Application of compound CCP-Cl as FGL1 binding inhibitor to preparation of medicine for treating renal fibrosis

Abstract

The invention discloses application of a compound CCP-Cl as an FGL1 binding inhibitor to preparation of a medicine for treating renal fibrosis. The invention finds that the compound CCP-Cl has an obvious effect of treating renal fibrosis; the invention also finds that the compound CCP-Cl is a binding inhibitor of the FGL1 protein and can play a role in treating renal fibrosis by inhibiting the FGL1 protein. Therefore, the compound CCP-Cl has the prospect of being developed into a medicine for treating renal fibrosis, has the prospect of being developed into a medicine for treating kidney diseases (such as chronic kidney diseases and diabetic nephropathy) accompanied by renal fibrosis, and has the prospect of being developed into an FGL1 protein binding inhibitor medicine. The application of the compound CCP-Cl is not disclosed in the prior art, and no technical enlightenment exists.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to new uses of known compounds, and specifically relates to the use of compound CCP-Cl as an FGL1 binding inhibitor and further for preparing a drug for treating renal fibrosis. Background Art

[0002] Renal fibrosis is the common basis for the progressive development of chronic kidney disease caused by various causes. It is characterized by the continuous deposition of extracellular matrix, which leads to renal tissue damage and impaired renal function, and eventually progresses to renal failure. At present, the clinical treatment measures for chronic kidney disease are mainly aimed at its primary disease, and there is no special drug for the treatment of renal fibrosis. The pathogenesis of renal fibrosis is complex. Due to the stimulation of various pathogenic factors such as trauma, infection, inflammation, blood circulation disorders, and immune response, the kidney's intrinsic cells are damaged. In the later stage of development, a large amount of collagen deposition and accumulation occur, causing the renal parenchyma to gradually harden and form scars until the kidney completely loses its organ function. The process of fibrosis and hardening of intrinsic cells in the kidney is also the process of renal fibrosis.

[0003] Fibrinogen-like protein 1 (FGL1), also known as hepatocyte-derived fibrinogen-related protein 1 (HFREP-1) or heparin (HPS), is a member of the fibrinogen family and a specific hepatocyte mitotic activity factor. It not only protects the liver, but also promotes the mitosis and proliferation of hepatocytes. FGL1 is located on chromosome 8p22-p21.3, and the gene cDNA length is 936bp. The gene can encode 312 amino acids, and the 1 to 22 amino acids at the N-terminus are signal peptide sequences. It usually exerts its biological activity in the form of homodimers.

[0004] The chemical name of CCP-Cl is 1-(2-carbamamido-2-oxoethyl)-3-carbamoylpyridin-1-ium--chloride, which is a chloride salt with the following cationic structure. Currently, there are no reports on the interaction of this compound with FGL1 protein or renal fibrosis:

[0005]

[0006] Based on the applicant's discovery, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a use of the compound CCP-Cl as a FGL1 binding inhibitor and further for preparing a drug for treating renal fibrosis.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] Use of a pharmaceutically acceptable salt of a cation of the following chemical structure for preparing a drug for treating renal fibrosis:

[0010]

[0011] Preferably, the anion in the pharmaceutically acceptable salt is a chloride ion.

[0012] More preferably, the drug uses a pharmaceutically acceptable salt of a cation of the chemical structure as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0013] Use of a pharmaceutically acceptable salt of a cation of the following chemical structure for preparing a drug for treating kidney disease associated with renal fibrosis:

[0014]

[0015] Preferably, the anion in the pharmaceutically acceptable salt is a chloride ion.

[0016] Preferably, the kidney disease accompanied by renal fibrosis includes chronic kidney disease.

[0017] More preferably, the drug uses a pharmaceutically acceptable salt of a cation of the chemical structure as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0018] The use of a pharmaceutically acceptable salt of a cation of the following chemical structure for preparing a drug of an FGL1 protein binding inhibitor, wherein the FGL1 protein binding inhibitor binds to the FGL1 protein and thereby inhibits the binding of the FGL1 protein to its receptor:

[0019]

[0020] Preferably, the anion in the pharmaceutically acceptable salt is a chloride ion.

[0021] More preferably, the drug uses a pharmaceutically acceptable salt of a cation of the chemical structure as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0022] Beneficial effects:

[0023] The present invention found that compound CCP-Cl has a significant effect in treating renal fibrosis; the present invention also found that compound CCP-Cl is a binding inhibitor of FGL1 protein, which may play a therapeutic role in renal fibrosis by inhibiting FGL1 protein. Therefore, compound CCP-Cl has the prospect of being developed into a drug for treating renal fibrosis, has the prospect of being developed into a drug for treating kidney diseases accompanied by renal fibrosis (such as chronic kidney disease, diabetic nephropathy), and has the prospect of being developed into a drug for FGL1 protein binding inhibitor. The prior art has not disclosed the above-mentioned uses of compound CCP-Cl, and there is no technical inspiration. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the expression level of fibrosis genes in each group of cells;

[0025] Figure 2 is the expression level of fibrotic proteins in each group of cells (quantified by Western Blot and bar graph);

[0026] Figure 3 A is the binding mode of compound CCP-Cl and FGL1 protein; B is the RMSD (root mean square deviation) of compound CCP-Cl binding to FGL1 protein; C is the RMSF (root mean square fluctuation) of compound CCP-Cl binding to FGL1 protein. DETAILED DESCRIPTION

[0027] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0028] 1. Experimental Materials

[0029] 1. Experimental cells

[0030] The human renal proximal tubule epithelial cell line (HK2) was cultured in DMEM / F12 medium containing double antibodies with 10% FBS, incubated in a 5% CO2, 37°C cell culture incubator, and grown to 70% confluence for subsequent experiments.

[0031] 2. Instruments and equipment

[0032] FRESCO17 high-speed refrigerated centrifuge (Thermo Fisher Scientific, USA); biological safety cabinet (Thermo Fisher Scientific, USA); Synergy2 multifunctional microplate reader (BioTek, USA); Thermomixer comfort metal bath heater (Eppendorf, Germany); PROTEAN-II XI Cell vertical electrophoresis tank (Bio-Rad, USA); Mini PROTEAN II (Bio-Rad, USA); PowerPac Basic electrophoresis instrument (Bio-Rad, USA); Tanon-520 Tianneng gel imaging system (Shanghai Tianneng Technology Co., Ltd.); real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, USA).

[0033] 3. Reagents

[0034] RIPA strong lysis buffer (Jiangsu Biyuntian Company); 30% acrylamide / methylene bisacrylamide solution purchased from Biotech (Shanghai Biotech Company); 0.5M pH=6.8 Tris-HCl buffer solution and 1.5M pH=8.8 Tris-HCl buffer solution (Shanghai Biotech Company); protein loading marker (Thermo Fisher Scientific, USA); antibody PAI-1 (CST, USA); antibody FN (abcam, USA); antibodies Vimentin, GAPDH (Wuhan Sanying Biotechnology Co., Ltd.); qPCR SYBR kit (Nanjing Novozymes Company).

[0035] CCP-Cl was purchased from ChemDiv, USA, with a purity of 99.9%.

[0036] 2. Experimental Methods

[0037] 1. Renal fibrosis modeling and drug intervention

[0038] (1) When HK2 cells grew to 70%, TGF-β modeling was performed. The culture medium was discarded, and the cells were washed once with sterilized 1× PBS. Then, the cells were starved for 4 hours with DMEM / F12 culture medium containing double antibodies without FBS. After starvation, the cells were replaced with DMEM / F12 culture medium containing double antibodies containing 1% FBS and 10 ng / mL TGF-β was added to the culture medium for fibrosis induction. At the same time, a control group without TGF-β was set up.

[0039] (2) 24 hours after modeling, 10 μM CCP-Cl was added to the drug group, and the corresponding volume of PBS was added to the control group and the model group. After further culture for 24 hours, the cells were sampled and subsequent experiments were performed.

[0040] 2. BCA protein quantification

[0041] (1) Preparation of protein standard solution: Add 1.2 mL of protein standard preparation solution to the protein standard, vortex centrifuge to prepare a 25 mg / mL protein standard solution, dilute with PBS to a final concentration of 0.5 mg / mL, and store at -20°C;

[0042] (2) Preparation of working solution: Prepare an appropriate amount of BCA working solution in a ratio of A solution: B solution = 50:1, mix it quickly and thoroughly before use;

[0043] (3) Draw a standard curve: add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard solution to a 96-well plate, fill up to 20 μL with PBS, and spot two replicates in each well;

[0044] (4) Add 1 μL of protein sample to a 96-well plate, fill to 20 μL with PBS, and spot two replicates per well;

[0045] (5) Add 200 μL BCA working solution to the above standard wells and sample wells and incubate in a 37°C oven for 20 minutes;

[0046] (6) Take out the 96-well plate, measure the absorbance of each well at a wavelength of 562 nm on an ELISA reader, draw a standard curve, and calculate the loading volume required for 40 μg of protein;

[0047] (7) Add 5× loading buffer to each protein sample, pipette evenly, and denature in a 99°C metal bath for 10 min. Take out, cool to room temperature, vortex, centrifuge, and store in a -20°C refrigerator.

[0048] 3. Western Blot

[0049] (1) Prepare separation gels of different concentrations and 5% stacking gel according to experimental requirements.

[0050] (2) Take out the protein sample and protein loading marker from -20℃, thaw them, vortex and centrifuge them, and carefully remove the spotting comb;

[0051] (3) Add 1× electrophoresis buffer to the glass bottle. Pour the electrophoresis solution into the electrophoresis tank and between the two glass plates, load the sample according to the calculated sample volume, and place different volumes of protein markers on the left and right sides of the gel plate to distinguish them;

[0052] (4) Place the electrophoresis tank in the electrophoresis apparatus and adjust the voltage to 80 V for 30 minutes. After the protein sample is concentrated and gelled into a straight line, adjust the voltage to 120 V and continue electrophoresis for 80 minutes;

[0053] (5) When the sample reaches the bottom of the electrophoresis, stop the electrophoresis;

[0054] (6) Add 1× wet transfer buffer to the glass bottle. Use a gel cutting board to cut the desired gel and place it in the transfer solution. Cut the PVDF membrane to the appropriate size and place the cut gel into the transfer folder: put the sponge, filter paper, gel, membrane, filter paper, and sponge in turn, layer by layer. Turn on the power, set the transfer time and current, 350 mA constant current, and transfer for 70 minutes;

[0055] (7) After the transfer, the membrane was placed in 5% skim milk prepared in TBST and blocked at room temperature for 2 hours;

[0056] (8) After blocking, place the membrane in the antibody diluent containing the primary antibody, place it on a rocking table, and incubate it in a refrigerator at 4°C overnight;

[0057] (9) The next day, incubate with secondary antibodies and wash the membrane again.

[0058] (10) The membrane was incubated with ECL luminescent solution and developed in a dark room.

[0059] 4. Real-time fluorescence quantitative PCR

[0060] 4.1 Total RNA extraction

[0061] (1) Add 500 μL Trizol lysis buffer to each well of a 6-well cell culture plate, pipette the cells off, and collect them in an RNase-free 1.5 mL centrifuge tube.

[0062] (2) Centrifuge at 12,000 rpm at 4°C for 15 min. Carefully pipette 600 μL of the upper aqueous phase into a new RNase-free 1.5 mL centrifuge tube. Add an equal volume of isopropanol, mix by inverting, and let stand at 4°C for 10 min.

[0063] (3) Centrifuge, discard the supernatant, add 500 μL of 75% ethanol, flick the bottom of the tube to suspend the precipitate, and invert it several times.

[0064] (4) Determine the RNA concentration and purity and calculate the amount required for 1000 ng RNA.

[0065] 4.2 RNA Reverse Transcription

[0066] Use two-step RT-PCR for reverse transcription and prepare 20 μL reaction system in an RNase free centrifuge tube:

[0067] Reverse transcription system

[0068]

[0069] After mixing the above reaction reagents, reverse transcription is performed. The procedure is as follows:

[0070] Reverse transcription procedure

[0071]

[0072] After the reaction, the template cDNA was obtained and stored at 4°C for future use.

[0073] 4.3 Real-time quantitative PCR (qRT-PCR)

[0074] (1) Add DNAForwardPrimer and Reverse Primer to the corresponding DEPC water, vortex and dissolve, mix in a 1:1 volume ratio to obtain the primer master solution (10×), dilute with DEPC water to 1× before use; add 80μL DEPC water to dilute the cDNA obtained by reverse transcription, vortex and centrifuge, and set aside; the system required for qRT-PCR (10μL) is: primer: cDNA: SYBR Green = 1:4:5, add them to the corresponding wells of the PCR white plate, stick the membrane and scrape it flat, centrifuge at 2000 rpm for 1 minute, put it into the qRT-PCR instrument and set the program for the experiment. The qRT-PCR program is as follows:

[0075]

[0076] (2) Primer sequence

[0077]

[0078] 5. Molecular dynamics simulation and molecular docking

[0079] The crystal structure of human FGL1 was predicted by AlphaFold 3 using Molecular docking of CCP-Cl and FGL1 was performed using Maestro software. Dynamics simulations were performed for 100 ns in an unconstrained NPT ensemble with periodic boundaries. The stability of each architecture was checked by analyzing the RMSD (root mean square deviation) and RMSF (root mean square fluctuation) of the dynamics trajectory using the CPPTRAJ program in AMBER.

[0080] 6. Data Analysis

[0081] Data were expressed as mean ± SEM. One-way ANOVA was used to analyze the statistical differences of multiple samples.

[0082] 3. Experimental Results

[0083] like Figure 1As shown in the figure, in HK2 cells, PBS was added to the control group, and TGF-β was added to the model group and the drug-treated group for stimulation for 24 hours. Among them, 10 μM CCP-Cl was added to the drug-treated group for intervention. The results showed that compared with the control group, under TGF-β stimulation, the expression of fibrosis genes COL1A1 (type I collagen), COL3A1 (type III collagen) and ACTA2 (actin α2) in the model group was significantly increased, indicating that the renal fibrosis model was successfully established; compared with the model group, after the intervention of compound CCP-Cl, fibrosis genes were significantly downregulated, indicating that compound CCP-Cl significantly improved renal fibrosis.

[0084] like Figure 2 As shown in the figure, in HK2 cells, PBS was added to the control group, TGF-β was added to the model group and the drug-treated group for stimulation for 24 hours, and 10 μM CCP-Cl was added to the drug-treated group for intervention. The results showed that compared with the control group, the expression of fibrosis proteins FN (fibronectin), VIMENTIN (vimentin) and PAI-1 (plasminogen activator inhibitor-1) in the model group under TGF-β stimulation was significantly increased, indicating that the renal fibrosis model was successfully established; compared with the model group, after the intervention of compound CCP-Cl, fibrosis proteins were significantly downregulated, indicating that compound CCP-Cl significantly improved renal fibrosis.

[0085] Figure 3 In the figure, A is the binding mode of compound CCP-Cl and FGL1 crystal structure, and the docking score is -7.87 kcal / mol. The results show that the binding sites of CCP-Cl and FGL1 are arginine at position 260 and aspartic acid at position 280. In order to verify the stability of the binding between CCP-Cl and FGL1, we conducted a 100 ns kinetic simulation of the small molecule-protein complex. Figure 3 Middle B shows that the backbone RMSD (root mean square deviation) of FGL1 converges to The trajectories all showed stable RMSD values, indicating that the binding between FGL1 protein and CCP-Cl had good stability. Figure 3 C is the calculated RMSF (root mean square fluctuation) value of the Cα atom of FGL1, and the results show that there is no obvious fluctuation in the binding pocket residues. The above indicates that compound CCP-Cl is stably bound to FGL1 protein, and compound CCP-Cl is a potential specific inhibitor of FGL1 protein.

[0086] In summary, compound CCP-Cl has a significant effect in treating renal fibrosis, and compound CCP-Cl is also a binding inhibitor of FGL1 protein, which may play a therapeutic role in renal fibrosis by inhibiting FGL1 protein. Therefore, compound CCP-Cl has the prospect of being developed into a drug for treating renal fibrosis, a drug for treating kidney diseases (such as chronic kidney disease and diabetic nephropathy) accompanied by renal fibrosis, and a drug for FGL1 protein binding inhibitor. The prior art has not disclosed the above-mentioned uses of compound CCP-Cl, and there is no technical inspiration.

[0087] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Use of a pharmaceutically acceptable salt of a cation of the following chemical structure for preparing a drug for treating renal fibrosis:

2. The use according to claim 1, wherein the anion in the pharmaceutically acceptable salt is a chloride ion.

3. The use according to claim 1 or 2, wherein the drug has a pharmaceutically acceptable salt of the cation of the chemical structure as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

4. Use of a pharmaceutically acceptable salt of a cation of the following chemical structure for preparing a drug for treating kidney disease associated with renal fibrosis:

5. The use according to claim 4, wherein the anion in the pharmaceutically acceptable salt is a chloride ion. The use according to claim 4 , wherein the kidney disease accompanied by renal fibrosis comprises chronic kidney disease.

7. The use according to any one of claims 4 to 6, wherein the drug has a pharmaceutically acceptable salt of the cation of the chemical structure as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

8. Use of a pharmaceutically acceptable salt of a cation having the following chemical structure for preparing a drug of an FGL1 protein binding inhibitor, wherein the FGL1 protein binding inhibitor binds to the FGL1 protein and thereby inhibits the binding of the FGL1 protein to its receptor:

9. The use according to claim 8, wherein the anion in the pharmaceutically acceptable salt is a chloride ion.

10. The use according to claim 8 or 9, wherein the drug has a pharmaceutically acceptable salt of the cation of the chemical structure as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

Images