Application of URB597 in preparation of medicine for treating chronic traumatic encephalopathy
By using the FAAH inhibitors URB597, inhibiting the death of neurons, solving the problem of insufficient CTE treatment methods, improved the improvement of CTE cognitive dysfunction, and provided new treatment ideas.
Patent Information
- Application Number
- CN202411733489.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-01-21
AI Technical Summary
There are no effective treatment methods for chronic traumatic encephalopathy (CTE) in the prior art, and the mechanism of ferrodystrophy in CTE has not been discussed in depth.
By using the FAAH inhibitor URB597, the death of neurons is suppressed, and the awareness dysfunction is reduced after CTE. Specific measures include reducing the expression of ACSL4, inhibiting the activity of hydrolytic enzymes in fatamide, increasing the level of endogenous marijuana AEA, and combining AEA and ACSL4's SER447 to inhibit the death of neurons.
URB597 provides new treatment ideas and methods by reducing neuronal ferroptosis and improving cognitive function in CTE patients.
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Figure CN119326745A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine, and in particular relates to the application of URB597 in preparing a medicine for treating chronic traumatic encephalopathy. Background Art
[0002] Chronic traumatic encephalopathy (CTE) is a chronic progressive neurodegenerative disease. Repetitive mild traumatic brain injury (rm-TBI) is the main cause of CTE. The incidence of CTE has increased significantly, especially in professional athletes (such as American football, ice hockey and boxing), veterans and elderly people with limited mobility. Studies have shown that its incidence can be as high as 30% to 50%. Therefore, CTE has become a serious public health problem. The main pathological feature of the disease is the abnormal aggregation of Tau protein. Over time, multiple pathological lesions accumulate, leading to neuronal damage and cognitive decline. Because the symptoms of CTE are usually mild and delayed in onset, they are often underestimated, leading to delayed therapeutic intervention. There is currently no effective treatment for CTE, but early intervention measures may help slow the course of the disease, improve symptoms, and even reverse cognitive decline in some cases. Therefore, it is particularly important to explore the pathogenesis of CTE and promising intervention measures. Research in this field can not only enhance public awareness of CTE and prevention, but also help to develop scientific intervention strategies to protect the health of high-risk populations.
[0003] During the pathological process of CTE, different forms of cell death occur, among which ferroptosis plays an important role in neuronal damage. Ferroptosis is a new form of programmed cell death caused by lipid peroxidation, and its biochemical characteristic is the excessive accumulation of lipid reactive oxygen species (ROS). The biochemical process driving ferroptosis involves the peroxidation of polyunsaturated fatty acid phospholipids, especially arachidonic acid (AA) and adrenic acid (AdA). In this process, acyl-CoA synthetase long-chain family member 4 (ACSL4), as an important regulator of fatty acid metabolism, participates in the regulation of AA and may affect the accumulation of intracellular iron, thus becoming a key determinant of susceptibility to ferroptosis. Therefore, in-depth research on the mechanism of ferroptosis in CTE is expected to provide new targets and methods for its treatment.
[0004] In addition, the endocannabinoid system (ECS) plays a wide range of regulatory roles in the nervous system, especially in cognitive function. The ECS is composed of endocannabinoids, synthases, and degrading enzymes, among which the main endocannabinoids include anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The enzymes responsible for degrading endocannabinoids include fatty amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Our proteomic sequencing results showed that the expression of FAAH in the CTE model was significantly reduced at 42 days, suggesting that it may play an important role in the pathological process of CTE. The endocannabinoid AEA can be degraded to AA by FAAH. URB597 (cyclohexylcarbamic acid 3'-carbamoyl-3-acyl ester) can effectively and selectively inhibit the activity of FAAH, thereby preventing the degradation of AEA to AA and increasing its level. Although URB597 has been shown to improve neurological impairment after stroke, its therapeutic potential in CTE has not been fully studied. At the same time, ferroptosis may play an important role in this process, but its specific mechanism still needs to be further explored.
[0005] Therefore, studying the interaction between endocannabinoids and ferroptosis, as well as FAAH as a potential target for improving CTE-related cognitive impairment, has important scientific significance and clinical value. This paper uses CTE animal models and in vitro experiments to deeply explore the mechanism by which the FAAH inhibitor URB597 inhibits CTE neuronal ferroptosis by reducing the degradation of the endocannabinoid AEA and its effect on CTE cognitive function. This study will provide new ideas and methods for the treatment of CTE. Summary of the invention
[0006] In view of this, the present invention aims to overcome the defects in the prior art and proposes the use of URB597 in the preparation of drugs for treating chronic traumatic encephalopathy.
[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:
[0008] The present invention proposes the use of URB597 in the preparation of a drug for treating chronic traumatic encephalopathy. URB597 alleviates cognitive dysfunction after chronic traumatic encephalopathy by inhibiting neuronal ferroptosis.
[0009] Furthermore, the URB597 inhibits neuronal ferroptosis by reducing the expression of ACSL4.
[0010] Furthermore, the URB597 increases the level of endogenous cannabinoid AEA by inhibiting the activity of fatty amide hydrolase.
[0011] Furthermore, the endocannabinoid AEA induces ACSL4 inactivation by directly binding to the Ser447 site of ACSL4, thereby inhibiting neuronal ferroptosis.
[0012] Furthermore, the URB597 reduces p-tau deposition and alleviates cognitive dysfunction by inhibiting fatty amide hydrolase.
[0013] The present invention also provides a pharmaceutical composition, which contains URB597; the URB597 alleviates cognitive dysfunction after chronic traumatic encephalopathy by inhibiting neuronal ferroptosis.
[0014] Furthermore, the URB597 inhibits neuronal ferroptosis by reducing the expression of ACSL4.
[0015] Furthermore, the URB597 increases the level of endogenous cannabinoid AEA by inhibiting the activity of fatty amide hydrolase; the endogenous cannabinoid AEA induces ACSL4 inactivation by binding to the Ser447 site of ACSL4, thereby inhibiting neuronal ferroptosis.
[0016] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable excipient or carrier; the pharmaceutical composition is in the form of a liquid preparation or a solid preparation; and the administration method of the pharmaceutical composition includes oral administration or injection.
[0017] The present invention also provides the application of the pharmaceutical composition, and the application of the pharmaceutical composition in preparing a medicine for treating chronic traumatic encephalopathy.
[0018] Compared with the prior art, the present invention has the following advantages:
[0019] URB597 described in the present invention can inhibit the activity of FAAH and increase the level of endogenous cannabinoid AEA in the brain, thereby improving the cognitive function of CTE patients; at the same time, AEA reduces neuronal ferroptosis by directly binding to and interacting with the Ser447 site of ACSL4.
[0020] The present invention reveals the key role of the endocannabinoid-ACSL4 axis in cognitive dysfunction after CTE, providing a new perspective and method to improve the cognitive function of CTE patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1Schematic diagram of FAAH expression level in the CTE model described in the embodiment of the present invention: wherein, Figure A is a heat map of differential protein expression after CTE; Figure B is a KEGG pathway enrichment analysis of differential genes between the CTE 42-day group and the control group; Figure C is a Western blot detection of FAAH at different time points in CTE, and quantitative analysis, and the protein level is standardized to GAPDH (n=6 per group); Figure D is a Western blot detection of FAAH levels in the brain tissue of mice in the URB597 group and the Vehicle group, and quantitative analysis, and the protein level is standardized to GAPDH (n=4 per group); Figure E is an ELISA detection of AEA content in the brain tissue of mice in the URB597 group and the Vehicle group (n=4 per group). ( * P<0.05, ** P<0.01, **** P<0.0001)
[0022] Figure 2 Schematic diagram of URB597 in an embodiment of the present invention that can improve cognitive impairment and reduce p-tau deposition in CTE mice: wherein, Figure A is the experimental design of the animal research part of the schematic diagram; Figure B is a Y-maze test on mice 36 days after CTE to detect alternating behavior (n=11 in each group); Figures C-H are water maze tests performed on days 37 to 42 after CTE (n=11 in each group): Figure C is the escape latency during water maze training; Figure D is a trajectory heat map of the water maze space exploration test; Figure E is the swimming speed of mice during the water maze space exploration test; Figure F is the number of times the mouse crosses the platform during the water maze space exploration test; Figure G is the latency time for the mouse to reach the platform for the first time during the water maze space exploration test; Figure H is the time the mouse stays in the target quadrant during the water maze space exploration test; Figure I is a Western blot of p-tau and tau46 levels in the brain tissue of mice in the Sham group, URB597 group and Vehicle group Blot detection and quantitative analysis were performed, and the protein level was normalized to GAPDH (n = 4 per group). * P<0.05, ** P<0.01, *** P<0.001)
[0023] Figure 3Schematic diagram of the fact that URB597 described in the embodiment of the present invention can significantly reduce the expression level of ACSL4 in neurons of CTE mice: wherein, Figures AB are immunofluorescence co-staining of ACSL4 and NeuN in the cortex and hippocampus, red indicates ACSL4 positive staining, green indicates NeuN positive staining, and blue indicates DAPI positive nuclear staining; scale bar = 25 μm; Figure C is Western blot detection of ACSL4 levels in brain tissues of mice in the Sham group, URB597 group and Vehicle group, and quantitative analysis, and the protein level is normalized to GAPDH (n=4 per group, ** P<0.01).
[0024] Figure 4 This is a schematic diagram showing that URB597 described in the embodiment of the present invention can reduce ACSL4-mediated neuronal ferroptosis: wherein, Figure A is a histogram and mean fluorescence intensity (MFI) of BODIPY lipid peroxidation; Figure B is a cell viability test for each group (n=3 for each group); Figure C is a Western blot test and quantitative analysis of ACSL4; the protein level is standardized to GAPDH (n=4 for each group); Figure E is a Western blot test of the expression level of ACSL4 at different virus transfection concentrations; Figure D is an Erastin-induced neuronal ferroptosis two days after transfection with LV-V or LV-A; Figure F is a histogram and mean fluorescence intensity of BODIPY lipid peroxidation after URB597 intervention; Figure G is a cell viability test for each group (n=3 for each group). ( * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001). E: Erastin; A: ACSL4; MOI: multiplicity of infection; LV-V: negative control for lentiviral transfection; LV-A: lentiviral overexpression of ACSL4.
[0025] Figure 5The molecular docking described in the embodiment of the present invention shows that the endocannabinoid AEA can directly bind to ACSL4: wherein, Figure AC is the potential site for AEA to bind to ACSL4, and forms a hydrogen bond interaction with the Ser 447 site of ACSL4: Figure A is a three-dimensional structural model of ACSL4 constructed according to its sequence information using the AlphaFold2 tool; Figure B is the potential binding site of ACSL4; Figure C is the complex binding mode of AEA and ACSL4; Figure D is the root mean square deviation (RMSD) of protein (blue) and small molecule (red) in 100ns molecular dynamics simulation; Figure E is the root mean square fluctuation (RMSF) diagram of small molecule AEA; Figure F is the root mean square fluctuation (RMSF) of protein in 20-100ns molecular dynamics simulation (green positions represent amino acids and small molecule and protein interactions); Figure G is a 2D schematic diagram of the binding mode of AEA and ACSL4 complex; Figure H shows the promoting effect of amino acids on small molecule binding.
[0026] Figure 6 Schematic diagram of the prediction of the key site of AEA-ACSL4 binding by alanine scanning and alanine mutation as described in the embodiment of the present invention is Ser 447: wherein, Figure A is a 2D schematic diagram of the binding mode of small molecules and proteins; Figure B is a scan of amino acids within 4A near AEA using Schordinger's residue scanning module; Figure C is a calculation of the change in binding energy before and after the amino acids within 4A of the small molecule AEA are mutated to alanine one by one; Figure D is an overlay of the AEA-ACSL4 complex structure before and after Ser447 mutation; Figure E is a comparison of the binding energy of the AEA-ACSL4 complex before and after mutation.
[0027] Figure 7 This is a schematic diagram of the inhibition of neuronal ferroptosis by the binding of the endogenous cannabinoid AEA to the Ser 447 site of ACSL4 as described in an embodiment of the present invention: wherein, Figure A is a binding curve of different concentrations of AEA and ACSL4 measured by surface plasmon resonance; Figure B is a BODIPY lipid peroxidation histogram and mean fluorescence intensity (MFI) of neurons treated with Erastin+URB597 (with or without AEA) two days after transfection with LV-A; Figure C is a neuronal cell viability test of each group (n=3 per group); Figure D is a BODIPY lipid peroxidation histogram and mean fluorescence intensity (MFI) before and after point mutation of ACSL4 protein; Figure E is a cell viability test before and after Ser 447 mutation of neuronal ACSL4 protein (n=3 per group). ( * P<0.05, ** P<0.01, *** P < 0.001, ****P<0.0001). LV-A: lentivirus overexpressed ACSL4; LV-A(S447A): lentivirus overexpressed ACSL4 protein with Ser447 mutated to Ala. DETAILED DESCRIPTION
[0028] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.
[0029] The present invention will be described in detail below with reference to the embodiments.
[0030] Example 1 Experimental method
[0031] 1. Animals and chronic traumatic encephalopathy models
[0032] The 8-12 week old male C57BL / 6 mice used in the present invention were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were raised under SPF conditions in the Animal Center of Tianjin Medical University, with an ambient temperature of 22±2°C, a humidity of 45%, and a light / dark cycle of 12h. Each mouse was raised individually in a cage and given free access to standard mouse food and sterilized water. The experimental procedures were carried out in accordance with the protocol approved by the Animal Care and Use Committee of Tianjin Medical University, following the Guidelines for the Care and Use of Laboratory Animals established by the National Institutes of Health (NIH).
[0033] We used the rm-TBI previously reported by our research group to prepare the CTE model. The mouse CTE model was established using controlled cortical impact (CCI). The mice were gas-anesthetized with 4.6% isoflurane for about 30 seconds. After waiting for the mice to enter the anesthetized state, the hair on the top of the mouse's head was shaved. The shoulders of the mice were then fixed to a homemade acrylic casting model with surgical tape. The model can maintain the position of the mouse's head during the blow and provide a 3.0mm buffer space under the mouse's head. A concave metal disk (diameter = 3mm) designed by our research group was attached to the mouse, located between the middle and front of the back of the skull. This design can transmit potential energy to the entire brain, causing mild diffuse brain injury (non-local brain injury). Subsequently, the mouse was hit on the head at a speed of 5.0m / s and a depth of 2.5mm. Each mouse was placed in a well-ventilated cage at 37℃ after the blow until it regained consciousness. The blow was performed once every 48 hours, for a total of 4 blows, to establish the CTE model. The mice in the sham group were subjected to the same operation except that no blows were given. Mice that showed movement disorders, eating difficulties, or skull fractures during or after the modeling period, as well as mice with severe injuries (such as visible brain hemorrhage) found during brain removal, were excluded from the study.
[0034] 2. Drug treatment
[0035] URB597 is a specific inhibitor of FAAH that can indirectly increase the level of AEA. URB597 was dissolved in DMSO and diluted with 5% Tween-80 and 30% PEG-300. CTE mice were intraperitoneally injected with URB597 (1 mg / kg) 2 hours after successful modeling, and then repeated every 2 days until day 28 after CTE. Mice in the Vehicle group received intraperitoneal injections of the vehicle solution (1 mg / kg).
[0036] 3. Morris Water Maze (MWM)
[0037] Morris water maze test was used to evaluate the memory and spatial learning ability of mice. MWM test was performed on day 42 after CTE to determine the changes in spatial memory and learning ability of the three groups of mice (Sham, CTE+Vehicle, CTE+URB597). Subsequently, all three groups of mice were trained to locate the hidden platform in an opaque pool. During the first 5 days of training, the platform (diameter = 10 cm) was placed 1 cm below the water surface of the pool. Each mouse was tested four times a day (each lasting 90 seconds), with the same starting quadrant and trial format for each trial, but the order of the trials was different for each mouse. The test evaluated the escape latency, the time required to find the hidden platform, as a measure of memory and spatial learning ability (maximum 90 seconds). Mice that did not find the platform within the first 90 seconds were gently guided to the platform. Once the mouse found the platform, it was allowed to stay on the platform for 15 seconds. On the sixth day, the spatial exploration test was performed. The platform was removed from the pool and each mouse swam for 90 seconds. Subsequently, the frequency of crossing the platform, the first latency to reach the platform location, and the swimming speed were determined using the Stoelting video tracking system.
[0038] 4. Y-maze test
[0039] The Y-maze was used to assess the short-term learning and memory function of mice. The Y-maze was made of blue opaque polyethylene plastic and consisted of three arms marked A, B, and C. The mice were placed at the central intersection of the three arms and allowed to explore freely for 8 minutes. Spontaneous alternation was recorded when the mice entered three different arms (such as ABC, CAB, ACB, BCA, CBA, and BAC) in succession. The percentage (%) of spontaneous alternation behavior was defined as the number of spontaneous alternation behaviors / (total number of entries into the arm-2) × 100.
[0040] 5. Proteomic analysis
[0041] Exosomes were isolated from brain tissues of sham and CTE mice, and exosome proteomics sequencing was performed using Echobiotech technology. Exosome protein concentration was determined using the Qubit fluorescence method (Invitrogen), and each sample (10 μg) was processed by 10% SDS-PAGE. Gel bands were excised and in-gel digestion was performed using a ProGest robot (DigiLab). Nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS / MS) analysis was performed using a Waters NanoAcquity high-performance liquid chromatography (HPLC) system interfaced with a ThermoFisher Q Exactive to analyze the differences in proteomic content between sham and CTE-42d. The raw data obtained by nanoLC-MS / MS were processed using Mascot (Matrix Science) software and searched in the database. Mascot Dial-A-Truck (DAT) files were parsed into the proteomics software Scaffold for verification, screening, and creation of non-redundant lists. The data were screened at a protein and peptide false discovery rate of 1%, and at least 2 specific peptides were required for each protein. Protein identification criteria were accepted if at least 2 specific peptides were detected or if one specific peptide was detected with a false discovery rate <1%.
[0042] 6. Western blot detection
[0043] Tissues or cells were lysed using protein lysis buffer (Keygen Biotech, Nanjing, China), and the supernatant was collected by centrifugation as protein extracts. Proteins were separated by 8% and 10% SDS-PAGE electrophoresis and then transferred to PVDF membranes (Merck Millipore). The membrane was blocked in 5% skim milk for 2 h and then incubated with anti-FAAH (CST, C8AF1, rabbit monoclonal antibody, 1:1000), NeuN (Abcam, ab279296, mouse monoclonal antibody, 1:1000), ACSL4 (Abcam, ab155282, rabbit monoclonal antibody, 1:1000), Gpx4 (Abcam, ab125066, rabbit monoclonal antibody, 1:1000), tau-46 (CST, 4019S, mouse monoclonal antibody, 1:1000), p-tau (CST, 49561S, rabbit monoclonal antibody, 1:1000) and GAPDH (CST, 14C10, rabbit monoclonal, 1:1000) for 1 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ABclonal, China, AS014, AS003, 1:5000). The next day, the membrane was washed with TBST and used Immunoblotting was performed using Maximum Sensitivity Substrate (Thermo Fisher Scientific). Densitometric measurements were performed using a ChemiDo XRS+ imaging system (Bio-Rad, CA, USA), and the average pixel density of each band was quantified using Quantity One software (Bio-Rad, CA, USA).
[0044] 7. Immunofluorescence
[0045] Brain tissues were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 12 h, then incubated in phosphate buffered saline containing 15% sucrose for 24 h at 4°C, and then incubated in phosphate buffered saline containing 30% sucrose for 48 h at 4°C. After precipitation, brain tissues were embedded in OCT compound and cut into 10 μm slices using a cryostat. Subsequently, the sections were treated with 0.3% Triton X-100 for 30 min at room temperature, blocked with phosphate buffered saline containing 10% goat serum for 1 h at room temperature, and incubated with primary antibodies against ACSL4 (1:200, Santa Cruz Biotechnology) and NeuN (1:500, Abcam) overnight at 4°C. After multiple washes with PBS, the sections were stained with secondary antibodies for 1 h. Nuclei were counterstained with DAPI (S2110, Solarbio). Colocalization was quantified using ImageJ Coloc2 and Pearson correlation coefficient (r) values. Imaging was performed using a Leica confocal microscope TCS SP8 or Nikon C2, and data analysis was performed using ImageJ software. ImageJColoc2 was used to quantify colocalization and calculate Pearson correlation coefficient (r) values to assess the correlation between colocalized voxels in different color channels.
[0046] 8. Cell culture and in vitro drug treatment
[0047] The mouse hippocampal HT22 neuronal cell line (provided by Wuhan Purcella Biotechnology Co., Ltd.) was cultured in high-glucose DMEM (Solarbio), supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Gibco), and cultured in a cell culture incubator at 37°C and 5% CO2. URB597 can inhibit the hydrolysis of the endogenous cannabinoid AEA catalyzed by FAAH. The concentration of URB597 used was 20uM. The concentration of AEA was 5uM. Erastin is a ferroptosis inducer, and its mechanism of inducing ferroptosis is related to ROS. The concentration of Erastin used was 2.5uM.
[0048] 9. Vectors and in vitro lentiviral transfection
[0049] For the experiments related to overexpression of neuronal ACSL4, the cDNA of mouse ACSL4 gene was cloned into GV341 lentiviral vector (Ubi-MCS-3FLAG-SV40-puromycin, GeneChem, Shanghai, China). The serine residue 447 of ACSL4 was mutated to alanine [A(S447A)]. For overexpression of point-mutated ACSL4, the cDNA of mouse ACSL4 gene with point mutation was cloned into GV341 lentiviral vector.
[0050] HT22 cell line was infected with lentivirus. Cell culture medium was replaced 48 h after lentivirus infection, and culture was continued for 24 h after medium replacement, and cells were selected with puromycin. After in vitro lentivirus infection, neurons stably overexpressing ACSL4 (LV-A) and neurons stably overexpressing ACSL4 with the 447th serine residue mutated to alanine (S447A) [LV-A(S447A)] were obtained for further analysis. Neurons were transfected with unloaded lentivirus (lentivirus, referred to as LV-V).
[0051] 10. Enzyme-linked immunosorbent assay (ELISA)
[0052] According to the instructions provided by the kit manufacturer, a mouse AEA (Elabscience, YJ330266) ELISA kit was used to determine the AEA content in the cell culture supernatant or tissue extract.
[0053] 11. Evaluation of C11-BODIPY591 / 581 on ferroptosis
[0054] To evaluate the occurrence of ferroptosis in cells, cells were treated with C11-BODIPY591 / 581. Flow cytometry was used to detect intracellular ferroptosis and lipid peroxidation levels based on the fluorescence emission peak of the C11-BODIPY591 / 581 probe. When ferroptosis occurs in the cell membrane, the fluorescence peak of the probe shifts from 510nm to 590nm. The degree of ferroptosis in the cell membrane is proportional to the degree to which the probe fluorescence peak shifts to the right.
[0055] Cells were cultured in 12-well plates and maintained at 37°C after treatment. Subsequently, cells were incubated with BODIPY (2 μM) for 30 minutes. Next, cells were detached by trypsinization, and then collected and washed with PBS. Cells were then resuspended in 500 μL PBS. BD FACSAria TM The cell fluorescence intensity was measured by a flow cytometer II and analyzed using FlowJo software.
[0056] 12. Cell Viability Assay
[0057] Cells were seeded onto 96-well plates (2000 cells per well) and treated with compounds [AEA (Selleck Chemical Company), Erastin (Selleck Chemical Company), DMSO, URB597 (Selleck Chemical Company)] after cell adhesion. Cell viability was assessed at various time intervals (usually 12 hours) after treatment using CCK8 cytotoxicity assay (GLPBIO, GK10001) according to the manufacturer's instructions of the Cell Counting Kit-8 (CCK-8) reagent.
[0058] 13. Modeling and Molecular Docking
[0059] Considering that the crystal structure of ACSL4 is still unclear, the present invention first uses the AlphaFold2 tool to construct a three-dimensional structural model of ACSL4 based on its sequence information. Subsequently, the online server POCASA (https: / / g6altair.sci.hokudai.ac.jp / ) was used to predict potential binding sites. In preparation for docking, we used AutodockTools to preprocess the model, including merging polar hydrogen atoms and saving the file in PDBQT format. Similarly, the structure of the small molecule AEA was drawn using ChemDraw, then converted using Chem3D, and then preprocessed using Autodock Tools and saved in PDBQT file format. Finally, the small molecule AEA was docked to the potential active site of ACSL4 using Autodock Vina software.
[0060] 14. Alanine Scan
[0061] According to the binding mode of the AEA-ACSL4 complex, amino acids within AEA 4A were selected for scanning. Using Schrodinger's Residue Scanning module, these amino acids were scanned for alanine and mutated to alanine (Ala) in sequence, and the changes in binding energy before and after mutation were calculated.
[0062] 15. Surface plasmon resonance (SPR)
[0063] SPR technology is the preferred assay method for kinetic and affinity analysis in basic life science research, drug discovery, and medical diagnostic analysis, and is also the "gold standard" for detecting biomolecular interactions.
[0064] AEA (MEC, HY-10863) and ACSL4 (ORIGENE, NM_207625) were purchased from the company. The purified recombinant proteins were immobilized on the sensor chip CM5. GE Healthcare Biacore S200 (GE, USA) was used to measure the binding affinity between small molecule compounds and recombinant proteins.
[0065] 16. Statistical analysis
[0066] Data are presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8. Prior to statistical analysis, the normal distribution of the data was assessed. If the data follow a normal distribution, a two-tailed unpaired t-test (two groups) or one-way analysis of variance (ANOVA) (more than two groups) will be used to assess differences between different groups. Otherwise, the Mann-Whitney test will be used to determine significance. Post hoc analysis of multiple group comparisons will be performed using two-way ANOVA and Tukey post hoc test. p values < 0.05 will be considered statistically significant.
[0067] Example 2 Experimental results
[0068] 1. Expression of FAAH in CTE model
[0069] To investigate the pathological mechanism of CTE, we performed proteomic sequencing. The results showed that compared with the Sham group, the CTE group showed downregulated proteins including FAAH, Gria3, Gria2, and Slc17a7 at 42 days. KEGG pathway enrichment analysis showed that these differentially expressed proteins were mainly enriched in the endocannabinoid signaling pathway ( Figure 1 A, B). We evaluated the levels of proteins related to the endocannabinoid system, including FAAH, at 1, 3, 7, 14, 28, and 42 days in the CTE and Sham groups. Figure 1 As shown in C, compared with Sham, FAAH levels were significantly increased on the 14th day of CTE. We further intervened CTE mice with FAAH inhibitor URB597, and found through Western blot analysis that FAAH expression in the brain of CTE mice injected with URB597 was significantly reduced compared with the Vehicle group ( Figure 1 D), while AEA levels increased significantly ( Figure 1 E). Based on this, we further explored whether inhibition of FAAH could improve the cognitive function of CTE mice.
[0070] 2. URB597 can reduce p-tau deposition and improve cognitive function in CTE mice
[0071] The main pathological feature of CTE is the deposition of p-tau. The present invention has carried out animal experiment design ( Figure 2 A), Western blot analysis of the effect of URB597 on CTE pathological protein deposition. The results showed that compared with the Sham group, the levels of pathological proteins p-tau and tau in the Vehicle group were significantly increased. In contrast, compared with the Vehicle group, the URB597 treatment group showed a significant decrease in p-tau and tau deposition ( Figure 2 I).
[0072] To explore whether URB597 can improve the cognitive function of CTE mice, we conducted Y-maze and water maze experiments on the mice to evaluate their memory and learning abilities.
[0073] We performed a Y-maze test on CTE mice on day 36 and evaluated the effect of URB597 on recognition memory in CTE mice by calculating alternation performance. Compared with the Sham group, the alternation behavior in the CTE+Vehicle group was significantly decreased, suggesting memory impairment. The URB597 group showed higher alternation performance compared with the CTE+Vehicle group. This suggests that URB597 treatment improved cognitive function in CTE mice ( Figure 2 B).
[0074] In the water maze test, the escape latency of mice in the CTE+Vehicle group was significantly longer than that in the Sham group during the training phase. In contrast, mice treated with URB597 showed a shorter escape latency ( Figure 2 C, D). In the exploration experiment, the number of times that mice in the CTE+Vehicle group crossed the target quadrant platform and the time they stayed in the target quadrant were significantly reduced compared with those in the Sham group ( Figure 2 F, H), while the delay to reach the first platform increased significantly ( Figure 2 G), indicating that CTE mice have impaired spatial learning and memory. In contrast, compared with Vehicle-treated mice, URB597-treated mice crossed the target quadrant platform more times and stayed in the target quadrant for a significantly longer time ( Figure 2 F, H), the delay time to reach the first platform is significantly shortened ( Figure 2 G), indicating that URB597 helps restore spatial learning and memory function in CTE mice. In addition, there was no significant difference in the average swimming speed of the four groups of mice ( Figure 2 E). In summary, URB597 can significantly improve cognitive impairment in CTE mice.
[0075] 3. URB597 can significantly reduce the expression of ACSL4 in neurons of CTE mice
[0076] To explore the relationship between FAAH and ACSL4, we treated CTE mice with the FAAH inhibitor URB597 and collected mouse brain tissue for Western blot and immunofluorescence detection 42 days after successful modeling. The results showed that URB597 could downregulate the expression level of ACSL4 in CTE mice ( Figure 3 AC).
[0077] To further investigate the effect of URB597 on neuronal ACSL4 expression, we performed immunofluorescence co-staining for ACSL4 and NeuN and assessed the number of ACSL4-positive cells. We found that the number of ACSL4-positive cells in the hippocampus and cortex increased significantly after CTE for 42 days ( Figure 3 A, B). URB597 can reduce the expression of ACSL4 in CTE mice. These results suggest that CTE leads to a significant increase in the expression level of ACSL4, while URB597 treatment significantly inhibits the upregulation of ACSL4 in CTE mice.
[0078] 4. URB597 reduces ACS14-mediated neuronal ferroptosis
[0079] To investigate the effect of URB597 on neuronal ferroptosis, we used C11-BODIPY591 / 581 staining for flow cytometry analysis. The BODIPY results showed that neurons in the Erastin+URB597 group showed stronger resistance to Erastin-induced ferroptosis compared with the Erastin group, and the occurrence of ferroptosis was reduced ( Figure 4 A), and cell viability was significantly enhanced ( Figure 4 B). Western blot results showed that compared with the control group, the expression of ACSL4 in neurons in the Erastin group was significantly increased, while that in the URB597-treated group was significantly decreased ( Figure 4 C).
[0080] ACSL4 has been shown to enhance the sensitivity of cells to ferroptosis, so we overexpressed ACSL4 in neurons. The results showed that we successfully constructed a neuronal ACSL4 overexpression model. At MOI = 20, ACSL4 protein was most significantly expressed in neurons ( Figure 4 E). BODIPY flow cytometry analysis showed that ACSL4 overexpression led to increased ferroptosis and decreased cell viability ( Figure 4 D, G). URB597 was found to attenuate the sensitivity of neurons to ferroptosis in neurons overexpressing ACSL4 ( Figure 4 F). In summary, URB597 can attenuate Erastin-induced neuronal ferroptosis by reducing the expression of ACSL4.
[0081] 5. Molecular docking showed that the endocannabinoid AEA can directly bind to ACSL4
[0082] In order to study whether AEA can directly bind to ACSL4, we performed molecular docking and molecular dynamics simulations. Considering that the crystal structure of ACSL4 is still unclear, the present invention firstly uses the AlphaFold2 tool to construct a three-dimensional structure model of ACSL4 based on its sequence information ( Figure 5 A). Subsequently, the online server POCASA (https: / / g6altair.sci.hokudai.ac.jp / ) was used to predict potential binding sites ( Figure 5 B). Finally, the small molecule AEA was docked to the potential active site of ACSL4 using Autodock Vina software ( Figure 5 C). The binding energy of the AEA-ACSL4 complex is -61.31 Kcal / mol.
[0083] The present invention analyzes the intermolecular stability and interaction between AEA and ACSL4 in a dynamic simulation environment through molecular dynamics simulation. The root mean square deviation (RMSD) of the protein backbone in the molecular dynamics simulation is used as the basis for whether the system is balanced. If the RMSD fluctuation of the protein backbone is very small within a certain range, the protein system is stable, indicating that the molecular dynamics simulation of the system is in equilibrium. If the RMSD curve ( Figure 5 As shown in D), the AEA-ACSL4 simulation time exceeds 20ns, and the curve reaches a dynamic equilibrium, indicating that the protein-small molecule complex is in a stable state. Therefore, we can perform the next step of sampling and analysis on the trajectory of 20-100ns. At the same time, we extract a superposition of 1 conformation every 100ns in the 100ns molecular dynamics simulation (a total of 100 conformations), as shown in Figure 5 As shown in D, the conformations of the protein and the small molecule have good superposition, and the small molecule can also stably bind to the active site of the protein.
[0084] The root mean square fluctuation (RMSF) of the amino acid residues of the receptor protein during the molecular docking process is used to analyze the freedom of movement of the amino acid residues, that is, flexibility. The RMSF of the trajectory protein and small molecules are extracted, and their B factors are calculated. Combining the RMSF and B factor graphs, it can be seen that the overall structural flexibility of the protein molecule is small (most of them are less than 2.0A), and the RMSF near the small molecule binding site is also small, indicating that the small molecule binding area is relatively stable. From the RMSF graph of the small molecule, it can be seen that most of the atoms of the small molecule are in a stable state, indicating that the small molecule can bind well to the active site of the protein ( Figure 5 E, F).
[0085] The present invention analyzes the interaction mode in the stable interval (20-100ns) of the kinetic trajectory. Figure 5 As shown in GH, the amino acids that play an important role in small molecule binding are mainly Pro 445, Ser 447 and Lys 593. Its main functions are hydrogen bonds, water bridges and hydrophobic interactions. Figure 5 H shows the percentage of interactions formed within 20-100 ns (number of frames in which interactions are formed / total number of frames). The binding of small molecules to proteins depends on two main interactions: 1. The hydroxyl bonds and amide bonds on the small molecules can directly interact with protein Pro445, Ser 447 and Lys 593 to form hydrogen bonds. 2. The hydrophobic side chains on the small molecules can form hydrophobic interactions with the hydrophobic amino acids in the active site. These interactions stabilize the binding of small molecules to proteins.
[0086] 6. Alanine scanning and alanine mutation predict that the key site for AEA-ACSL4 binding is Ser 447
[0087] In order to further reveal the amino acid sites that play a key role in the binding process of AEA and ACSL4, the present invention conducted a point mutation experiment. We selected amino acids within 4A near AEA for alanine scanning. The key sites for AEA to bind to ACSL4 are Pro 445, Ser 447 and Lys 593. The hydroxyl and amide bonds on small molecules can directly form hydrogen bonds with proteins, such as Pro 445, Ser 447 and Lys 593 ( Figure 6 A). The residue scanning module of Schordinger was used to scan the amino acids within 4A near AEA ( Figure 6 B), mutate these amino acids to alanine one by one, and calculate the change in binding energy before and after the mutation. After the 447th Ser of ACSL4 mutated to Ala, the binding ability of AEA and ACSL4 was significantly reduced ( Figure 6 C). The superposition of the AEA-ACSL4 complex structure before and after the Ser447 mutation shows that the position of the small molecule has hardly changed before and after the mutation, but the structure of the hydroxyl segment has changed ( Figure 6 D). The binding energy of the AEA-ACSL4 complex was -61.31 Kcal / mol before the mutation and -36.80 Kcal / mol after the mutation. Compared with before the mutation, the binding energy between AEA and ACSL4 decreased after the mutation, indicating that the binding energy between the two was weakened ( Figure 6 E).
[0088] 7. Endocannabinoid AEA binds to Ser 447 of ACSL4 to inhibit neuronal ferroptosis
[0089] In order to further clarify the interaction between AEA and ACSL4, the present invention conducted a surface plasmon resonance (SPR) experiment. We found that AEA and ACSL4 can stably bind to form a complex. Single concentration experiments showed that AEA highly binds to the target protein ACSL4 at 500μM, 250μM, 125μM, and 62.5μM ( Figure 7 A).
[0090] URB597 inhibits FAAH activity and indirectly increases the content of endogenous cannabinoid AEA in neurons. We analyzed neuronal cell viability and BODIPY lipid peroxidation. The results showed that compared with the ACSL4 overexpression group, the URB597 treatment group had enhanced cell viability and reduced lipid peroxidation ( Figure 7 B, C). To further investigate the direct effect of AEA on neuronal ACSL4, we treated neurons with AEA under the premise of blocking AEA dissociation by URB597. The results showed that LV-A+E+URB597+AEA could significantly reduce neuronal ferroptosis compared with the LV-A+E+URB597 group ( Figure 7 B, C). Therefore, our results suggest that AEA alleviates Erastin-induced neuronal ferroptosis by directly binding to ACSL4.
[0091] In vitro point mutation experiments showed that the mutation of Ser 447 to Ala in the neuronal ACSL4 protein could significantly inhibit neuronal ferroptosis and enhance cell activity ( Figure 7 D, E). These results indicate that AEA can inhibit neuronal ferroptosis by binding to Ser 447 of ACSL4 protein.
[0092] The present invention illustrates that in chronic traumatic encephalopathy, increased FAAH levels lead to a weakened inhibitory effect of the endocannabinoid AEA on neuronal ferroptosis, thereby promoting neuronal ferroptosis, which in turn leads to cognitive dysfunction after CTE. We found that URB597 can reduce the expression of ACSL4 and effectively inhibit neuronal ferroptosis. In addition, the present invention also confirms that URB597 significantly increases the level of the endocannabinoid AEA, and AEA directly binds to the ACSL4 protein to inhibit its activity, thereby reducing neuronal ferroptosis. Further studies show that the key site for AEA to bind to ACSL4 and exert its effect is the Ser 447 site on ACSL4. AEA binding to this site can induce ACSL4 inactivation, thereby inhibiting neuronal ferroptosis, reducing the deposition of p-Tau, and improving cognitive impairment in CTE. In summary, the present invention reveals the key role of the endocannabinoid-ACSL4 axis in cognitive impairment caused by CTE, as well as the potential application of URB597 in CTE therapy, providing a new perspective and method for improving the cognitive function of CTE patients.
[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. The use of URB597 in the preparation of a drug for treating chronic traumatic encephalopathy, characterized in that: URB597 alleviates cognitive dysfunction after chronic traumatic encephalopathy by inhibiting neuronal ferroptosis.
2. The use according to claim 1, characterized in that: The described URB597 inhibits neuronal ferroptosis by reducing the expression of ACSL4.
3. The use according to claim 2, characterized in that: The URB597 increases the level of endogenous cannabinoid AEA by inhibiting the activity of fatty amide hydrolase.
4. The use according to claim 3, characterized in that: The endocannabinoid AEA induces ACSL4 inactivation by directly binding to the Ser447 site of ACSL4, thereby inhibiting neuronal ferroptosis.
5. The use according to claim 1, characterized in that: The URB597 reduces p-tau deposition and alleviates cognitive dysfunction by inhibiting fatty amide hydrolase.
6. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises URB597; the URB597 alleviates cognitive dysfunction after chronic traumatic encephalopathy by inhibiting neuronal ferroptosis.
7. The pharmaceutical composition according to claim 6, characterized in that: The described URB597 inhibits neuronal ferroptosis by reducing the expression of ACSL4.
8. The pharmaceutical composition according to claim 7, characterized in that: The URB597 increases the level of endogenous cannabinoid AEA by inhibiting the activity of fatty amide hydrolase; the endogenous cannabinoid AEA induces ACSL4 inactivation by binding to the Ser447 site of ACSL4, thereby inhibiting neuronal ferroptosis.
9. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier; the pharmaceutical composition is in the form of a liquid preparation or a solid preparation; and the administration method of the pharmaceutical composition comprises oral administration or injection administration.
10. Use of the pharmaceutical composition according to any one of claims 6 to 9, characterized in that: The application of the pharmaceutical composition in preparing a drug for treating chronic traumatic encephalopathy.