Application of Hispidin in the Preparation of FXR Antagonists

Abstract

This invention discloses the application of Hispidin in the preparation of FXR antagonists, belonging to the field of biomedical technology. This invention provides the application of Hispidin in the preparation of FXR antagonists. Hispidin can target and bind to FXR and possesses FXR antagonistic activity, thereby reversing the pathological signaling pathways caused by excessive FXR activation. This invention expands the new pharmacological effects and applications of Hispidin, not only providing new candidates for the regulation of FXR-related signaling pathways, but also offering new research directions for the development of drugs for the prevention and treatment of bile acid metabolism disorders, lipid metabolism disorders, glucose metabolism disorders, and related diseases, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of Hispidin in the preparation of FXR antagonists. Background Technology

[0002] The farnesoid X receptor (FXR, NR1H4) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. It is primarily highly expressed in the liver and small intestine and is a key target for regulating bile acid homeostasis, glucose and lipid metabolism, inflammation, and liver fibrosis. FXR signaling disorders are closely associated with the development and progression of various metabolic diseases, including non-alcoholic steatohepatitis (NASH), cholestatic liver disease, obesity, type 2 diabetes, and atherosclerosis. Therefore, the development of drugs targeting FXR has become a core research direction in the treatment of metabolic diseases.

[0003] Currently, drug development targeting FXR mainly focuses on FXR agonists, such as obeticholic acid (OCA). These drugs can improve cholestasis and liver fibrosis, but they generally suffer from problems such as skin itching, dyslipidemia, and insufficient safety profiles with long-term use, limiting their further application. Furthermore, multiple studies have shown that FXR antagonists have significant effects in improving cholestatic liver injury, hypercholesterolemia, hyperglycemia, and fatty liver, and have also shown potential value in the treatment of esophageal cancer and pancreatic cancer. Compared to chemically synthesized small molecules, FXR antagonists derived from natural products generally have better pharmacological activity and safety, thus showing promising application prospects in the regulation of metabolic diseases and the development of drugs for related diseases.

[0004] Hispidin is a styrylpyranone-based natural polyphenol primarily derived from fungi such as *Phellinus linteus*. Previous studies have confirmed that Hispidin is a potent and selective PKC inhibitor, particularly showing significant inhibitory activity against PKCβ. However, there are currently no reports on Hispidin regulating FXR. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides the application of Hispidin in the preparation of FXR antagonists. Hispidin has a good binding capacity to FXR in the liver or intestine, and can inhibit the transcriptional activity of FXR receptors, thereby reversing the pathological signaling pathways caused by excessive FXR activation.

[0006] The technical solution of the present invention is as follows: The first aspect of this invention protects the use of Hispidin in the preparation of FXR antagonists.

[0007] Preferably, Hispidin comprises at least one of the following: (1) Hispidin monomer obtained by chemical preparation; (2) The Hispidin monomer obtained by separation and extraction; (3) Fermentation broth of Phellinus linteus fungi with Hispidin as the main active ingredient; (4) Extracts of Phellinus linteus fungi with Hispidin as the main active ingredient; (5) Fermentation broth of Phellinus linteus fungi with Hispidin polymer as the main active ingredient; (6) Extracts of Phellinus linteus fungi with Hispidin polymer as the main active ingredient.

[0008] Preferably, the application includes Hispidin inhibiting FXR transcriptional activity.

[0009] A second aspect of this invention protects the use of Hispidin in the preparation of medicaments for the prevention and / or treatment of FXR-related diseases.

[0010] Preferably, the related disease includes at least one of the following: (1) Diseases associated with FXR overactivation; (2) Diseases related to FXR signal disorder.

[0011] Preferably, the related diseases include at least one of the following: hyperuricemia, non-alcoholic steatohepatitis, cholestatic liver disease, obesity, type 2 diabetes, and atherosclerosis.

[0012] Preferably, Hispidin comprises at least one of the following: (1) Hispidin monomer obtained by chemical preparation; (2) The Hispidin monomer obtained by separation and extraction; (3) Fermentation broth of Phellinus linteus fungi with Hispidin as the main active ingredient; (4) Extracts of Phellinus linteus fungi with Hispidin as the main active ingredient; (5) Fermentation broth of Phellinus linteus fungi with Hispidin polymer as the main active ingredient; (6) Extracts of Phellinus linteus fungi with Hispidin polymer as the main active ingredient; Preferably, the drug includes a drug that antagonizes FXR.

[0013] The beneficial technical effects of this invention are as follows: This invention discloses that Hispidin can target and bind to FXR, exhibiting FXR antagonistic activity, thereby reversing pathological signaling pathways caused by excessive FXR activation. This invention expands the pharmacological effects and applications of Hispidin, providing not only new candidates for the regulation of FXR-related signaling pathways but also new research directions for the development of drugs for the prevention and treatment of bile acid metabolism disorders, lipid metabolism disorders, glucose metabolism disorders, and related diseases, demonstrating promising application prospects. Attached Figure Description

[0014] Figure 1 The results are from the cell thermal displacement experiment in Example 1 of this invention.

[0015] Figure 2 The results are from molecular dynamics simulations in Example 2 of this invention; In the figure: the top left shows the RMSD curves of proteins, ligands, and protein-ligand complexes; The top right corner shows the RMSF curve of the residues in the complex system; The lower left is a graph of the radius of rotation (Rg) of the complex system; The bottom right shows the soluble surface area (SASA) curve of the complex system.

[0016] Figure 3 This shows the mRNA expression of FXR in each group of cells in Example 3.

[0017] Figure 4 This is a graph showing the Western Blot detection results in Example 4. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0019] The Hispidin used in the following embodiments of the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] The culture medium used in the following embodiments of the present invention is Gibco DMEM high-glucose medium.

[0021] It is understood that the dosage form of the drug described in this invention is not limited, and the dosage form can be tablets, lozenges, capsules and soft capsules, granules, syrups, micro-pellets, microspheres, pills, liquids, etc. Different administration methods can be formulated according to clinical needs, including intravenous, oral, subcutaneous, intra-arterial, or local administration for patient treatment.

[0022] Example 1: Cell thermal displacement experiment Caco2 cells were obtained from the Institute of Biochemistry and Cell Biology (SIBS, CAS) in Shanghai, China. Caco2 cells were cultured in DMEM medium containing 20% ​​fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0023] Caco2 cells were seeded in 96-well plates at a density of 1 × 10⁻⁶ cells / well. 4 / well, DMEM containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin. To assess the effect of Hispidin on Caco2 cell activity, Caco2 cells were first treated with 6.25, 12.5, 25, 50, 100, and 200 μM Hispidin for 24 hours, respectively. Then, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for 2 hours. The absorbance at 450 nm was then measured.

[0024] To assess the binding of Hispidin to FXR, Caco2 cells were seeded in 6-well plates at a density of 1 × 10⁻⁶ cells / well. 5 / well, DMEM containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin. Caco2 cells in the Hispidin group were treated with 50 μM Hispidin for 24 hours, while Caco2 cells in the control group were treated with 0.1% DMSO for 24 hours. Cells were then collected. Cells were evenly separated and heated at 40, 45, 50, 55, 60, and 65°C for 3 minutes each. After heat treatment, cells were lysed, and proteins were collected by centrifugation. Western blotting was then performed to determine protein content.

[0025] The results showed that Hispidin directly binds to FXR (Nr1h4) in Caco2 cells, thereby improving protein stability. First, CCK8 assays confirmed that Hispidin at concentrations up to 200 μM did not exhibit significant cytotoxicity in Caco2 cells, thus ruling out interference from cell death or abnormal cell states in subsequent assays.

[0026] Further thermal migration analysis (CETSA) data showed that, compared with the DMSO control group, the melting curve of Nr1h4 treated with Hispidin changed significantly, with an increased melting temperature (Tm) of +6.7°C. Figure 1Meanwhile, this significant stabilizing effect was supported by protein blot analysis. Western blot results showed that, under higher temperature conditions, the retention of soluble Nr1h4 protein in the Hispidin-treated group was higher than that in the DMSO control group. That is, in the Hispidin-treated sample, the retention rate of soluble Nr1h4 protein was higher at higher temperatures, and Hispidin treatment enhanced the thermal stability of Nr1h4 protein.

[0027] This strongly suggests the existence of direct ligand-target interactions in the native cellular environment, meaning that Hispidin can bind to FXR(Nr1h4) in the native cellular environment and enhance its protein stability, indicating that FXR(Nr1h4) is the target of Hispidin.

[0028] Example 2: Molecular Dynamics Simulation To further evaluate the stability of the interaction between Hispidin and FXR (Nr1h4) at the molecular level, molecular dynamics simulation analysis was performed on the Hispidin-Nr1h4 complex.

[0029] Specifically as follows: Protein (FXR) information was obtained from the uniprot website (https: / / www.uniprot.org / uniprotkb), and the protein structure was obtained from the SCSB PDB database (https: / / www.rcsb.org / ). The obtained structure was imported into Discovery Studio 2019 for protein structure optimization. This process mainly includes dehydration, hydrogenation, charge completion, amino acid completion, and side chain completion of the protein. Finally, the optimized protein structure was obtained and output as a pdb file.

[0030] The structure of the small molecule (Hispidin) was obtained from the PubChem website, and energy minimization was performed using Discovery Studio 2019, saving it as a PDB file. The protein and small molecule PDBQT files were prepared using Autodock 4.0, and docking was performed using Autodock Vina 1.2.6. Finally, visualization analysis was performed using PyMOL 3.1 and Discovery Studio 2019.

[0031] Combined pattern analysis revealed that the small molecule is anchored in the protein's active pocket through multiple non-covalent interactions, with hydrogen bonding being the core driving force: the phenolic hydroxyl group of the small molecule forms a conventional hydrogen bond with the protein's ASN-448 residue with a bond length of 2.2 Å, and the carbonyl oxygen on the pyranone ring forms a conventional hydrogen bond with the GLN-400 residue with a bond length of 2.0 Å. These two high-energy hydrogen bonds (both with bond lengths less than 2.5 Å) provide the complex with crucial binding affinity and structure specificity.

[0032] In addition to hydrogen bonds, the system also exhibits various auxiliary interactions: the small molecule benzene ring forms a Pi-Sigma interaction with the VAL-329 residue, and the pyranone ring forms a Pi-Cation interaction with the ARG-399 residue, further enhancing binding stability. Simultaneously, multiple residues such as LEU-455, TRP-473, and LYS-325 interact hydrophobically with the small molecule through van der Waals forces, filling the spatial gaps in the active pocket, optimizing the shape matching between the small molecule and the pocket, and avoiding conformational fluctuations in binding.

[0033] A 100 ns molecular dynamics simulation of the protein-ligand complex was performed using Gromacs 2025. The specific procedure is as follows: the protein was simulated using AMBER99SB-ILDN force field parameters, and the ligand topology was constructed using GAFF2 force field parameters. The protein and ligand topology files were merged to ensure no atomic type conflicts. Periodic boundary conditions (PBC) were applied, placing the complex in a cubic box with a minimum distance of 1.2 nm from the complex surface to the box wall. The solvent was filled using a TIP3P water model, and Na was added. + / Cl - The net charge of the counter-ion neutralization system was maintained at 0.15 M to simulate a physiological environment. Energy minimization was performed first, followed by 1,000,000 steps of isothermal and isochoric ensemble equilibrium and isothermal and isobaric ensemble equilibrium simulations, with a coupling constant of 0.1 ps and a duration of 2 ns. Temperature / pressure coupling parameters were the same as in the equilibrium phase, and the trajectory was saved every 1000 steps for subsequent analysis. Finally, free molecular dynamics simulations were run, totaling 50,000,000 steps at a step size of 2 fs, for a total duration of 100 ns. Finally, molecular dynamics simulations were performed at constant temperature (300 K) and constant pressure (1 bar) using Gromacs 2025, for a total duration of 100 ns. The binding free energy of the system after stabilization was calculated using MMPBSA.

[0034] Molecular dynamics simulations showed that the root mean square deviation (RMSD) of the Hispidin-Nr1h4 complex stabilized after initial fluctuations during the 100 ns simulation, indicating that the overall conformation of the complex did not undergo significant shift. The root mean square fluctuation (RMSF) of residues showed that most amino acid residues exhibited only limited fluctuations, indicating that the overall protein structure remained stable. Meanwhile, the radius of rotation (Rg) and soluble surface area (SASA) remained within a relatively stable range during the simulation, indicating that the complex possesses good folding and structural integrity. Figure 2 ).

[0035] Example 3: Effect of Hispidin on FXR expression in hyperuricemia-induced Caco2 cells Caco2 cells were obtained from the Institute of Biochemistry and Cell Biology (SIBS, CAS) in Shanghai, China, and were cultured in DMEM medium containing 20% ​​fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0036] Caco2 cells were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. 5 / hole. After incubation for 24 hours, they were divided into the following four groups: Blank control group: treated with complete culture medium containing 0.1% DMSO for 36 hours.

[0037] High uric acid treatment group: Pretreated with complete culture medium containing 0.1% DMSO for 12 hours, and then treated with 12.5 mg / L uric acid for 24 hours.

[0038] Hispidin group: Pretreated with 10 μM Hispidin (containing 0.1% DMSO) for 12 hours, followed by treatment with 12.5 mg / L uric acid for 24 hours.

[0039] FXR antagonist group: Pretreated with 2 μM DY268 (FXR antagonist, containing 0.1% DMSO) for 12 hours, followed by treatment with 12.5 mg / L uric acid for 24 hours.

[0040] Subsequently, cells from each group were collected and RNA was extracted. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to measure the mRNA expression of FXR in each group of cells. Results are as follows: Figure 3As shown, compared with the blank control group (NC), the FXR level in the hyperuricemia treatment group (HUA) was significantly increased, while the FXR expression level was significantly decreased after Hispidin pretreatment (His), and its trend was basically consistent with that of the FXR antagonist DY268 group. This indicates that Hispidin can effectively reverse the abnormal increase of FXR induced by hyperuricemia, and that Hispidin can effectively regulate the expression level of FXR.

[0041] Example 4: Investigation of the inhibitory activity of Hispidin on FXR in hyperuricemic mice Eighteen KM mice were randomly selected and divided into a control group, a model group, and a Hispidin group, with six mice in each group. After one week of acclimatization feeding: Control group: Mice were given 0.1 ml of distilled water daily, followed by 0.1 ml of distilled water 1 hour later, for 4 consecutive weeks. During this period, the mice were not restricted in their diet and were given a regular diet.

[0042] Model group: Mice were given 0.1 ml of potassium oxonate (500 mg / kg) solution daily, followed by 0.1 ml of distilled water 1 hour later, for 4 consecutive weeks. During this period, the mice were not restricted in their diet and were given a high-protein diet supplemented with 0.15% adenine and 10% yeast powder.

[0043] Hispidin group: Mice were given 0.1 ml of potassium oxonate (500 mg / kg) solution daily, followed by 0.1 mL of Hispidin solution (9.4 mg / kg) 1 hour later, for 4 consecutive weeks. During this period, the mice were not restricted in their diet and were given a high-protein diet supplemented with 0.15% adenine and 10% yeast powder.

[0044] Mice in each group were fasted for 12 hours after the last administration of the drug, and liver and kidney tissues were collected.

[0045] Total RNA was extracted from the tissue samples mentioned above, and RNA quality detection, mRNA enrichment, fragmentation, reverse transcription, PCR amplification and sequencing analysis were performed in sequence. The expression level of FXR in the liver and intestinal tissues of mice in the control group, model group and Hispidin group was calculated and compared by FPKM method. The detection results are shown in Table 1.

[0046] Table 1: Effects of Hispidin on FXR expression in liver and intestinal tissues of hyperuricemia mice

[0047] Simultaneously, total protein was extracted from the above tissue samples, and Western blot (WB) was performed using GADPH as an internal control. The results were analyzed using ImageJ image analysis software. (See attached image). Figure 4 .

[0048] The results showed that, compared with the control group, the expression of FXR in the liver and intestine of mice in the model group was significantly increased, while the expression of FXR in the liver and intestine of mice was significantly decreased after Hispidin treatment, indicating that Hispidin can inhibit the abnormally high expression of FXR caused by hyperuricemia in vivo and has FXR antagonistic activity.

[0049] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. Application of Hispidin in the preparation of FXR antagonists.

2. The application according to claim 1, characterized in that, Hispidin includes at least one of the following: (1) Hispidin monomer obtained by chemical preparation; (2) The Hispidin monomer obtained by separation and extraction; (3) Fermentation broth of Phellinus linteus fungi with Hispidin as the main active ingredient; (4) Extracts of Phellinus linteus fungi with Hispidin as the main active ingredient; (5) Fermentation broth of Phellinus linteus fungi with Hispidin polymer as the main active ingredient; (6) Extracts of Phellinus linteus fungi with Hispidin polymer as the main active ingredient.

3. The application according to claim 1, characterized in that, The application includes Hispidin inhibiting FXR transcriptional activity.

4. The use of Hispidin in the preparation of drugs for the prevention and / or treatment of FXR-related diseases.

5. The application according to claim 4, characterized in that, The relevant diseases include at least one of the following: (1) Diseases associated with FXR overactivation; (2) Diseases related to FXR signal disorder.

6. The application according to claim 4, characterized in that, The relevant diseases include at least one of the following: hyperuricemia, non-alcoholic steatohepatitis, cholestatic liver disease, obesity, type 2 diabetes, and atherosclerosis.

7. The application according to claim 4, characterized in that, Hispidin includes at least one of the following: (1) Hispidin monomer obtained by chemical preparation; (2) The Hispidin monomer obtained by separation and extraction; (3) Fermentation broth of Phellinus linteus fungi with Hispidin as the main active ingredient; (4) Extracts of Phellinus linteus fungi with Hispidin as the main active ingredient; (5) Fermentation broth of Phellinus linteus fungi with Hispidin polymer as the main active ingredient; (6) Extracts of Phellinus linteus fungi with Hispidin polymer as the main active ingredient.

8. The application according to claim 4, characterized in that, The drugs include those that antagonize FXR.

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