reverse fatty liver through lifestyle intervention for more than half a year, but compliance and sustainability of lifestyle intervention treatment are currently difficult points in the treatment of MAFLD.

[0004] The main clinical treatment options for MAFLD are liver protection and lipid-lowering. Among them, liver protection drugs (such as bicyclol, bifendate, adenosine triphosphate, etc.) are mainly used for patients with fatty hepatitis such as abnormal liver function, and lipid-lowering drugs (such as statins, fibrates, niacin, etc.) are mainly used for patients with combined hyperlipidemia, cardiovascular and cerebrovascular diseases, etc.; in the early stages of MAFLD, although there is lipid deposition and abnormal lipid metabolism in the liver, blood lipid indicators such as triglycerides and cholesterol in the peripheral blood and liver function indicators such as ALT and AST have not yet shown abnormalities, and traditional liver protection and lipid-lowering drugs are not applicable.

[0005] With the continuous development of molecular biology technology, exploring target genes or cell subpopulations closely related to the disease has become an important approach for targeted therapy research. For most patients with early MAFLD who have not yet developed liver inflammation, fibrosis, and abnormal liver function, it is of great significance to explore targets to intervene in the treatment strategy of early MAFLD progression. However, no target genes or target cell populations that can be used for the treatment of MAFLD have been reported so far. Providing a target that can effectively improve liver lipid accumulation is an urgent problem to be solved. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides the use of ANKRD22 in the preparation of a drug for treating metabolic-associated fatty liver disease. The present invention first discovered that there is a correlation between ANKRD22 and metabolic-associated fatty liver disease (MAFLD). ANKRD22 can be used as a target for treating MAFLD and can meet the needs of early treatment of MAFLD.

[0007] The specific technical scheme of the present invention is:

[0008] In a first aspect, the present invention provides the use of ANKRD22 as a target in the preparation of a drug for treating metabolism-related fatty liver disease.

[0009] ANKRD22 protein is a small mitochondrial protein with four ankyrin repeat motifs (ANK). Its application in the diagnosis and treatment of tuberculosis has been reported, but its association with metabolic-related fatty liver disease has not been reported.

[0010] The present invention found that there is a correlation between ANKRD22 and metabolic-related fatty liver disease. Long-term loss of ANKRD22 can cause the occurrence and development of MAFLD, while normal expression of ANKRD22 helps to avoid the occurrence and development of MAFLD. Specifically, after long-term normal diet feeding, the weight and body fat rate of ANKRD22-negative mice increased significantly, subcutaneous and visceral lipids accumulated, especially lipid deposition in the liver, and liver triglyceride levels increased; while the weight and body fat rate of ANKRD22-positive mice increased relatively less, and there was less lipid deposition in the liver, and the increase in liver triglyceride levels was also relatively small. Therefore, ANKRD22 can be used as a target for the development of drugs to treat MAFLD.

[0011] In addition, in the early stages of MAFLD, although there is lipid deposition and abnormal lipid metabolism in the liver, blood lipid indicators such as triglycerides and cholesterol in the peripheral blood and liver function indicators such as ALT and AST have not yet shown abnormalities, and traditional liver protection and lipid-lowering drugs are not applicable. Therapeutic drugs targeting ANKRD22 can inhibit lipid deposition in the liver and increase the level of triglycerides in the liver, so they can meet the needs of early treatment of MAFLD and intervene in the early development of MAFLD, which is expected to break through the current situation where there is no specific early treatment plan for MAFLD.

[0012] Furthermore, the drug is a drug that reduces lipid accumulation in the liver, or a drug that reverses or blocks abnormal liver lipid metabolism.

[0013] Furthermore, the drug is one or more of chemical drugs, nucleic acids, polypeptides and proteins.

[0014] Furthermore, the chemical drugs include one or more of the following substances:

[0015] (a) a compound represented by structural formula (I) to (IV);

[0016] (b) derivatives or mixtures of the compounds represented by structural formulas (I) to (IV) in which one or more groups are substituted and / or one or more covalent bonds are broken; the derivatives or mixtures contain at least four hydrophobic ring structures;

[0017]

[0018]

[0019] In a second aspect, the present invention provides the use of an agent for increasing the expression level of ANKRD22 in the preparation of a drug for treating metabolism-related fatty liver disease.

[0020] Furthermore, the agent for increasing the expression level of ANKRD22 is an agent for increasing the expression level of ANKRD22 mRNA and / or ANKRD22 protein in monocytes and / or macrophages.

[0021] The inventor team found that ANKRD22 is hardly expressed in liver tissue cells. In the case of long-term loss of ANKRD22, abnormal lipid metabolism (liver lipid deposition) may be mediated by monocytes-macrophages (including liver Kupffer cells), that is, the occurrence and development of MAFLD is caused by the disorder of ANKRD22 expression in monocytes-macrophages. Therefore, by increasing the expression level of ANKRD22 in monocytes and / or macrophages, it can be used to treat MAFLD and intervene in the occurrence and development of MAFLD.

[0022] Furthermore, the agent for increasing the expression level of ANKRD22 is one or more of a polypeptide, a protein, a chemical drug and a gene editing system.

[0023] Currently, effective means to increase the expression level of ANKRD22 mRNA or protein include peptides and proteins (including antibodies), as well as chemical drugs and gene editing systems. Increasing the expression level of ANKRD22 by these means can be used as a way to treat MAFLD.

[0024] Furthermore, the agent for increasing the expression level of ANKRD22 includes interferon-γ and / or its derivatives.

[0025] Experiments have confirmed that interferon-γ (IFN-γ) can activate macrophages and promote the expression of ANKRD22 in them. Therefore, interferon-γ can be used as one of the treatments for MAFLD.

[0026] Furthermore, the chemical drugs include one or more of the following substances:

[0027] (a) a compound represented by structural formula (I) to (IV);

[0028] (b) derivatives or mixtures of the compounds represented by structural formulas (I) to (IV) in which one or more groups are substituted and / or one or more covalent bonds are broken; the derivatives or mixtures contain at least four hydrophobic ring structures;

[0029]

[0030] The experiments showed that the four compounds shown in structures (I) to (IV) can promote the expression of ANKRD22 and are expected to be used in the treatment of MAFLD.

[0031] Furthermore, the gene editing system includes reagents for delivering the ANKRD22 gene and / or ANKRD22 protein into monocytes and / or macrophages or Kupffer cells.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) The present invention is the first to discover that there is a correlation between ANKRD22 and MAFLD. ANKRD22 can be used as a target for the treatment of MAFLD and can meet the needs of early treatment of MAFLD.

[0034] (2) The present invention provides four small molecule compounds that can target ANKRD22 and promote the expression of ANKRD22, which have important clinical potential application prospects for the treatment of MAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The organ tissues of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) fed with a normal diet for 32 days (8W) and 180 days (180d). Figure 1 (A) is a photo of each organ tissue; Figure 1 (B) is a microscopic examination of liver tissue pathological sections.

[0036] Figure 2 The figure shows the body weight changes of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) during 0 to 184 days of feeding with a normal diet.

[0037] Figure 3 Body fat percentage of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) fed with a normal diet for 32 days (8W) and 180 days (180d).

[0038] Figure 4These are the magnetic resonance imaging images of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) fed with a normal diet for 32 days (8W) and 180 days (180d).

[0039] Figure 5 These are the results of lipid-related index detection in liver tissue of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) when they were fed a normal diet for 32 days and 180 days.

[0040] Figure 6 The lipid levels in liver tissue of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) fed with a normal diet for 180 days. Figure 6 (A)~ Figure 6 (F) The levels of cholesterol (CHO), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), apolipoprotein (APOB) and lipoprotein a (LPa) in liver tissue, respectively.

[0041] Figure 7 The expression of ANKRD22 in liver and gastric mucosa. Figure 7 (A) ANKRD22 protein expression in liver and gastric mucosa; Figure 7 (B) The expression of ANKRD22 in liver and gastric mucosa.

[0042] Figure 8 The effect of cytokine (IFN-γ, IL-4 and IL-10) stimulation on the expression of ANKRD22. Figure 8 (A) Effects of cytokine stimulation on ANKRD22 expression in THP-1 cells; Figure 8 (B) Effects of cytokine stimulation on the expression of ANKRD22 in ImKC cells; Figure 8 (C) Effects of cytokine stimulation on ANKRD22 protein expression in Lo2 cells, Lx2 cells and THP-1 cells.

[0043] Fig. 9 The results of monocyte detection in ANKRD22-positive mice (WT) and ANKRD22-negative mice (KO) fed with a normal diet for 32 days (8W+) and 180 days (180d+). Fig. 9 (A) is the percentage of mononuclear cells (MO%); Fig. 9 (B) is the mononuclear cell count (MO#).

[0044] Fig.10Immunohistochemical staining of CD68-positive macrophages in the liver of ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) fed with a normal diet for 32 days (8W) and 180 days (180d).

[0045] Fig.11 The effects of four small molecule compounds on the expression level of ANKRD22 in LS-174T cells.

[0046] Fig.12 Effects of four small molecule compounds on Ca2+ in THP-1 cells 2+ The influence of concentration. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the embodiments.

[0048] The terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the scope of protection of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0049] Example 1: ANKRD22-negative mice can cause the development of MAFLD

[0050] The animals were housed in a 12-hour light-dark cycle housing room at a constant temperature and humidity. Feed and water were available ad libitum. All animal procedures were performed in accordance with procedures approved by the Animal Welfare Ethics Committee.

[0051] ANKRD22-positive mice (ANKRD22-WT): obtained from the Experimental Animal Center of Zhejiang University of Chinese Medicine, with normal expression of ANKRD22 gene; mice were fed a normal diet after birth, with at least 6 mice in each group.

[0052] ANKRD22-negative mice (ANKRD22-KO): commissioned by Saiye (Guangzhou) Biotechnology Co., Ltd. to construct, with ANKRD22 gene deletion; mice were fed a normal diet after birth, with at least 6 mice in each group of experiments.

[0053] The body weight of mice was measured and recorded twice a week from 0 to 184 days when they were fed a normal diet. Figure 2 .from Figure 2It can be seen that during long-term normal diet feeding, the weight gain rate of ANKRD22-negative mice was significantly higher than that of ANKRD22-positive mice.

[0054] On the 32nd day (8W) and 180th day (180d) of normal diet feeding, the body composition of mice was tested, and the measured body fat percentage was shown in Figure 3 .from Figure 3 It can be seen that during long-term normal diet feeding, the body fat percentage of ANKRD22-negative mice increased significantly, while the increase in body fat percentage of ANKRD22-positive mice was relatively small.

[0055] On the 32nd day (8 weeks) and 180th day (180 days) of normal diet feeding, small animal MRI was performed. The results are shown in Figure 4 .from Figure 4 It can be seen that during long-term normal diet feeding, ANKRD22-negative mice showed obvious accumulation of subcutaneous fat and visceral fat throughout the body, while ANKRD22-positive mice had relatively less fat accumulation in their bodies.

[0056] On the 32nd day (8W) and 180th day (180d) of normal diet feeding, the mice were anesthetized with CO2 gas, and the organs and tissues were separated after being killed. The results are shown in Figure 1 (A) Subsequently, part of the tissue was fixed in 4% paraformaldehyde overnight, and part was quickly frozen in liquid nitrogen and stored at -80°C. The liver tissue was sliced ​​and examined under a microscope after immunohistochemical staining. The results are shown in Figure 1 (B) From Figure 1 It can be seen that during long-term normal diet feeding, ANKRD22-negative mice had obvious lipid deposition in the liver and large fat vacuoles formed in the liver, while ANKRD22-positive mice had less fat deposition in the liver and no obvious large fat vacuoles were observed.

[0057] Conclusion: Long-term deficiency of ANKRD22 can cause the occurrence and development of MAFLD in mice, while normal expression of ANKRD22 can help avoid the occurrence and development of MAFLD.

[0058] Example 2: Abnormal lipid metabolism in the liver of ANKRD22-negative mice

[0059] The liver tissues of ANKRD22-positive and ANKRD22-negative mice fed with normal diet on the 32nd and 180th day in Example 1 were taken for non-targeted lipid mass spectrometry detection. The results are shown in Figure 5 . Figure 5 The figure shows the changes in lipid composition in mouse liver, from which it can be seen that long-term loss of ANKRD22 can cause upregulation of triglyceride levels in mouse liver.

[0060] After the liver tissue of mice fed with a normal diet for 180 days was homogenized, the levels of lipids such as triglycerides and cholesterol in the tissue were detected. Figure 6 .from Figure 6 It can be seen that during long-term normal diet feeding, the triglyceride level in the liver of ANKRD22-negative mice was significantly upregulated, while the increase in ANKRD22-positive mice was relatively small.

[0061] Conclusion: Long-term deficiency of ANKRD22 will cause abnormal lipid metabolism and increased triglyceride levels in the liver of mice, while normal expression of ANKRD22 can inhibit abnormal lipid metabolism and increased triglyceride levels in the liver.

[0062] Example 3: Macrophages mediate lipid deposition in ANKRD22-negative mice

[0063] Human and mouse liver tissues and gastric mucosal tissues (as positive controls) were obtained, ground and then protein was extracted and lysed. The expression of ANKRD22 was detected by immunoblotting and RT-qPCR. The results are shown in Figure 7 .from Figure 7 It can be seen that ANKRD22 is highly expressed in the stomach and almost not expressed in the liver.

[0064] Human macrophages THP-1, mouse liver Kupffer cells ImKC, human hepatocytes Lo2, and human hepatic stellate cells Lx2 were stimulated with cytokines (IFN-γ, IL-4, and IL-10), i.e., 20 ng / mL of cytokines (IFN-γ, IL-4, and IL-10) were added to the cell culture medium for 48 h; the expression level of ANKRD22 was detected by immunoblotting and RT-qPCR. The results are shown in Figure 8 .from Figure 8 It can be seen that after IFN-γ stimulation, macrophages and Kupffer cells were activated, and the expression level of ANKRD22 was upregulated, while there was no obvious change in the expression of ANKRD22 in hepatocytes and hepatic stellate cells.

[0065] In Example 1, on the 32nd day (8W) and the 180th day (180d) of normal diet feeding, ANKRD22-positive mice (ANKRD22-WT) and ANKRD22-negative mice (ANKRD22-KO) were anesthetized with CO2 gas, blood was collected from the eyeballs, and blood routine and blood biochemistry were tested. The results are shown in Table 1, where the monocyte percentage (MO%) and monocyte count (MO#) are shown in Table 1. Fig. 9 From Table 1 and Fig. 9 It can be seen that long-term deficiency of ANKRD22 will cause a significant decrease in the absolute number and percentage of peripheral blood monocytes in mice.

[0066] Table 1

[0067]

[0068] On the 32nd day (8W) and 180th day (180d) of normal diet feeding, ANKRD22-positive mice and ANKRD22-negative mice were killed and liver tissues were obtained for immunohistochemical staining of CD68 markers. The results are shown in Fig.10 .from Fig.10 It can be seen that long-term deficiency of ANKRD22 will cause an increase in the number of CD68-positive macrophages in the liver of mice.

[0069] Conclusion: Long-term deficiency of ANKRD22 can lead to a decrease in peripheral blood monocytes and an increase in CD68-positive macrophages in the liver of mice; in addition, ANKRD22 is almost not expressed in liver tissue, but ANKRD22 expression is upregulated after macrophages are activated. Therefore, long-term deficiency of ANKRD22 may be caused by abnormal lipid metabolism (liver lipid deposition) mediated by macrophages (including liver Kupffer cells).

[0070] Example 4: Small molecule compounds that promote ANKRD22 expression

[0071] The four small molecule compounds in Table 2 were respectively acted on LS-174T cells (in which ANKRD22 background expression was high) for 24 h. After protein extraction and lysis, the changes in ANKRD22 expression levels after the action of small molecule compounds were detected by immunoblotting. The results are shown in Fig.11 .from Fig.11 It can be seen that these four small molecules can promote the expression of ANKRD22.

[0072] The four small molecule compounds in Table 2 were respectively treated with THP-1 cells for 24 h, and the intracellular Ca2+ was detected by Fluo-4 flow cytometry. 2+ The concentration of FITC was changed and the average fluorescence intensity of FITC was measured. The results are shown in Fig.12 .from Fig.12 It can be seen that the four small molecule compounds can increase Ca2+ in THP-1 cells. 2+ concentration, among which compounds F637-1182 and Y504-9387 increased Ca in THP-1 cells at lower concentrations. 2+ concentration.

[0073] Table 2

[0074]

[0075]

[0076] Conclusion: The four small molecule compounds in Table 2 can promote the expression of ANKRD22 and are expected to be used in the treatment of MAFLD.

[0077] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of compound I in the preparation of a drug for treating metabolic-related fatty liver disease, characterized in that: The structural formula of the compound I is as follows: 。 2. The use according to claim 1, characterized in that The drug is a drug for reducing lipid accumulation in the liver.

3. The use according to claim 1, characterized in that The drug is a drug for reversing abnormal liver lipid metabolism.

4. The use according to claim 1, characterized in that The drug is a drug for blocking abnormal liver lipid metabolism.

5. The use according to claim 1, characterized in that The drug is a drug for increasing the expression level of ANKRD22.

6. The use according to claim 1, characterized in that The drug is a drug for inhibiting the increase of triglyceride level.

7. The use according to claim 1, characterized in that The medicine is a chemical medicine.

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