Application of virtual screening compound in preparation of farnesol X receptor stimulant and medicine

Abstract

According to the invention, a virtual screening method based on molecular docking is adopted to obtain 14 Miao compounds with relatively strong binding capacity with a farnesol X receptor. A series of in-vitro experiments are carried out on 14 Miao compounds, including a farnesol X receptor agonistic activity experiment, detection of the cytotoxicity of the compounds by an MTT method, and testing of the influence of the compounds on the triglyceride level of free fatty acid induced HepG2 cells by a GPO-PAP method. Experimental results show that the 14 compounds all have obvious agonistic activity on farnesoid X receptors, and have no cytotoxicity on HepG2 cells and L02 cells. Particularly, the compound 1, the compound 2 and the compound 3 can obviously reduce the content of triglyceride in HepG2 cells with fatty degeneration. Experimental results show that the 14 compounds can be further developed into drugs for treating farnesoid X receptor mediated diseases such as non-alcoholic fatty liver diseases, non-alcoholic steatohepatitis, cholestatic liver diseases, hepatic fibrosis and the like.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to applications of virtual screening compounds in the preparation of farnesoid X receptor agonists and medicines. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by fatty degeneration and lipid storage of hepatocytes, excluding other clear liver damage factors such as excessive drinking, drug effects and chronic liver disease. The pathological evolution of NAFLD is complex, and its disease spectrum includes simple fatty liver, non-alcoholic steatohepatitis (NASH), cirrhosis, etc. NAFLD is highly correlated with obesity. In recent years, the number of obese people in my country has continued to rise, and the prevalence of NAFLD has also increased accordingly. According to statistics in 2018, the prevalence of NAFLD in my country has reached 29.6%. At present, there is no specific drug for the treatment of NAFLD on the market.

[0003] Farnesoid X receptor (FXR) is a bile acid-activated receptor, a member of the nuclear receptor superfamily. It is present in multiple organs of the human body, including the kidney, intestine, liver, etc., and plays an important role in regulating bile acid metabolism, lipid metabolism, and glucose metabolism in the human body. FXR is closely related to diseases such as NAFLD, NASH, liver fibrosis, cholestatic liver disease, primary biliary cholangitis, and lipid metabolism disorders. In particular, it is of great significance for controlling the occurrence and development of NAFLD and treating NAFLD. Studies have found that activation of FXR can induce the small heterodimer partner to inhibit cholesterol 7α-hydroxylase and promote cholesterol metabolic transformation. Activation of FXR can also inhibit the expression of sterol regulatory element binding protein 1, downregulate hepatocyte triglyceride levels, and reduce liver lipid deposition. FXR agonists are a potential method for treating NAFLD. Currently, many mature FXR agonists have entered the clinical trial stage, such as obeticholic acid (OCA), EDP-305, Tropifexor, MET-409, Nidufexor, etc. However, the above-mentioned FXR agonists were found to be prone to side effects such as itching, increased low-density lipoprotein levels, and decreased high-density lipoprotein levels during clinical trials (Adorini L, Trauner M. J Hepatol. 2023, 79 (5): 1317-1331), which often results in the inability to obtain satisfactory therapeutic effects in the treatment of NAFLD. To this end, it is necessary and meaningful to develop new, efficient and low-toxic FXR agonists to reduce adverse reactions in response to the existing deficiencies of FXR agonists.

[0004] Virtual screening is an important method for computer-aided drug design. It uses molecular docking technology to find ligand compounds that have an ideal binding mode with the target receptor, evaluates the quality of the docking results through a scoring function, screens out lead compounds, and combines the results of in vitro biological experiments to further develop lead compounds into lead compounds to accelerate the process of new drug research and development. At present, virtual screening is widely used and mature in the early stages of drug research and development. It has the advantages of low cost and high efficiency. Using molecular docking technology to virtually screen FXR agonists has positive significance for the development of new NAFLD therapeutic drugs. Summary of the invention

[0005] The present invention discloses an application of a virtual screening compound in preparing a farnesoid X receptor agonist and a drug, selects the farnesoid X receptor as a target, screens out a compound with farnesoid X receptor agonist activity, and provides a broader idea for preparing the farnesoid X receptor agonist.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] Use of a virtual screening compound in the preparation of a farnesoid X receptor agonist, wherein the compound is any one of the following:

[0008] Furthermore, the farnesoid X receptor agonist is used to treat and / or prevent farnesoid X receptor-mediated diseases.

[0009] Furthermore, the disease mediated by farnesoid X receptor is one or a combination of two or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, primary biliary cholangitis, and lipid metabolism disorder.

[0010] A drug for treating and / or preventing diseases mediated by farnesoid X receptor, wherein any one of the following compounds is an active ingredient:

[0011]

[0012] Furthermore, the above-mentioned drug is composed of any one of Compound 1 to Compound 14 and a pharmaceutically acceptable carrier.

[0013] The above-mentioned compounds 1 to 14 are obtained by the following screening method:

[0014] (1) Obtain the protein crystal complex of farnesoid X receptor from the Protein Data Bank, optimize the protein structure and define the active site operations, and establish a molecular docking model;

[0015] (2) Obtain the structural information of the compounds from the ChemDiv, Drugbank, and ZINC databases, perform hydrogenation, structural optimization, and energy optimization on the compounds, perform drug-like screening according to the Lipinski rule, and construct a database of compounds to be screened that meet drug-like properties;

[0016] (3) Discovery Studio 2020 and Autodock Vina molecular docking software were used to perform three rounds of molecular docking on the database compounds, namely LibDock, CDOCKER, and Autodock Vina, to virtually screen for lead compounds with potential FXR agonist activity.

[0017] Furthermore, in step (1), the PDB ID of the FXR protein crystal complex is: 3OLF.

[0018] Furthermore, in step (1), the molecular docking model is established by using the Prepare protein module of DiscoveryStudio 2020 software to delete redundant side chains, delete water molecules, and hydrogenate the protein crystal complex, and define the active site with the position of the protein co-crystal ligand small molecule as the center. Use Pymol software to delete redundant side chains, delete water molecules, and hydrogenate the protein crystal complex, and import it into Autodock software. The active site is defined as: X=13.354, Y=21.993, Z=4.063, and set a Molecular docking box.

[0019] Furthermore, in step (2), the construction of a database of compounds to be screened that meet drug-like properties is specifically carried out as follows: the compounds are imported into the Discovery Studio 2020 software, and the Prepare ligand and Fullminimization modules are used to perform structural optimization and energy minimization on the compounds in the database; the Filtered by Lipinski and Veber rules module is used to exclude the compounds in the database that do not meet the drug-likeness rules according to the Lipinski drug-likeness five rules and Veber rules in the drug-likeness rules, and the remaining compounds are used to construct the database of compounds to be screened.

[0020] The present invention adopts a virtual screening method based on molecular docking to obtain 14 compounds with strong binding ability to farnesoid X receptor, and conducts in vitro farnesoid X receptor agonist activity experiments, MTT method to detect compound cytotoxicity, and GPO-PAP method to test the effect of compounds on the triglyceride content of HepG2 cells induced by free fatty acids. The experimental results show that the above 14 compounds have significant agonist activity on farnesoid X receptor and are non-cytotoxic to HepG2 cells and L02 cells. In particular, compounds 1, 2, and 3 can significantly reduce the triglyceride content in fatty degenerated HepG2 cells. The experimental results show that these 14 compounds can be further developed into drugs for treating farnesoid X receptor-mediated related diseases such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, primary biliary cholangitis, and lipid metabolism disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the molecular docking interaction model diagram of compound 1 and FXR protein;

[0022] Figure 2 It is the molecular docking interaction model diagram of compound 2 and FXR protein;

[0023] Figure 3 It is the molecular docking interaction model diagram of compound 3 and FXR protein;

[0024] Figure 4 is the agonistic effect curve of compound 1 on FXR;

[0025] Figure 5 is the agonistic effect curve of compound 2 on FXR;

[0026] Figure 6 is the agonistic effect curve of compound 3 on FXR;

[0027] Figure 7 The results show the effects of different concentrations of compounds 1, 2, and 3 on the triglyceride content of HepG2 cells induced by free fatty acids. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0029] Example 1

[0030] A method for preparing a farnesoid X receptor agonist compound by virtual screening based on molecular docking technology comprises the following steps:

[0031] (1) Acquisition and processing of the farnesoid X receptor (FXR) protein structure

[0032] The protein crystal structure of FXR (PDBID: 3OLF) was downloaded from the RCSB PDB database (https: / / www.rcsb.org / ). The Prepare Protein module in Discovery Studio 2020 software was used to delete water molecules from the FXR protein, retain the A chain, remove the B, C, and D chains, and add hydrogen. According to the position of the co-crystal ligand OLF bound to FXR in the protein crystal structure, the active site was defined as X = 12.408263, Y = 23.008263, Z = 3.149421, and a radius of The molecular docking cavity constructed by this method is used for molecular docking in LibDock and CDOCKER.

[0033] The FXR protein (PDB ID: 3OLF) was imported into the Pymol software and processed as follows: the B, C, and D chains were deleted, and the A chain was retained; water removal and hydrogenation were performed to extract the cocrystal ligand OLF of the protein and save it as a pdb format file. Subsequently, the AutoDock molecular docking software was opened, and the FXR protein pdb format file processed and saved by Pymol was imported to add atom types and charge processing. The active site was defined as: X = 13.354, Y = 21.993, Z = 4.063, and a The molecular docking box is saved in pdbqt format after processing. The molecular docking model constructed by this method is used for Autodock Vina molecular docking.

[0034] (2) Source and processing of compound database

[0035] About two million compounds were obtained from ChemDiv, Drugbank, and ZINC databases and imported into DiscoveryStudio 2020 software. In the Small Molecules module, the Filter by Lipinski and VeberRules function was used to change the parameter of the Number of Violations Allowed item to 0 for drug-likeness screening. For compounds that meet drug-like properties, in the Small Molecules module, Prepare Ligands was run for hydrogenation and conformation optimization; then, Full Minimization was run to minimize the energy of the compounds based on the CHARMM force field. After the processing was completed, the compounds were saved in pdb and pdbqt format files, thereby constructing a database of compounds to be screened that meet drug-like properties.

[0036] (3) Virtual screening based on molecular docking

[0037] Molecular docking model was constructed. To ensure the reliability of the model, the co-crystal ligand OLF of the pre-processed FXR protein (PDB ID: 3OLF) was taken out, and the co-crystal ligand was re-docking into the set FXR protein molecular docking cavity using three molecular docking methods: LibDock, CDOCKER, and Autodock Vina. The results showed that the root mean square deviation (RMSD) values ​​of the three molecular docking models were all less than It showed good docking reproducibility and can be used for subsequent molecular docking virtual screening. The constructed molecular docking model and the database of compounds to be screened were imported into the Discovery Studio2020 software. The LibDock molecular docking method was first used for initial screening, and the compounds ranked in the top 5% were selected with the LibDock Score docking score as a reference. Then, the CDOCKER molecular docking method was used for the second round of screening, and the -CDOCKER InteractionEnergy docking score was used as a reference to select the compounds ranked in the top 5% for the next round of screening. The AutodockVina molecular docking method was used to perform a third round of screening on the top 5% of the compounds ranked in CDOCKER molecular docking. Finally, based on the Binding Affinity docking score and the binding conformation with the key amino acid residues of FXR, the compounds ranked high in molecular docking were selected as lead compounds with potential FXR agonist activity for subsequent biological activity verification.

[0038] The compounds specifically include:

[0039] Compound 1

[0040] English name: Kaempferol 3-O-gentiobioside

[0041] Chinese name: Kaempferol-3-O-gentiobioside, Cassia leaf glycoside

[0042] CAS No.: 22149-35-5

[0043] Chemical Structure:

[0044]

[0045] Compound 2

[0046] English name: Tiliroside

[0047] Chinese name:Tilia ginsenoside

[0048] CAS No.: 20316-62-5

[0049] Chemical Structure:

[0050]

[0051] Compound 3

[0052] English name: Dehydroandrographolide succinate Chinese name: Dehydroandrographolide succinate CAS number: 786593-06-4

[0053] Chemical Structure:

[0054]

[0055] Compound 4

[0056] English name: Apigenin 8-C-neohesperidoside Chinese name: Vitexin rhamnoside

[0057] CAS No.: 64820-99-1

[0058] Chemical Structure:

[0059]

[0060] Compound 5

[0061] English name: Luteolin 7-glucuronide Chinese name: Luteolin-7-glucuronide CAS number: 29741-10-4

[0062] Chemical Structure:

[0063]

[0064] Compound 6

[0065] English name: Genipin 1-β-D-gentiobioside Chinese name: Genipin-1-β-D-gentiobioside CAS number: 29307-60-6

[0066] Chemical Structure:

[0067]

[0068] Compound 7

[0069] English name: Glomeratose A

[0070] Chinese name: Globuloside A

[0071] CAS No.: 202471-84-9 Chemical Structure:

[0072]

[0073] Compound 8

[0074] English name: Isochlorogenic acid C Chinese name: Isochlorogenic acid C CAS number: 57378-72-0 Chemical structure:

[0075]

[0076] Compound 9

[0077] English name: Orientin

[0078] Chinese name: orientin CAS number: 28608-75-5 Chemical structure:

[0079]

[0080] Compound 10

[0081] English name: Aurantiamide Chinese name: Orange-yellow piperonamide CAS number: 58115-31-4 Chemical structure:

[0082]

[0083] Compound 11

[0084] English name: Matairesinol Chinese name: Matairesinol CAS number: 580-72-3 Chemical structure:

[0085]

[0086] Compound 12

[0087] English name: Cyclocurcumin

[0088] Chinese name: Cyclocurcumin CAS number: 153127-42-5

[0089] Chemical Structure:

[0090]

[0091] Compound 13

[0092] English name: 8α-Hydroxypinoresinol Chinese name: 8α-Hydroxypinoresinol CAS number: 81426-17-7

[0093] Chemical Structure:

[0094]

[0095] Compound 14

[0096] English name: L-(-)-1-Methyltryptophan Chinese name: Abrusine CAS number: 21339-55-9

[0097] Chemical Structure:

[0098]

[0099] The docking scores of compounds 1-14 are shown in Table 1:

[0100] Table 1 Docking scores of the top 14 compounds in molecular docking

[0101]

[0102] a LibDock molecular docking scoring function in Discovery Studio 2020 software.

[0103] b CDOCKER molecular docking scoring function in Discovery Studio 2020 software.

[0104] c Autodock Vina molecular docking scoring function.

[0105] d OCA and GW4064 are positive control compounds, both of which are FXR agonists.

[0106] As shown in Table 1, after three rounds of molecular docking, 14 lead compounds were obtained through virtual screening, and their molecular docking scores were higher than those of positive compounds OCA and GW4064, indicating that the lead compounds obtained through virtual screening have good binding ability with FXR protein. Specifically, the molecular docking interaction patterns of compounds 1, 2, and 3 with FXR protein can be referred to Figure 1 , Figure 2 , Figure 3 .

[0107] Example 2: In vitro biological activity test

[0108] 1. Dual luciferase reporter gene assay

[0109] The present invention evaluates the agonist activity of the lead compound on FXR by a dual luciferase reporter gene assay. HEK293T cells were seeded at a density of 20,000 / well in a 96-well plate. Take 0.15 μL of Polyethylenimine Linear, add 50 ng each of pRL-TK, pGL3-BASIC-FXR, and Pcdna3.1-NR1H4, add DMEM culture medium to a final volume of 30 μL, and prepare a transfection complex solution. Add 100 μL of DMEM culture medium to each well of the 96-well plate, then add 30 μL of transfection complex solution, mix well, and transfer to an incubator for incubation for 6 hours. After the incubation is completed, replace with fresh complete culture medium and continue incubation for 24 hours. After the incubation is completed, remove the old culture medium, add culture medium containing different concentrations of the drug to be tested, and incubate for another 24 hours. After 24 hours, transfer the cultured cells to a 1.5 mL centrifuge tube for lysis and centrifugation. The firefly luciferase signal and the Renilla luciferase signal of the sample were detected using the dual luciferase reporter gene detection kit from UElandy. The signal value of the compound is the ratio of the luciferase signal to the Renilla luciferase signal, which represents the transcriptional activity of the compound.

[0110] The formula for calculating the FXR agonist rate is: Activation rate % = (compound signal value - background signal value) / (maximum signal value of positive compound - background signal value) × 100%. The half effective concentration (EC) of the compound agonist for FXR was calculated by nonlinear regression (curve fitting) method. 50 ). The results are shown in Table 2.

[0111] 2. Cell Viability Detection

[0112] The present invention uses the MTT method to detect the effect of the compound on the cell viability of HepG2 cells (human liver cancer cells) and L02 cells (normal human liver cells) to investigate the cytotoxicity of the compound. HepG2 and L02 cells are uniformly inoculated in a 96-well plate, and when the cells grow to a confluence of 80%, they are divided into a drug group and a control group. Complete medium containing 6.25, 12.5, 25, 50, 100, and 150 μM concentrations of drugs is added to each well of the drug group, and the control group is treated with a complete medium without drugs. The well plate is placed in an incubator and cultured for 24 hours. After the cell culture is completed, 20 μL of MTT solution is added to each well and incubated in the dark for 4 hours. After completion, the culture fluid in the well is aspirated, 150 μL of DMSO is added to each well, and after low-speed shaking, the absorbance value of each well is measured at a wavelength of 490 nm. The calculation formula for cell viability is: Cell viability (%) = (OD value of the drug group-OD value of the blank group) / (OD value of the control group-OD value of the blank group) × 100. The half inhibitory concentration (IC) of the compound on cells was calculated by nonlinear regression (curve fitting) method. 50 ). The results are shown in Table 2.

[0113] Table 2 FXR agonist activity and cytotoxicity of 14 compounds

[0114]

[0115] a The data are expressed as the average of 3 parallel experiments.

[0116] b OCA and GW4064 are positive control compounds, both of which are FXR agonists.

[0117] The results of dual luciferase reporter gene assay and MTT assay are shown in Table 2. EC values ​​of 14 compounds obtained by virtual screening for FXR stimulation 50 <500nM, and the agonist activity was better than that of the positive control compound OCA (EC 50 =558.6 nM) and GW4064 (EC 50 =468.8nM), all of which showed significant FXR agonist activity. In addition, the results in Table 2 also showed that the 14 compounds had an inhibitory IC of 1. 50 The values ​​were all greater than 100 μM, and the cell viability of HepG2 cells and L02 cells was slightly affected, indicating that these 14 compounds had basically no cytotoxicity to HepG2 cells and L02 cells. The agonist effect curves of compounds 1, 2, and 3 on FXR are shown in Figure 4 , Figure 5 , Figure 6 .

[0118] Example 3: Effects of compounds on triglyceride content in HepG2 cells induced by free fatty acids

[0119] The GPO-PAP method was used to determine the effect of compounds on triglyceride content in HepG2 cells induced by free fatty acids. First, HepG2 cells grown to the logarithmic phase were digested and counted, and then seeded in a 6-well plate at an appropriate density. When the cells were confluent to 80-90%, the old culture medium was discarded, and DMEM culture medium containing 5% BSA was added. The cells were starved for 16 hours and the old culture medium was discarded. After that, the old culture medium was discarded again, and DMEM culture medium containing 0.5% BSA and 0.6mmol free fatty acids was added to induce for 12 hours to establish a cell model of fatty degeneration. After the model was established, the cells were cultured for 24 hours with different concentrations of compounds (including 0.025μM compound 1, 0.25μM compound 1, 0.025μM compound 2, 0.25μM compound 2, 0.025μM compound 3, 0.25μM compound 3, 10μM OCA) (three replicates for each concentration). After the compound treatment is completed, add 1mL PBS to each well to rinse twice, then add 0.25% trypsin, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant of the centrifuge tube. Add 1% Triton X-100 to lyse the cells for 30 minutes to extract intracellular triglycerides. After the lysis is completed, use the triglyceride kit for detection. The specific steps are: take 2.5μL of the supernatant after lysis, add it to 250μL of triglyceride detection working solution, and incubate at 37°C in the incubator for 10 minutes. Finally, the reaction solution is placed at a wavelength of 510nm to detect the absorbance value to quantitatively evaluate the triglyceride content.

[0120] The results of the test on the triglyceride content of HepG2 cells induced by free fatty acids by compounds 1, 2, and 3 are shown in Figure 7 Compared with the model group, compounds 1, 2, and 3 can significantly reduce the triglyceride level in HepG2 cells induced by free fatty acids, and the effect is better than the positive control drug OCA, *P≤0.05, **P≤0.01, ***P≤0.001.

Claims

1. Application of virtual screening compounds in the preparation of farnesoid X receptor agonists, characterized in that: The compound is any one of the following:

2. The use according to claim 1, characterized in that: The farnesoid X receptor agonist is used for treating and / or preventing diseases mediated by farnesoid X receptor.

3. The use according to claim 2, characterized in that: The disease mediated by farnesoid X receptor is one or a combination of two or more of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, primary biliary cholangitis, and lipid metabolism disorder.

4. A drug for treating and / or preventing a disease mediated by farnesoid X receptor, characterized in that: Any of the following compounds is the active ingredient:

5. The drug according to claim 4, characterized in that: It consists of any one of Compound 1 to Compound 14 and a pharmaceutically acceptable carrier.

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