Pharmaceutical application of nobiletin as a small molecule agonist of EPOR in hypoxic injury
By developing Chuanchen Piosin as an EPOR small molecular agonist, the existing EPO and RHEPO have the problems of inconvenience and adverse events that are inconvenient and possibly caused by hypoxia damage in the treatment of hypoxia damage. Essence
Patent Information
- Application Number
- CN202310352838.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-04
- Publication Date
- 2025-01-24
- Estimated Expiration
- 2043-04-04
AI Technical Summary
Existing EPO and rhEPO have problems with inconvenient administration, harsh storage conditions and possible adverse events such as hypertension, thrombosis and tumor progression in the treatment of hypoxia injury.
Nobiletin was developed as an EPOR small molecule agonist. By specifically binding to the EPOR protein, it activates EPOR and triggers an increase in downstream calcium signals and cAMP levels, thereby protecting glomerular mesangial cells from hypoxic damage.
Chuanchenpi can significantly improve the growth rate of glomerular cell cell cells under hypoxia damage, reduce apoptosis, and protect cells from damage, and provide a novel drug for treating hypoxia damage.
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Figure CN116159050B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to pharmaceutical application of nobiletin as a small molecule agonist of EPOR in hypoxic injury. Background Art
[0002] Erythropoietin (EPO) is an acidic glycoprotein hormone that is mainly secreted by the kidneys. It exerts a wide range of biological effects by binding to the erythropoietin receptor (EPOR) in the body. EPO is one of the essential hormones involved in human hematopoiesis. It can promote the differentiation and proliferation of erythroid progenitor cells, increase hemoglobin levels, and improve anemia symptoms. EPO is mostly used in the treatment of renal anemia and tumor-related anemia in clinical practice. In 1989, recombinant human erythropoietin (rhEPO) developed by gene recombination technology began to be used in clinical practice. It is widely used in the treatment of renal anemia, tumor chemotherapy-related anemia, perioperative anemia, and anemia caused by bone marrow failure. However, both EPO and rhEPO need to be injected, and patient compliance is low. As a large molecule protein drug, there is a problem of harsh storage conditions. In addition, it also has the disadvantage of easily causing adverse events such as hypertension, thrombosis, and tumor progression.
[0003] In recent years, due to the widespread discovery of EPOR in non-hematopoietic tissues, the physiological effects of EPO other than hematology have been widely concerned, such as anti-apoptosis, angiogenesis, immunomodulation, neuroprotection, etc. Therefore, the development of effective therapeutic drugs in the EPO-EPOR cycle pathway has great development value and clinical significance. Studies have shown that the activation of EPOR is a necessary pathway to trigger its downstream signaling pathways. Therefore, the development of EPOR small molecule agonists with small molecular weight, oral administration, and blood-brain barrier crossing has great development potential and clinical value. However, there are few studies on EPOR small molecule agonists. Only reports have shown that ingenol-3,20 dibenzoic acid diethylene glycol ester is a potential EPOR small molecule agonist. It binds to EPOR through physical adsorption, forms an agonist effect with the receptor at low doses, and produces an antagonist effect at high doses. However, its binding with EPOR and the analysis of its action force still need to be studied.
[0004] Tangerine peel (Citrus Reticulata) is the dried mature peel of the citrus plant and its cultivated varieties of the Rutaceae family. It is mainly produced in Fujian, Zhejiang, Guangdong and other places in my country. It tastes bitter and spicy, and is warm in nature. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. It is a commonly used Chinese medicine and a good food for both medicine and food. Tangerine peel has a wide range of pharmacological effects and plays an active role in protecting the liver, anti-tumor, and anti-respiratory diseases. The active Chinese medicine ingredient chuanxiongpi extracted from tangerine peel has multiple pharmacological effects such as anti-tumor, hypoglycemic, inhibition of oxidative stress, inhibition of inflammatory response, and protection of cardiovascular system. However, there is no relevant report on the role of chuanxiongpi as a small molecule agonist of EPOR in the treatment of renal hypoxia. Summary of the invention
[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide pharmaceutical application of nobiletin as a small molecule agonist of EPOR in hypoxic injury.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] The invention discloses the use of nobiletin as an EPOR small molecule agonist in the preparation of a therapeutic drug for treating diseases caused by hypoxia damage. The drug specifically binds to the EPOR protein.
[0008] Preferably, the drug is a drug that can specifically bind to the ARG178 site of the EPOR protein.
[0009] Preferably, the drug is a drug targeting EPOR protein.
[0010] Further preferably, the drug is a drug that specifically activates EPOR to trigger downstream calcium signals and increase cAMP levels.
[0011] Preferably, the drug is a drug for hypoxic damage to the whole body or local organs.
[0012] More preferably, the drug is a drug for kidney damage caused by hypoxia.
[0013] More preferably, the drug is a drug that increases the proliferation rate and reduces the apoptosis rate of glomerular mesangial cells under hypoxic injury conditions.
[0014] More preferably, the drug is a drug that protects glomerular mesangial cells from hypoxic damage.
[0015] The invention also discloses a medicine for improving cell damage caused by hypoxia, wherein the medicine is a preparation made of nobiletin and pharmaceutically acceptable auxiliary materials.
[0016] Preferably, the drug is a drug targeting hypoxic injury to glomerular mesangial cells.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] The present invention discloses a novel pharmaceutical use of nobiletin as a small molecule agonist of EPOR in hypoxic injury. The present invention discloses for the first time that nobiletin can specifically bind to EPOR and bind to EPOR through SiO 2 -GLP-1R / CMC model analysis determined the binding specificity of Nobiletin to EPOR; molecular docking technology was used to identify the binding site of Nobiletin to EPOR; frontier analysis technology and nonlinear chromatography analysis were used to identify the interaction between Nobiletin and EPOR; fluorescent calcium imaging technology and cAMP level determination were used to identify the intrinsic activity of Nobiletin on EPOR; cell activity experiments and proliferation and apoptosis experiments were used to identify the protective effect of Nobiletin on hypoxia-induced cell damage. The above research results fully confirmed the protective effect of Nobiletin as a small molecule agonist of EPOR on hypoxia-induced kidney damage. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The computer-assisted molecular docking experiment simulates the binding of EPO and nobiletin to EPOR protein;
[0020] Figure 2 The results of the interaction between Nobiletin and EPOR determined by frontal analysis (ab) and nonlinear chromatography (cd); (a) Nobiletin on SiO 2 -Representative breakthrough curves on EPOR / CMC column; (b) Nobiletin on SiO 2 -EPOR / CMC column 1 / mLapp and 1 / [A] regression curve; (c) is the SiO2 column with a series of concentrations of nobiletin injected directly 2 -EPOR / CMC column chromatogram overlay; (d) The experimental determination curve (upper) and nonlinear chromatographic fitting curve (lower) of 2.0 mmol / L nobiletin fitted by Peak Fit 4.12 software;
[0021] Figure 3 The cell activity assay was used to determine the effects of EPO and nobiletin on CoCl 2 The effect of CoCl on the proliferation of glomerular mesangial cells induced by hypoxia. 2 Effects on the proliferation of glomerular mesangial cells; (b) Effects on the proliferation rate of glomerular mesangial cells after treatment with EPO and nobiletin;
[0022] Figure 4The results of the fluorescent calcium imaging technology and cAMP level determination experiment were used to explore the effects of EPO and nobiletin on the calcium signal and cAMP level of EPOR-HEK293 cells. (a) The time-fluorescence intensity curve of the fluorescence calcium signal intensity of EPOR-HEK293 cells treated with different concentrations of EPO; (b) The quantitative statistical results of the relative fluorescence calcium signal intensity (peak fluorescence signal minus valley fluorescence signal intensity) and concentration of EPOR-HEK293 cells treated with different concentrations of EPO; (c) The results of the cAMP release level of EPOR-HEK293 cells stimulated by different concentrations of EPO; (d) The time-intensity curve of the fluorescence calcium signal intensity of EPOR-HEK293 cells treated with different concentrations of nobiletin; (e) The quantitative statistical results of the relative fluorescence calcium signal intensity and concentration of EPOR-HEK293 cells treated with different concentrations of nobiletin; (f) The results of the cAMP release level of EPOR-HEK293 cells stimulated by different concentrations of nobiletin.
[0023] Figure 5 shows the effects of EPO and nobiletin on CoCl 2 The effect of EPO and nobiletin on apoptosis of mesangial cells caused by hypoxia. (a) The flow cytometry results of the Annexin V-FITC / PI apoptosis kit were used to measure the effect of EPO and nobiletin on mesangial cells; (b) The statistical results of the apoptosis rate of mesangial cells after drug treatment. DETAILED DESCRIPTION
[0024] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
[0025] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.
[0026] The present invention is further described in detail below in conjunction with the accompanying drawings:
[0027] The experimental materials involved in the present invention are as follows:
[0028] The molecule of nobiletin is C 21 H 22 O 8 , CAS No. 478-01-3, purchased from Chengdu Pufeide Biotechnology Co., Ltd.; the structural formula is as follows:
[0029]
[0030] EPOR high-expressing cells are obtained by transfecting HEK293 cells with a lentiviral plasmid vector containing the full-length human EPOR gene sequence and performing a 14-day resistance screening with puromycin.
[0031] Example 1: Specific binding of nobiletin to EPOR protein
[0032] Cell membrane chromatography (CMC) is an affinity chromatography that uses the cell membrane of cells that overexpress specific receptors to prepare a stationary phase and utilizes the binding characteristics of drugs to receptors to achieve high-throughput screening of membrane receptor-specific binding components in vitro. By constructing a stable cell line with high EPOR expression and expanding its culture, the cell membrane of cells that overexpress specific receptors is extracted and mixed with activated silica gel to prepare a cell membrane chromatography stationary phase. SiO 2 -EPOR / CMC column, and HPLC detection was performed. SiO 2 -NC-HEK293 / CMC column and SiO 2 - HEK293 / CMC column was used as negative control. The results are shown in Table 1 below:
[0033] Table 1 Retention time of nobiletin on EPOR high expression cell membrane chromatography
[0034]
[0035] It can be seen that nobiletin is retained on the EPOR high-expression cell membrane column, but not on the negative control column. The above results show that nobiletin has a good specific binding effect with EPOR protein.
[0036] Example 2: Investigation of protein binding sites of nobiletin and EPOR
[0037] The molecular docking study was conducted using AutoDock Vina software. The protein X-ray crystal structure (PDB code: 1ERN) was input. Water molecules were removed from the protein structure and hydrogen atoms were added, and the energy was minimized using the Tripos force field and Pullman charge. The structure of Nobiletin was drawn using ChemDraw 19.0, and the structure was optimized using ChemDraw 3D software. The ligand structure was optimized using AutoDock software, the charges in the ligand residues were dispersed, the root of the ligand was determined, and the torsion bonds were selected. AutoDock software was run in the Linux terminal, and the optimized protein and energy-optimized Nobiletin were loaded in turn. The grid energy was calculated using Autogrid, and the Lamarckian genetic algorithm was used to evaluate the binding of the ligand to the receptor using the free energy method. The results of molecular docking were visualized using pymol, and the results of molecular docking were displayed in different modes.
[0038] Use pymol to open the molecular docking results and visualize them. Figure 1 The docking results of EPO and nobiletin with EPOR are shown in Figure 1. The binding energy of nobiletin with EPOR is lower than that of EPO, and the ARG178 amino acid is the common binding site of EPO and nobiletin.
[0039] Example 3: Investigation of the interaction between nobiletin and EPOR
[0040] Frontier analysis is a classic chromatographic method for studying drug-receptor interactions. The present invention uses frontier analysis to determine the equilibrium dissociation constant ( K D The specific implementation method is as follows: a SiO 2 -EPOR / CMC column, configured with mobile phase (mobile phase A: 10 mmol / L sodium dihydrogen phosphate, pH 7.4; mobile phase B: containing 1 × 10 -6 mol / L nobiletin (10 mmol / L), sodium dihydrogen phosphate (pH 7.4), flow rate 0.2 mL / min, column temperature 37°C. First, phase A was used to balance SiO 2 -EPOR / CMC column for 1 h, and then the lowest concentration of the drug solution to be tested was continuously introduced into the SiO 2 -EPOR / CMC, form a breakthrough curve and stop after reaching the platform, then switch to phase A buffer to continue balancing SiO 2 -EPOR / CMC column, after reaching equilibrium, the next concentration of the drug is measured, and the breakthrough curve is recorded and the breakthrough time is calculated to obtain the concentration of nobiletin at different concentrations on SiO 2-Breakout curve chart on EPOR / CMC column, the result is as follows Figure 2 As shown in (a), it can be seen that the inflection point time of the breakthrough curve advances with the increase of the analyte concentration in the mobile phase, and the degree of the advance is related to the number of EPOR receptors on the cell membrane stationary phase surface, the concentration of the ligand in the mobile phase, and the size of the dissociation constant of the ligand-receptor affinity interaction. According to the chromatogram obtained by the cell membrane front analysis of Nobiletin, the first-order derivative of each chromatographic curve is calculated to obtain the breakthrough time, and then the saturated adsorption amount is calculated, and finally the interaction between Nobiletin and EPOR is calculated. K D Among them, the saturated adsorption amount is equal to the product of the breakthrough time and the drug concentration and flow rate in the mobile phase. The results are shown in Table 2.
[0041] The equilibrium dissociation constant of the drug was calculated according to the following formula (1): K D value).
[0042] (1)
[0043] Where:
[0044] m Lapp — The number of moles of ligand required to reach the midpoint of the breakthrough curve at a given molar concentration of ligand / mol;
[0045] [A] — concentration of ligand in mobile phase / mol / L;
[0046] m L —Total number of moles of bound receptors on the cell membrane chromatography column / mol;
[0047] K D — Equilibrium dissociation constant / mol / L.
[0048] According to the breakthrough curve obtained, formula m can be calculated Lapp value, and then according to formula (1-1), 1 / m Lapp Plotting 1 / [A], the derivative of the intercept of the regression curve is mL, and the ratio of the slope to the intercept is K D This value can reflect the strength of the binding ability between the ligand and the receptor. Generally, the smaller the value, the stronger the interaction between the ligand and the receptor.
[0049] Table 2 Frontier analysis results of nobiletin and EPOR
[0050]
[0051] The reciprocal of the saturated adsorption amount of nobiletin in Table 2 was used to perform linear regression on the reciprocal of the analyte concentration in the mobile phase to obtain a regression curve. The results are as follows: Figure 2 As shown in (b), the regression curve shows that the reciprocal of the breakthrough volume in the front analysis of nobiletin has a good linear relationship with the reciprocal of the analyte concentration in the mobile phase, and the regression equation is: 1 / m Lapp =1.870 / [A] + 1.612, and the linear correlation coefficient r is 0.9538. According to formula (1), the number of binding sites between nobiletin and EPOR is 1.56×10 -8 mol / L; Binding of nobiletin and EPOR K D The value is 1.160×10 -7 mol / L. This indicates that nobiletin has a strong interaction with EPOR.
[0052] Nonlinear chromatography (NLC) is an ideal method for characterizing on-column separation of drugs, especially in the presence of tailing peaks. NLC can be used to simultaneously determine the kinetic parameters and thermodynamic parameters of the interaction between ligand and receptor. This method assumes that the shape of the chromatographic peak is mainly affected by the binding and dissociation kinetics of the interaction between the component to be tested and the immobilized protein, and different binding and dissociation rates are the main factors for peak broadening and peak deformation. In affinity chromatography, due to the rapid association and slow dissociation of drugs and proteins during the action process, the chromatographic peak is a tailing chromatographic peak, and nonlinear chromatography is particularly suitable for the analysis of tailing chromatographic peaks. A series of concentrations of nobiletin were injected into SiO 2 -EPOR / CMC columns, retention behavior is Figure 2 As shown in (c), the results show that the retention time of nobiletin on the EPOR / CMC column gradually decreases with the increase of drug concentration. Figure 2 (d) Experimental determination curve (upper) and nonlinear chromatographic fitting curve (lower) of 2.0 mmol / L nobiletin fitted by Peak Fit 4.12 software. The chromatographic peaks were fitted to obtain relevant parameters: peak area parameter (a 0 ), kinetic capacity factor parameter (a 1 ), peak broadening parameter (a 2 ), peak deformation parameter (a 3 ), see Table 3, by a 2 The dissociation rate constant of nobiletin and EPOR can be obtained K d ; by a 3 The equilibrium constant of nobiletin and EPOR can be obtained K A , K a = K d ×K A We calculate K A , K a and K d , see Table 4.
[0053] Table 3 Nonlinear chromatographic determination parameters of nobiletin at different concentrations
[0054]
[0055] Table 4 Interaction parameters between nobiletin and EPOR determined by nonlinear chromatography
[0056]
[0057] Example 4: Investigation of the agonistic effect of nobiletin on EPOR
[0058] Calcium ion is an important intracellular second messenger, involved in many important cellular physiological activities and pathological processes. Monitoring the changes in intracellular calcium ion levels can reflect the binding of extracellular signals to cell surface receptors. We used intracellular calcium imaging technology to measure the effects of positive drugs EPO and nobiletin on intracellular Ca 2+ The effect of nobiletin on calcium flux in EPOR-HEK293 cells was investigated. Figure 4 As shown in the figure, both Nobiletin and the positive drug EPO can increase the fluorescent calcium signal intensity of EPOR-HEK293 cells, and the agonist effect shows a significant dose-dependency. cAMP is another important second messenger signal molecule in cells. EPOR-HEK293 was treated with different concentrations of EPO and Nobiletin for 48 hours, and the cAMP level in the cell culture buffer was measured using an enzyme-linked immunosorbent assay kit (ELISA kit). As shown in the figure, Nobiletin and EPO have similar agonist effects, which is manifested in that both can promote the release of cAMP.
[0059] Example 5: Effect of Nobiletin on the Survival Rate of Hypoxic Damaged Glomerular Mesangial Cells
[0060] Hypoxia is a common pathogenesis of chronic kidney disease, and cobalt dichloride (CoCl 2 ) was applied to glomerular mesangial cells (HMC) for 24 h, 48 h, and 72 h to construct a cell model of hypoxia injury. HMC cells were cultured at 3×10 3 The cells were seeded in a 96-well plate and each well was filled with culture medium to 100 μL. Incubate at 37°C with 5% CO 2Culture in the incubator for 24 h to adhere to the wall. Aspirate the culture medium and add different concentrations of CoCl 2 100 μL of serum-free medium was added and cultured at 37°C for 24 h, 48 h or 72 h. After the preset time, 10 μL of 5 mg / mL MTT solution was added to the culture medium and incubated at 37°C for 4 h. The culture medium was discarded and 100 μL of DMSO solution was added. The culture medium was placed on a shaker for 15 min and the absorbance of the solution was measured at 490 nm on a microplate reader. The CoCl 2 The effects of concentration and action time on cell survival rate, the lowest drug concentration and shortest action time that caused obvious damage were selected as the subsequent modeling conditions.
[0061] The original culture medium of HMC cells after adhesion was discarded, and the control group cells were given 100 μL of serum-free culture medium, and the model group and the drug group were given a culture medium containing a preset concentration of Cocl 2 The cells were then treated with 10 μL of culture medium, 10 μL of blank culture medium, and different concentrations of nobiletin and EPO solution for modeling time. MTT assay was then performed. After treatment, the cell survival rate was calculated according to formula (2).
[0062] (2)
[0063] It can be seen that nobiletin and EPO have similar effects and can significantly improve CoCl 2 On the survival rate of glomerular mesangial cells.
[0064] Example 6 Effect of Nobiletin on the Apoptosis Rate of Hypoxic Damaged Glomerular Mesangial Cells
[0065] Glomerular mesangial cells were cultured at 5×10 4 The cells were inoculated into 6-well plates and cultured for 24 h. After the cells attached to the wall, intervention was given. The control group was treated with blank culture medium, and the model group and drug treatment group were treated with 200 μM CoCl 2 Serum-free culture medium was added, the EPO group was treated with 5, 10 and 20 IU / mL of EPO, and the Nobiletin group was treated with 4, 8, 16 and 32 μM Nobiletin, and culture was continued for 48 h. The original culture medium was discarded, and the cells were washed twice with pre-cooled PBS. The cells were digested with EDTA-free trypsin, and the cells were collected by centrifugation at 300 g. The cells were washed twice with pre-cooled PBS, and centrifugation was required for each wash. Then, the cells were stained in the dark using the Annexin V-FITC / PI apoptosis detection kit, stored on ice, and detected by flow cytometry within 1 h. The experimental results are shown in Figure 5 As shown, CoCl 2Treatment would increase the apoptosis rate of glomerular mesangial cells, while EPO and nobiletin would significantly reduce the apoptosis rate of cells.
[0066] The above contents are only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.
Claims
1. Use of nobiletin as a small molecule agonist of EPOR in the preparation of a drug for treating kidney cell damage caused by hypoxic injury, characterized in that: The drug specifically binds to the EPOR protein.
2. The use according to claim 1, characterized in that The drug is a drug that can specifically bind to the ARG178 site of the EPOR protein.
3. The use according to claim 1, characterized in that The drug is a drug that takes EPOR protein as a target.
4. The use according to claim 3, characterized in that The drug is a drug that specifically activates EPOR to trigger downstream calcium signals and increase cAMP levels.
5. The use according to claim 1, characterized in that The drug is a drug that increases the proliferation rate of glomerular mesangial cells under hypoxic injury conditions and reduces the apoptosis rate.
6. The use according to claim 1, characterized in that The medicine is a medicine for protecting glomerular mesangial cells from hypoxic damage.