Use of small molecule compounds that inhibit hnf4a degradation in the preparation of medicaments for treating liver cancer

Abstract

The application discloses application of a small-molecule compound for inhibiting HNF4 alpha degradation in preparation of a liver cancer treatment drug. The structural general formula of the small-molecule compound for inhibiting HNF4 alpha degradation is shown in the following formula: the small-molecule compound for inhibiting HNF4 alpha degradation provided by the application can play the biological effects of correcting glycolipid metabolism disorder of the liver, reducing liver fibrosis and inhibiting malignant phenotypes of liver cancer, and is applied to clinical treatment of chronic liver diseases and liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of a small molecule compound that inhibits the degradation of HNF4α in the preparation of drugs for treating liver cancer. Background Technology

[0002] Hepatocyte nuclear factor-4α (HNF4α) is a highly conserved ligand-dependent transcription factor in the nuclear receptor superfamily. It is expressed in tissues such as the liver, kidney, pancreas, and intestine, but primarily in mature hepatocytes. In mature hepatocytes, HNF4α binds to promoters of approximately 12% of intracellular genes, participating in the maintenance of important functions such as lipid metabolism, albumin synthesis, drug detoxification, energy metabolism, and bile acid synthesis. It plays a crucial regulatory role in hepatocyte differentiation and maintaining hepatocyte function. HNF4α also regulates the development of kidney and intestinal tissues and regulates insulin production in pancreatic tissue. Previous studies have shown that HNF4α expression is decreased in various epithelial-derived tumors, including hepatocellular carcinoma (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), pancreatic cancer, colorectal cancer, and renal cancer. Overexpression of HNF4α can inhibit tumor cell proliferation and metastasis and promote tumor cell apoptosis, suggesting that HNF4α is a potential target for tumor therapy. The use of adenovirus vectors to mediate HNF4α overexpression in liver cancer cells has been reported, and the therapeutic effect of HNF4α on tumors has been clarified. Meanwhile, previous studies have also shown that HNF4α expression is significantly decreased in various chronic liver diseases, including fatty liver and liver fibrosis. In animal experiments, upregulating HNF4α expression in damaged hepatocytes during fatty liver and liver fibrosis using genetic engineering can promote hepatocyte function and alleviate hepatocyte fat deposition and the progression of liver fibrosis, demonstrating the important therapeutic value of HNF4α for chronic liver diseases. These studies clearly indicate that increasing HNF4α expression has a therapeutic effect on chronic liver diseases and liver cancer.

[0003] Tripartite motif (TRIM) proteins belong to the E3 ubiquitin ligase subfamily and participate in various biological processes such as intracellular signal transduction, apoptosis, autophagy, and immunity by regulating the ubiquitination of target proteins. Among them, TRIM47 protein is closely related to the development and progression of chronic liver disease and liver cancer. TRIM47, as an E3 ubiquitin ligase, has been found to interact with HNF4α through its SPRY domain and mediate its ubiquitination and degradation, thereby reducing HNF4α levels. Therefore, blocking the TRIM47-HNF4α interaction to stabilize HNF4α is an important direction for developing HNF4α-based drugs against chronic liver disease and liver cancer. Therefore, screening small molecule compounds targeting the three-dimensional conformation of the TRIM47-HNF4α binding region holds promise for inhibiting the ubiquitination and degradation of HNF4α by TRIM47.

[0004] There are currently no research reports on the application of blocking or inhibitory methods that affect the binding of TRIM47 to HNF4α protein. Summary of the Invention

[0005] The purpose of this invention is to provide the application of a small molecule compound that inhibits the degradation of HNF4α in the preparation of drugs for treating liver cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of a small molecule compound or a pharmaceutical salt thereof that inhibits the degradation of HNF4α in the preparation of a drug for treating liver cancer, wherein the general structural formula of the small molecule compound that inhibits the degradation of HNF4α is shown below:

[0008]

[0009] Wherein, R1 is selected from C1~C20 alkyl groups, ;

[0010] R2 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0011] R3 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0012] R4 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0013] R5 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0014] R6 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0015] R7 is selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy;

[0016] R8 is selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy;

[0017] R9 is selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy;

[0018] R 10 Selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy groups;

[0019] R 11 Selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy groups;

[0020] R 12 Selected from hydrogen and C1-C20 alkyl groups;

[0021] Alternatively, R4 and R5 can form five-membered or six-membered rings with carbon and oxygen (e.g., R4 and R5). (The dashed line indicates the junction with the benzene ring).

[0022] Preferably, in the small molecule compound that inhibits HNF4α degradation,

[0023] R1 is selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl,

[0024]

[0025] R2 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0026] R3 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0027] R4 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0028] R5 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0029] R6 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0030] Alternatively, a five-membered ring composed of R4, R5, carbon, and oxygen. ;

[0031] Alternatively, a six-membered ring composed of R4, R5, carbon, and oxygen. The dashed line indicates the junction with the benzene ring.

[0032] Most preferably, the small molecule compound that inhibits HNF4α degradation is selected from one of the following structures:

[0033]

[0034] In this application, a small molecule compound or its pharmaceutical salt that inhibits the degradation of HNF4α is used as the sole active ingredient.

[0035] In a second aspect, the present invention provides the use of a small molecule compound or a pharmaceutical salt thereof that inhibits the degradation of HNF4α in the preparation of a medicament for treating chronic liver disease.

[0036] The general structural formula of the small molecule compound that inhibits HNF4α degradation is shown below:

[0037]

[0038] Wherein, R1 is selected from C1~C20 alkyl groups, ;

[0039] R2 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0040] R3 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0041] R4 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0042] R5 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0043] R6 is selected from hydrogen, C1~C20 alkyl, C1~C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine). ;

[0044] R7 is selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy;

[0045] R8 is selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy;

[0046] R9 is selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy;

[0047] R 10 Selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy groups;

[0048] R 11 Selected from hydrogen, C1~C20 alkyl, and C1~C20 alkoxy groups;

[0049] R 12 Selected from hydrogen and C1-C20 alkyl groups;

[0050] Alternatively, R4 and R5 can form five-membered or six-membered rings with carbon and oxygen (e.g., R4 and R5). (The dashed line indicates the junction with the benzene ring).

[0051] Preferably, in the small molecule compound that inhibits HNF4α degradation,

[0052] R1 is selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl,

[0053]

[0054] R2 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0055] R3 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0056] R4 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0057] R5 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0058] R6 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentoxy, fluorine, chlorine, bromine, ;

[0059] Alternatively, a five-membered ring composed of R4, R5, carbon, and oxygen. ;

[0060] Alternatively, a six-membered ring composed of R4, R5, carbon, and oxygen. The dashed line indicates the junction with the benzene ring.

[0061] Most preferably, the small molecule compound that inhibits HNF4α degradation is selected from one of the following structures:

[0062] .

[0063] The chronic liver diseases mentioned are selected from non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and liver failure.

[0064] In this application, a small molecule compound or its pharmaceutical salt that inhibits the degradation of HNF4α is used as the sole active ingredient.

[0065] A third aspect of the present invention provides a pharmaceutical formulation made from the small molecule compound that inhibits HNF4α degradation or a pharmaceutical salt thereof and medically acceptable excipients.

[0066] The dosage forms of the pharmaceutical preparations include injections, capsules, tablets, granules, pills, microcapsule preparations, microsphere preparations, and nano-preparations.

[0067] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the small molecule compound that inhibits HNF4α degradation or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and a medicament for improving chronic liver disease or treating liver cancer.

[0068] The drugs used to improve chronic liver disease are selected from ursodeoxycholic acid, polyene phosphatidylcholine, glutathione, etc.

[0069] The drugs used to treat liver cancer are selected from lenvatinib, donafenib, regorafenib, atezolizumab, nivolumab, cisplatin, paclitaxel, etc.

[0070] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0071] This invention discovers that small molecule compounds that inhibit HNF4α degradation directly target intracellular HNF4α protein, promote the transcriptional activity of HNF4α protein, and upregulate the protein level of HNF4α in liver cancer cells.

[0072] This invention utilizes a series of protein function research techniques, such as protein interaction, docking, and site mutation, combined with in vitro and in vivo experiments, to determine that TRIM47 is an E3 ubiquitin ligase that binds to HNF4α and promotes its ubiquitination and degradation. It also identifies the key amino acid sites that affect the binding of TRIM47 to HNF4α protein and further screens out small molecule drugs that can inhibit the binding of TRIM47-HNF4α and maintain the stability of HNF4α protein.

[0073] The small molecule compound provided by this invention that inhibits the degradation of HNF4α can exert biological effects such as correcting liver glucose and lipid metabolism disorders, reducing liver fibrosis, and inhibiting the malignant phenotype of liver cancer. It can be applied to the clinical treatment of chronic liver disease and liver cancer.

[0074] This invention is the first to discover that TRIM47 protein can directly bind to HNF4α protein and mediate HNF4α protein degradation, thereby promoting the progression of liver cancer. Through a series of protein-protein interaction techniques, the key amino acid sites affecting the binding of TRIM47 and HNF4α proteins were identified as His534 / His600 for TRIM47 and Trp349 / Glu353 / Pro342 for HNF4α. Mutations at these sites in both proteins significantly inhibited the binding of TRIM47 to HNF4α and promoted the stability and transcriptional activity of HNF4α protein. Molecular docking experiments and MOE-Site Finder software prediction revealed that compound CZ2401 can bind to HNF4α. Furthermore, compound CZ2401 and its analogues CZ-2401-1, CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9, and CZ2401-10 can upregulate the activity of the HNF4α reporter gene and the expression level of HNF4α protein in hepatocellular carcinoma cells. Pharmacological activity experiments revealed that the preferred compound CZ2401 can inhibit hepatocyte fat deposition, alleviate the progression of CCl4-induced liver fibrosis in mice, significantly inhibit the proliferation, migration, invasion, and colony formation of hepatocellular carcinoma cells, and inhibit the growth of subcutaneous tumors implanted in mice with hepatocellular carcinoma cells.

[0075] This invention discovers that a series of small-molecule compounds, including CZ2401, can upregulate the protein level of HNF4α by inhibiting the interaction between TRIM47 and HNF4α. Specifically, compound CZ2401 directly binds to HNF4α, blocking the interaction between HNF4α and TRIM47, thereby inhibiting the ubiquitination and degradation of HNF4α. Adding compound CZ2401 during hepatocellular carcinoma cell culture can inhibit the malignant phenotype of hepatocellular carcinoma cells, promote the transcriptional activity of HNF4α, and upregulate the expression of its downstream genes. Further in vivo experiments show that compound CZ2401 can inhibit the growth of subcutaneous hepatocellular carcinoma tumors in mice, indicating that compound CZ2401 has potential therapeutic effects on tumors. On the other hand, compound CZ2401 can also inhibit hepatocyte fat deposition and alleviate liver fibrosis in mice by upregulating HNF4α, indicating that compound CZ2401 also has potential therapeutic effects on chronic liver diseases. These research results indicate that CZ-2401 and its analogues have potential therapeutic effects on chronic liver diseases and hepatocellular carcinoma, and have very good clinical application prospects. Attached Figure Description

[0076] Figure 1 A schematic diagram illustrating how TRIM47 protein knockdown significantly inhibits HNF4α protein degradation and promotes the expression of downstream liver function-related genes.

[0077] Figure 2 This is a schematic diagram showing the interaction between TRIM47 and HNF4α proteins.

[0078] Figure 3 A schematic diagram illustrating potential amino acid sites that affect the binding of TRIM47 to HNF4α protein using molecular docking technology.

[0079] Figure 4 This is a schematic diagram illustrating how the mutation at the TRIM47-related site inhibits its binding to the HNF4α protein.

[0080] Figure 5 This diagram illustrates how mutations at HNF4α-related sites inhibit their binding to the TRIM47 protein.

[0081] Figure 6 This is a schematic diagram of the predicted HNF4α ligand binding pocket.

[0082] Figure 7 This is a schematic diagram illustrating the results of a dual-fluorescent reporter gene system detecting the effect of a compound on enhancing HNF4α transcriptional activity.

[0083] Figure 8 This is a schematic diagram showing the effect of Western blotting on the increase of HNF4α protein levels by the compound.

[0084] Figure 9 This is a schematic diagram showing the results of the micro-thermal surge assay (MST) for detecting the binding of the preferred compound CZ2401 to the HNF4α protein.

[0085] Figure 10 This is a schematic diagram showing the effect of compound CZ2401 in significantly inhibiting the binding of TRIM47 to HNF4α protein.

[0086] Figure 11 This is a schematic diagram illustrating the effect of a structural analogue of compound CZ2401 on enhancing HNF4α transcriptional activity, as detected by a luciferase reporter gene system.

[0087] Figure 12 This is a schematic diagram showing the effect of Western blotting on the increase of HNF4α protein levels by compound CZ2401 and its structural analogues.

[0088] Figure 13 This is a schematic diagram showing the effect of compound CZ2401 in significantly inhibiting hepatocellular lipotoxicity induced by oleic acid (OA).

[0089] Figure 14 This is a schematic diagram showing how compound CZ2401 can significantly alleviate the progression of CCl4-induced liver fibrosis in mice.

[0090] Figure 15 This is a schematic diagram showing the effect of compound CZ2401 in significantly inhibiting the proliferation, migration, invasion and colony formation of Hep3B liver cancer cells.

[0091] Figure 16 This is a schematic diagram showing the effect of compound CZ2401 in significantly inhibiting tumor progression in a mouse model of subcutaneous xenografts of Hep3B liver cancer cells. Detailed Implementation

[0092] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0093] Compound CZ2401 (Compound ID G856-9858), CZ2401-1 (Compound ID G856-9872), CZ2401-2 (Compound ID G856-9887), CZ2401-3 (Compound ID G856-9873), CZ2401-4 (Compound ID G856-9856), CZ2401-5 (Compound ID G856-9857), CZ2401-6 (Compound IDG856-9861), CZ2401-7 (Compound ID G856-9859), CZ2401-8 (Compound ID G856-9860), CZ2401-9 (Compound ID G856-9837) and CZ2401-10 (Compound ID (G856-9921), all of the above compounds were purchased from Chemdiv INC.

[0094] Example 1

[0095] Wild-type Hep3B liver cancer cells were divided into groups of 5 × 10 5 Cells were seeded at a density of 60 mm in culture dishes, transfected with small interfering TRIM47 to knock down TRIM47 expression, and proteins were collected using protein lysis buffer after 48 hours. After high-temperature denaturation, Western blotting was performed. At the same time, RNA was extracted from cells by lysing them with TRIZOL, and the expression level of downstream genes of HNF4α was detected by qRT-PCR. Figure 1This diagram illustrates how TRIM47 knockdown significantly inhibits HNF4α protein degradation and promotes the expression of downstream liver function-related genes. Figure A shows the results of Western blot analysis, and figure B shows the results of qRT-PCR. As can be seen from the figure, TRIM47 knockdown significantly upregulates the protein level of HNF4α. Figure 1 (As shown in A) and promotes the expression of downstream liver function-related genes ( Figure 1 (As shown in B).

[0096] Example 2

[0097] Huh7 hepatocellular carcinoma cells were transfected with a TRIM47 overexpression plasmid tagged with Flag and an HNF4α overexpression plasmid tagged with V5. After 24 hours, MG132 was added for 6-8 hours of treatment. Upon sample collection, IP lysis buffer (containing phosphatase and protease inhibitors) was added, and the cells were lysed on ice for 30 minutes before protein extraction. After centrifugation at 12000 rpm for 15 minutes, the supernatant was collected for protein quantification. Anti-Flag or anti-V5 affinity gels were added, and the cells were incubated overnight at 4°C. The following day, the cells were washed three times with 100 mM Wash Buffer, denatured at high temperature, and then subjected to Western blotting. Results are as follows: Figure 2 As shown, Figure 2 This diagram illustrates the interaction between TRIM47 and HNF4α proteins. As can be seen from the figure, TRIM47 can bind to HNF4α.

[0098] Example 3

[0099] Protein-protein docking experiments were performed using the HNF4α-LBD protein domain (PDB: 4IQR) and the three-dimensional structure of TRIM47 predicted by Alphafold. The results are as follows: Figure 3 As shown, Figure 3 This diagram illustrates the screening of potential amino acid sites affecting the binding of TRIM47 and HNF4α proteins using molecular docking technology. As shown in the diagram, HNF4α and TRIM47 proteins form a strong interaction in the complex. The key amino acids for HNF4α are Pro342, Trp349, and Glu353, while the key amino acids for TRIM47 are His534 and His600.

[0100] Example 4

[0101] Mutant TRIM47 plasmids expressing the Flag tag were constructed (including TRIM47-K534A, TRIM47-K600A, and double mutant TRIM47-2A (K534A / K600A)). HNF4α overexpression plasmids with the V5 tag were co-transfected into 293T cells with either wild-type or mutant TRIM47 plasmids with the Flag tag. Cells were lysed and proteins were collected after 48 hours. Anti-Flag affinity gels were added and incubated overnight at 4°C. Cells were washed three times with 100 mM Wash Buffer (20 mM Tris-HCl, 4 mM NaCl, 10% Triton X-100, NP40, and glycerol, in doses of 1 mL, 1.25 mL, 2.5 mL, 2.5 mL, and 6.25 mL, respectively, and 36.5 mL of ultrapure water). After high-temperature denaturation, Western blotting was performed. Results are as follows: Figure 4 As shown, Figure 4 This diagram illustrates how mutations at TRIM47-related sites inhibit its binding to HNF4α protein. As can be seen from the figure, the TRIM47-2A mutation significantly inhibits the binding of TRIM47 to HNF4α.

[0102] Example 5

[0103] Mutant HNF4α plasmids were constructed, including overexpression plasmids of K342A, W349A, E353A, and HNF4α-3A (K342A / W349A / E353A). Wild-type TRIM47 plasmids with the Flag tag were co-transfected into 293T cells with either wild-type or mutant HNF4α plasmids with the V5 tag. Cells were lysed and proteins were collected after 48 hours. Anti-Flag affinity gels were added, and the cells were incubated overnight at 4°C with rotation. The cells were washed three times with 100 mM Wash Buffer, and after high-temperature denaturation, Western blotting was performed. Results are as follows: Figure 5 As shown, Figure 5 This diagram illustrates how mutations at HNF4α-related sites inhibit its binding to the TRIM47 protein. As can be seen from the figure, the HNF4α-3A mutation significantly inhibits the binding of HNF4α to TRIM47.

[0104] Example 6

[0105] Molecular docking of HNF4α with TRIM47 revealed that Pro342, Trp349, and Glu353 of HNF4α are important binding sites. Further binding pocket prediction was performed using MOE-Site Finder software. The results are as follows... Figure 6 As shown, Figure 6The diagram shows the predicted HNF4α ligand binding pockets. The left image shows the top 5 candidate pockets predicted, and the right image is a partial diagram of the preferred binding pocket, Pocket 5. As can be seen from the diagram, among the top 5 candidate binding pockets predicted (shown in the left image), the preferred binding pocket is Pocket 5, which contains Pro342, Gln345, Ser346, Trp349, Gln350, and Glu353, covering three key amino acids. Subsequent virtual screening of compounds will be performed based on this pocket (shown in the right image).

[0106] Example 7

[0107] Hep3B liver cancer cells were treated at a rate of 8×10 3 / wells were seeded into 96-well plates and transfected with pGL3-NINJ1-9p (HNF4α reporter gene) and pRL-SV40 plasmid. The medium was changed after 8 hours. Twenty candidate TRIM47-HNF4α binding inhibitors (CZ2401 to CZ2420, working concentration 10 µM) were added and treated for 24 hours. Samples were collected using the Dual-Luciferase Reporter kit, and OD135nm values ​​were read using a TECAN Infinite F200 microplate reader to obtain the relevant values ​​of firefly luciferase and Renilla luciferase. Results are as follows: Figure 7 As shown, Figure 7 This is a schematic diagram illustrating the effect of a compound on HNF4α transcriptional activity as detected by a dual-fluorescent reporter gene system (*P<0.05, **P<0.01, ***P<0.001). The figure shows that compound CZ2401 significantly promotes HNF4α protein transcriptional activity.

[0108] The structure of compound CZ2401 is shown below:

[0109]

[0110] Example 8

[0111] Hep3B liver cancer cells were treated at a rate of 3×10 5 Cells were seeded per well in 6-well plates, and the medium was changed after cell adhesion. Twenty candidate TRIM47-HNF4α binding inhibitors (CZ2401 to CZ2420, working concentration 10 µM) were added for 24 hours. Protein was extracted using protein lysis buffer (containing PMSF), denatured at high temperature, and then subjected to Western blotting to detect HNF4α protein expression levels. Results are as follows: Figure 8 As shown, Figure 8This is a schematic diagram showing the effect of Western blotting on the increase of HNF4α protein levels by the compound. The diagram shows that compound CZ2401 significantly increases HNF4α protein levels.

[0112] Example 9

[0113] The binding affinity between the preferred compound CZ2401 and HNF4α protein was determined using micro-thermophoresis (MST). Compound CZ2401 was used as the ligand, and HNF4α protein as the target. The protein was labeled using a RED-NHS protein labeling kit. The small molecule reaction buffer was serially diluted 16 times (50 mM HEPES buffer [pH = 7.4], containing 0.05% Tween 20). After equal-volume mixing, the mixture was incubated at room temperature for 20 minutes. The thermophoretic signal was measured using a Monolith NT.115 (Nano Temper), and the Kd value was calculated using Nano Temper analysis software. Results are shown below. Figure 9 As shown, Figure 9 This is a schematic diagram of the binding results of the preferred compound CZ2401 to HNF4α protein detected by the micro-thermal surge method (MST). In the diagram, A is a schematic diagram of the binding of HNF4α and CZ2401, and B is a schematic diagram of the MST experimental results of HNF4α and CZ2401. As can be seen from the figure, the binding constant of compound CZ2401 to HNF4α is Kd = 5.17 ± 0.57 µM.

[0114] Example 10

[0115] Hep3B liver cancer cells were transfected with an HNF4α overexpression plasmid carrying the V5 tag. Cells were treated with different concentrations of compound CZ2401 (1 μM and 10 μM) for 6-8 hours, followed by cell lysis and protein collection. Anti-V5 affinity gel was added, and the cells were incubated overnight at 4°C by rotation. After washing three times with 100 mM Wash Buffer and denaturing at high temperature, Western blotting was performed. Results are as follows: Figure 10 As shown, Figure 10 This diagram illustrates the significant inhibition of TRIM47-HNF4α protein binding by compound CZ2401. As shown in the figure, compound CZ2401 can inhibit the binding of HNF4α and TRIM47 in a dose-dependent manner.

[0116] Example 11

[0117] Hep3B liver cancer cells were treated at a rate of 8×10 3 / wells were seeded into 96-well plates and transfected with pGL3-NINJ1-9p (HNF4α reporter gene) and pRL-SV40 plasmid. The medium was changed after 8 hours. Compound CZ2401 and its analogues (CZ2401-1 to CZ2401-10, working concentration 10 µM) were added and treated for 24 hours. Samples were collected using a Dual-Luciferase Reporter kit, and OD135nm values ​​were read using a TECAN Infinite F200 microplate reader to obtain the relevant values ​​of firefly luciferase and Renilla luciferase. Results are as follows: Figure 11 As shown, Figure 11 A schematic diagram illustrating the effects of structural analogs of compound CZ2401 on HNF4α transcriptional activity as detected by a luciferase reporter gene system (*P<0.05, **P<0.01, ***P<0.001). The figure shows that compound CZ2401 and its structural analogs CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9, and CZ2401-10 significantly promoted HNF4α protein transcriptional activity.

[0118] The structures of compound CZ2401 and its structural analogues CZ2401-1, CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9 and CZ2401-10 are shown below:

[0119] .

[0120] Example 12

[0121] Hep3B liver cancer cells were treated at a rate of 3×10 5 Cells were seeded per well in 6-well plates, and the medium was changed after cell adhesion. The preferred compound CZ2401 and its analogues (CZ2401-1 to CZ2401-10, working concentration 10 µM) were added for 24 hours. Protein was extracted using protein lysis buffer (containing PMSF), denatured at high temperature, and then subjected to Western blotting to detect the protein expression level of HNF4α. Results are as follows: Figure 12 As shown, Figure 12This is a schematic diagram showing the effects of Western blotting on the increase of HNF4α protein levels by compound CZ2401 and its structural analogs. As can be seen from the figure, compound CZ2401 and its structural analogs CZ2401-1, CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9, and CZ2401-10 significantly increased HNF4α protein levels.

[0122] Example 13

[0123] HepG2 liver cancer cells were divided into groups of 3 × 10⁻⁶. 5 Cells were seeded at a density of [insert density here] in 6-well plates. After cell adhesion, the medium was changed, and cells were stimulated with 0.8 mM oleic acid (OA) solution for 24 hours. After treatment with compound CZ2401 (10 µM) or DMSO for 48 hours, cells were fixed with 4% PFA (paraformaldehyde) and stained with Oil Red O. Results are as follows: Figure 13 As shown, Figure 13 This diagram illustrates the significant inhibition of oleic acid (OA)-induced hepatocellular lipotoxicity by compound CZ2401. The figure shows that compound CZ2401 can significantly inhibit OA-induced lipotoxicity, suggesting that CZ2401 can inhibit hepatocellular fat deposition and may have a role in alleviating fatty liver disease.

[0124] Example 14

[0125] Five- to six-week-old male C57BL / 6 mice were acclimatized in an SPF-grade animal facility for one week and then randomly divided into a control group (Control), a model group (CCl4 + DMSO), and a treatment group (CCl4 + CZ2401), with three mice in each group. Liver fibrosis models were established in the model and treatment groups by intraperitoneal injection of 10% CCl4, twice weekly. Four weeks after CCl4 injection modeling, mice in the treatment group were administered compound CZ2401 (8 mg / kg) intraperitoneally once daily, while the control group was given physiological saline. CCl4 was continuously used to induce the model during the treatment period. Figure 14 (As shown in Figure A). After 4 weeks of continuous drug administration, the experiment was terminated and the mice were sacrificed. Liver tissue was separated, weighed, and photographed. Figure 14 (As shown in B and C). The results are as follows: Figure 14 As shown, Figure 14This diagram illustrates how compound CZ2401 significantly alleviated the progression of CCl4-induced liver fibrosis in mice. A shows a schematic of the mouse model; B shows a photograph of the liver tissue of the modeled mice; C shows the liver weight of the mice; D shows the liver weight-to-body weight ratio of the mice; E shows the detection of serum alanine aminotransferase (ALT) levels in mice; F shows the detection of serum aspartate aminotransferase (AST) levels in mice; G shows the detection of α-SMA gene levels in liver tissue by qRT-PCR; H shows the detection of COL1A1 gene levels in liver tissue by qRT-PCR; and I shows the results of H&E staining, Sirius red staining, and immunohistochemistry to detect the degree of fibrosis in liver tissue of different groups of mice (*P<0.05, **P<0.01, ***P<0.001). The results show that CZ2401 reduced CCl4-induced liver enlargement and significantly decreased the liver weight-to-body weight ratio in mice. Figure 14 As shown in Figure D). Small animal biochemical analyzer measurements of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice showed that the ALT and AST levels in the CZ2401 treatment group were lower than those in the model control group, indicating that CZ2401 can inhibit CCl4-induced liver inflammation (…). Figure 14 (As shown in E and F); qRT-PCR experiments showed that CZ2401 inhibited the expression of α-SMA, an indicator of activated hepatic stellate cells, and the collagen COL1A1 gene in fibrotic liver tissue. Figure 14 As shown in G and H), H&E staining and Sirius red staining showed that CZ2401 reduced collagen deposition in fibrotic liver, and immunohistochemistry also showed that CZ2401 reduced the expression of the fibrosis marker α-SMA. Figure 14 (As shown in Figure I). These results indicate that CZ2401 can significantly alleviate the progression of CCl4-induced liver fibrosis in mice.

[0126] Example 15

[0127] Hep3B liver cancer cells were treated at a rate of 8×10 3 Cells were seeded at a density of [number] cells / well in 96-well plates. After complete cell adhesion, the cells were treated with compound CZ2401 (10 μM) or DMSO, and the complete medium containing the drug was changed every 2 days. For assay, following the CellCounting Kit-8 instructions, the medium in the test wells was aspirated, and the cells were washed once with PBS buffer. CCK8 working solution was prepared by mixing serum-free DMEM medium and CCK8 stock solution at a volume ratio of 10:1. 100 µL / well of CCK8 working solution was added to each test well, and the cells were incubated at 37°C for 1 hour. OD at 450 nm was measured using a multi-mode microplate reader. Cell growth curves were plotted after 7 consecutive days of monitoring. Results are as follows: Figure 15 As shown, Figure 15 This diagram illustrates the results showing that compound CZ2401 significantly inhibits the proliferation, migration, invasion, and colony formation of Hep3B liver cancer cells. In the diagram, A shows the inhibitory effect of CZ2401 on liver cancer cell proliferation as detected by CCK8 assay; B shows the inhibitory effect of CZ2401 on liver cancer cell migration and invasion as detected by Transwell assay; C and D show the statistical results of migration and invasion; E shows the effect of CZ2401 on the colony formation ability of liver cancer cells as verified by colony formation assay; and F shows the statistical results of colony formation (*P<0.05, **P<0.01). The results are as follows... Figure 15 As shown in Figure A, compound CZ2401 can significantly inhibit the proliferation of Hep3B liver cancer cells.

[0128] Hep3B liver cancer cells were digested and resuspended at a concentration of 2×10⁻⁶. 5 / mL, add 200 μL to a transwell chamber or a transwell chamber with added matrix gel, place the chamber in a 24-well plate, and add 500 μL of DMEM complete culture medium containing 10% FBS to the bottom layer of the chamber. Add compound CZ2401 (10 µM) or DMSO, with 3 replicates for each treatment, and incubate at 37°C for 72 hours before detecting cell migration or invasion. For detection, aspirate the culture medium inside the chamber, fix the chamber in 4% PFA for 20 minutes, and gently rinse the chamber 3 times with PBS buffer. Gently wipe the cells from the inside of the polycarbonate membrane of the chamber with a cotton swab, then place it in a new 24-well plate and stain with 500 μL of crystal violet staining solution for 30 minutes. After washing the chamber, observe it under a microscope, and take pictures of five fields of view (top, middle, bottom, left, and right) for recording. Use ImageJ software to count the proportion of positively stained areas of migrating cells. Figure 15 B in the diagram is a representative example. Figure 15 Figures C and D show that compound CZ2401 can significantly inhibit the migration and invasion of Hep3B liver cancer cells.

[0129] Hep3B liver cancer cells were treated at a rate of 1×10 3Cells were seeded at a density of 1 cell / well in 6-well plates. After overnight adhesion, compound CZ2401 (10 µM) or DMSO was added, with 3 replicates for each treatment. Cells were incubated at 37°C for approximately 2 weeks, with the complete culture medium containing the drug changed every 3 days. The size and number of cell clones were continuously observed during culture. For assay, the cells were washed twice with PBS buffer, fixed with 2 mL / well of 4% PFA for 20 minutes, and then washed with PBS buffer to remove residual PFA. The cells were then stained with 2 mL / well of crystal violet for 30 minutes, repeatedly rinsed with PBS buffer to remove excess crystal violet stain, dried in an oven, and photographed. The number of cell clones formed in each well was counted using ImageJ software. The clone formation is shown in the figure. Figure 15 As shown in E, Figure 15 E and F in the middle section show that compound CZ2401 reduces the clonogenic ability of Hep3B liver cancer cells.

[0130] The results show that in vitro treatment with compound CZ2401 can significantly inhibit malignant phenotypes such as proliferation, migration, invasion, and colony formation of liver cancer cells.

[0131] Example 16

[0132] 4-5 week old male BALB / c nude mice were subcutaneously injected with Hep3B hepatocellular carcinoma cells (4 × 10⁻⁶) into the right axilla. 6 (Each xenograft was used to establish a subcutaneous xenograft model.) The size of the xenograft was observed and the long and short diameters of the tumor were measured every two days. The average volume of the xenograft reached approximately 100 mm. 3 Nude mice were randomly divided into a model control group (VEH) and a treatment group (CZ2401), with 7 mice in each group. Mice in the treatment group were administered compound CZ2401 (8 mg / kg) via intraperitoneal injection once daily, while mice in the model control group were given physiological saline (…). Figure 16 (As shown in Figure A). Tumor volume was measured daily after drug administration, and a tumor growth curve was plotted. Figure 16 As shown in Figure B), the experiment was terminated and the mice were sacrificed after 14 days of continuous administration. The subcutaneous tumors were isolated, weighed, and photographed (as shown in Figures C and D in Figure 16).

[0133] The results are as follows Figure 16 As shown, Figure 16This diagram illustrates the significant inhibition of tumor progression in a mouse model of subcutaneous xenograft Hep3B liver cancer cells by compound CZ2401. A shows the modeling process; B shows the tumor volume measured every two days and the tumor growth curve plotted; C shows a gross photograph of the subcutaneous tumor in the mouse; D shows the tumor weight in the mouse; E shows the results of Western blotting detection of HNF4α protein expression; F shows the results of statistical analysis of protein expression levels using ImageJ software; G shows the results of immunohistochemical detection of HNF4α and the proliferation index Ki67 expression levels in liver cancer tissue; and H shows the statistical results of Ki-67 (*P<0.05).

[0134] The results showed that compound CZ2401 inhibited the growth of subcutaneous tumors of hepatocellular carcinoma in mice. After extracting proteins from a portion of the tumor tissue, the HNF4α protein level was detected by Western blotting. Figure 16 As shown in Figure E), the relative expression level was statistically analyzed using ImageJ software. Figure 16 As shown in Figure F), the results showed that compound CZ2401 significantly upregulated the expression of HNF4α in hepatocellular carcinoma (HCC) tumor tissues. Further immunohistochemical staining was used to detect the expression levels of HNF4α and the proliferation index Ki67 in HCC tissues. Figure 16 As shown in G), and the detection results are statistically analyzed. Figure 16 As shown in Figure H), the results further indicate that compound CZ2401 can upregulate the expression of HNF4α in hepatocellular carcinoma xenograft tissues and inhibit the proliferation of hepatocellular carcinoma cells. These results demonstrate that compound CZ2401 can significantly inhibit tumor progression in a subcutaneous xenograft mouse model.

[0135] The experimental results above demonstrate that, through a series of protein function research techniques, including protein interaction, docking, and site mutation, combined with in vitro and in vivo biochemical experiments, TRIM47 protein has been identified as a key molecule that binds to HNF4α and inhibits its transcriptional activity. Furthermore, the key amino acid sites affecting the binding of TRIM47 to HNF4α and the function of HNF4α protein have been identified. Small molecule drugs containing these key sites were then screened to inhibit TRIM47-HNF4α binding and protect HNF4α protein activity. These small molecule drugs exert biological effects both in vitro and in vivo, correcting hepatic glucose and lipid metabolism disorders, alleviating liver fibrosis, and inhibiting the malignant phenotype of liver cancer. This invention lays the foundation for the development of small molecule drugs for the treatment of chronic liver diseases and liver cancer, and their widespread application in adjuvant therapy.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of a small molecule compound that inhibits HNF4α degradation in the preparation of drugs for treating liver cancer, characterized in that, The small molecule compound that inhibits HNF4α degradation is selected from one of the following structures:

2. The application of the small molecule compound that inhibits HNF4α degradation according to claim 1 in the preparation of drugs for treating liver cancer, characterized in that, In this application, a small molecule compound that inhibits the degradation of HNF4α is used as the sole active ingredient.

3. The application of a pharmaceutical preparation in the preparation of a drug for treating liver cancer, characterized in that, The pharmaceutical preparation is made from the small molecule compound that inhibits HNF4α degradation as described in claim 1 or 2 and medically acceptable excipients.

4. The use of a pharmaceutical composition in the preparation of a drug for treating liver cancer, characterized in that, The pharmaceutical composition is made from the small molecule compound that inhibits HNF4α degradation as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

Images