Application of depression treatment target TMEM132E in reutilization of depression drugs

Abstract

The invention discloses application of a depression treatment target TMEM132E in reutilization of depression drugs, and relates to the technical field of crude drugs and molecular biology. According to the invention, TMEM132E is taken as a core target spot, and marketed drugs capable of targeting interaction of TMEM132E and TrkB are screened and verified, namely pimozide, lybramer and risperidone. The three can significantly enhance the formation of a TMEM132E-TrkB compound, restore the activity of a BDNF / TrkB signal pathway, activate a downstream ERK signal, and improve the pathologic state related to depression. According to the invention, TMEM132E is determined as a precise target for reutilization of the depression drug for the first time, the related technical blank is filled, the safety of the selected drug is clinically verified, the research and development period can be greatly shortened, the cost is reduced, a new path is developed for treatment of depression, and meanwhile, the synergistic potential of combined application of the drug and the existing antidepressant drug is prompted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crude medicine and molecular biology, and particularly relates to application of a depression treatment target TMEM132E in drug repurposing for depression. BACKGROUND

[0002] Depression is a high-incidence mental illness worldwide, and its core symptoms include persistent low mood and decreased interest, often accompanied by anxiety, sleep disorders and impaired cognitive function, and can even lead to suicidal behavior, causing heavy burden to patients' families and society. According to statistics of the World Health Organization, the incidence of depression worldwide is increasing year by year. However, the pathogenesis of depression is extremely complex, involving neural circuit abnormalities, synaptic dysfunction, neurotransmitter imbalance and signal pathway abnormalities, and has not been fully elucidated, which greatly hinders the development of efficient targeted therapeutic drugs.

[0003] In recent years, with the in-depth research, it is found that transmembrane protein 132E (TMEM132E) gene may play a role in the occurrence of depression. Studies have found that TMEM132E is significantly down-regulated in the cerebral cortex and hippocampus of anxiety and depression-like mouse models. By constructing a TMEM132E knockout mouse model, it is found that the absence of Tmem132e can cause anxiety and depression-like behaviors in mice, which suggests that abnormal function of TMEM132E is likely to be involved in the pathological process of depression. Further studies have shown that the absence of TMEM132E can reduce the expression level of TrkB in the brain, thereby inhibiting the BDNF-TrkB signaling pathway and hindering the normal maintenance and repair of neural plasticity, ultimately leading to anxiety and depression-like behaviors. However, the current research on the specific mechanism of TMEM132E in depression is still in its infancy, and its potential value in the treatment of depression needs to be explored.

[0004] From the treatment status, the traditional antidepressants have unsatisfactory efficacy. Traditional drugs represented by selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) generally have the problems of slow onset (usually 2-4 weeks to show efficacy) and low response rate (only 50%-60% of patients are effective), and are difficult to effectively improve the damage of neural plasticity in patients. At the same time, the development of new drugs for depression is facing the difficulties of high cost, long cycle and high failure rate. Drug repurposing as an innovative strategy can shorten the development cycle, but due to the lack of understanding of the precise mechanism of the marketed drugs in the molecular pathways related to depression, most drugs are difficult to achieve effective "old drug new use". SUMMARY

[0005] The application aims to provide an application of a depression treatment target TMEM132E in drug repositioning for depression, so as to screen and verify marketed drugs capable of enhancing the interaction by means of drug repositioning technology, solve core technical problems such as limited efficacy of traditional drugs, unknown TMEM132E related mechanism, lack of precise target for new use of old drugs, and open up a new path for depression treatment.

[0006] To achieve the above-mentioned object, the application provides an application of a depression treatment target TMEM132E in drug repositioning for depression.

[0007] Preferably, the drug is a drug targeting the interaction between TMEM132E and TrkB.

[0008] Preferably, the drug is pimozide, rebotie or risperidone.

[0009] Therefore, the application of the depression treatment target TMEM132E in drug repositioning for depression has the following technical effects: (1) TMEM132E is firstly determined as a precise target for drug repositioning for depression, which fills the blank of unknown depression related molecular pathway targets in the new use of old drugs; (2) The screened pimozide, rebotie and risperidone are marketed drugs, the safety of which has been clinically verified, so that the research and development cycle of antidepressants can be greatly shortened and the research and development cost can be reduced; (3) The mechanism of the three drugs for activating downstream signals by enhancing the interaction between TMEM132E and TrkB is clarified, which provides a new action pathway for the targeted treatment of depression, and suggests that the combination of the three drugs with existing antidepressants may have a synergistic effect, further improving the treatment efficiency.

[0010] The technical solutions of the application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0011] To more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the description of the embodiments of the application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative labor on the basis of these drawings.

[0012] Figure 1 The results of Western blot analysis of SH-SY5Y cells stably expressing shNC or shTMEM132E after BDNF treatment, wherein A is the result of Western blot analysis of the TrkB downstream signaling pathway, and B is the quantitative analysis of the protein band gray value based on the blot data; Figure 2 Western blot analysis results of SH-SY5Y cells stably expressing shNC or shTMEM132E after 7,8-DHF treatment, wherein A is the results of analyzing TrkB downstream signaling pathway by Western blot, and B is quantitative analysis of protein band gray value based on blot data; Figure 3 Flow chart of AutoDockVina virtual screening; Figure 4 Binding of pimozide, risperidone and risperidone to target TMEM132E-TrkB, wherein (a) is the docking results of pimozide and TMEM132E-TrkB complex, (b) is the docking results of risperidone and TMEM132E-TrkB complex, and (c) is the docking results of risperidone and TMEM132E-TrkB complex; Figure 5 Co-immunoprecipitation (CoIP) results of HEK293T cells stably expressing empty vector or TMEM132E-HA after treatment with pimozide, risperidone and risperidone, wherein A is the results of pimozide treatment, B is the results of risperidone treatment, and C is the results of risperidone treatment; Figure 6 Co-localization of TMEM132E-HA (red fluorescence) and TrkB (green fluorescence) after transfection of plasmid encoding TMEM132E-HA in different cells, wherein A is HeLa cells, and B is HEK293 cells; Figure 7 Analysis of changes in the expression levels of p-ERK / ERK downstream of TrkB in HEK293T cells stably expressing empty vector or TMEM132E-HA after treatment with pimozide, risperidone and risperidone, wherein A is the Western blot data after pimozide treatment, B is quantitative analysis of protein band gray value based on the Western blot data of A; C is the Western blot data after risperidone treatment, D is quantitative analysis of protein band gray value based on the Western blot data of C; E is the Western blot data after risperidone treatment, and F is quantitative analysis of protein band gray value based on the Western blot data of E. DETAILED DESCRIPTION

[0013] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0014] In order to make the purpose, technical scheme and advantages of the present application more clear, thorough and complete, the technical scheme of the present application is described clearly and completely by means of the drawings and examples below. The following detailed description is a description of the examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0015] The instrument equipment and reagent materials used in the examples are obtained through commercial channels.

[0016] Example 1 Brain-derived neurotrophic factor (BDNF) and its high-affinity receptor TrkB are key molecular mediators of neural plasticity and antidepressant efficacy. Various antidepressants, including fluoxetine and other traditional selective serotonin reuptake inhibitors, imipramine and other tricyclic compounds, and ketamine and other fast-acting NMDA receptor antagonists, can directly bind to the transmembrane domain of TrkB, enhancing BDNF signaling through allosteric regulation. Based on this, in the human neuroblastoma cell line SH-SY5Y cell line with TMEM132E deletion, BDNF and TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) were added for drug intervention to evaluate their therapeutic potential.

[0017] Specifically, the following steps are included: (1) In order to study the biological function of TMEM132E, TMEM132E-targeted shRNA (shTMEM132E) and negative control shRNA (shNC) lentiviral plasmids were prepared, and corresponding lentiviral liquid was obtained by lentiviral packaging. The prepared shTMEM132E or shNC lentiviral liquid was used to infect human neuroblastoma cell line SH-SY5Y cells, and Puro puromycin screening was performed to construct shTMEM132A stably expressed SH-SY5Y cell strain and control group shNC stably expressed SH-SY5Y cell strain.

[0018] (2) Drug treatment: ①Stably express shNC or shTMEM132E SH-SY5Y cells were treated with 50 ng / mL brain-derived neurotrophic factor (BDNF) at 37°C for 30 minutes.

[0019] ②Stably express shNC or shTMEM132E SH-SY5Y cells were treated with 10 μM 7,8-DHF at 37°C for 2 hours.

[0020] (3) Western blot detection was performed on BDNF or 7,8-DHF drug treatment groups, and TrkB downstream signaling pathways were analyzed by Western blotting: Cells were collected, lysed with RIPA lysis buffer containing protease and phosphatase inhibitors, and proteins were extracted by ultrasonic disruption. The obtained proteins were quantified using the BCA method, and then denatured. PAGE gel was prepared, 1x SDS was used as the electrophoresis liquid, and electrophoresis was performed at 80V. The membrane transfer condition was voltage 100V for 1 hour. 5% skim milk was used for antigen blocking. After washing, antibody incubation was performed. The primary antibodies TMEM132E and GAPDH were purchased from Sino Biological, and the rest were purchased from Cell Signaling Technology, Inc. The dilution ratio of the GAPDH antibody was 1:100,000, and the dilution ratio of the rest of the antibodies was 1:2000. The primary antibody was incubated at 4°C on a shaker for 12-16 hours. The strip was washed with TBST, and the secondary antibody was incubated at room temperature for 1 hour. Then the strip was washed with TBST, and the strip was developed using chemiluminescence solution.

[0021] The test results of the BDNF treatment group are shown in Figure 1 , where A is the protein band, and B is the quantitative analysis based on the gray value of the protein band in A. The test results of the 7,8-DHF treatment group are shown in Figure 2 , where A is the protein band, and B is the quantitative analysis based on the gray value of the protein band in A.

[0022] The results show that the TrkB downstream signaling pathway is inhibited after TMEM132E knockdown. The addition of BDNF or 7,8-DHF treatment in control cells can enhance the activation of the TrkB downstream signaling pathway, but in the TMEM132E knockdown group, the addition of BDNF or 7,8-DHF treatment still keeps the TrkB downstream signaling pathway in an inhibited state, indicating that BDNF and 7,8-DHF cannot reverse the TrkB signaling defects induced by the absence of TMEM132E, which suggests that TMEM132E plays an indispensable role in maintaining the integrity of the TrkB pathway.

[0023] Example 2 Virtual screening and binding experiments were performed using AutoDock Vina to screen small molecule compounds targeting the TMEM132E-TrkB binding interface, as shown in the flowchart Figure 3 , which specifically includes the following steps: (1) 1193 drugs in the U.S. Food and Drug Administration (FDA) approved drug database were used as candidate ligands. Ligand files were converted to PDBQT format using AutoDock Vina 1.2.2 for docking with protein files. To ensure the rationality of the ligand structure, energy minimization was performed to remove possible high-energy conformations and add polar hydrogen atoms.

[0024] (2) Set a 40A x 40A x 40A cubic region centered at the TMEM132E-TrkB binding site (center_x=35.151, center_y=22.244, center_z=1.317) and ensure that this region covers all possible binding sites. The grid resolution is set to 0.05 nm to ensure high spatial resolution. Use AutoDockTools to create the grid required for docking and perform ligand docking in this grid. The size and position of the grid are optimized by the surface characteristics of the protein and known binding information. Ensure that all potential binding regions of the target are covered during the docking process.

[0025] (3) Use AutoDock Vina 1.2.2 for molecular docking. During the docking process, the ligand is rotated and displaced multiple times within the set grid region, and Vina optimizes the binding energy of the ligand to the protein by simulating different binding modes. Docking calculations include setting the maximum number of rotations and the maximum number of dockings to ensure a comprehensive search for possible binding conformations. During the docking process, the parameters of AutoDock Vina are set: maximum search number: 50, maximum rotation number: 10. AutoDock Vina outputs multiple ligand-binding conformations of the target protein and their corresponding binding energies (affinity). To select the most likely binding mode, select the conformation with the lowest binding energy, usually the top 5 conformations for subsequent analysis.

[0026] (4) Use PyMOL 2.5 to visualize the protein-ligand complex and analyze the position, orientation and binding mode of the ligand on the protein surface. Through structure optimization of the docking conformation, the best binding site is further confirmed. Use PLIP (Protein-Ligand Interaction Profiler) and LigPlus to analyze the binding interface of the protein-ligand complex in detail. Mainly analyze the hydrogen bonds, hydrophobic interactions, salt bridges and other interaction forces between the protein and the ligand, which play an important role in the process of molecular recognition.

[0027] Through this analysis, the top 19 compounds with binding energy ≤-9.6 kcal / mol are selected for further verification.

[0028] The top 19 compounds are shown in Table 1 below: Table 1 Top 19 compounds with docking energy ≤-9.6 kcal / mol

[0029] (3) Through the systematic screening of candidate drugs for the treatment of mental illness, pimozide, lemborexant and risperidone were selected as three psychoactive therapeutic drugs.

[0030] The binding of the three drugs to the target TMEM132E-TrkB was analyzed by molecular docking experiments, and the results are shown in FIG. 1, wherein (a) is the docking result of pimozide and the TMEM132E-TrkB complex, wherein pimozide is shown in pink, TMEM132E is shown in yellow, NTRK2 is shown in purple, and the binding affinity score is -10.9 kcal / mol; (b) is the docking result of reboroise and the TMEM132E-TrkB complex, wherein reboroise is shown in pink, TMEM132E is shown in yellow, NTRK2 is shown in purple, and the binding affinity score is -9.7 kcal / mol; and (c) is the docking result of risperidone and the TMEM132E-TrkB complex, wherein risperidone is shown in pink, TMEM132E is shown in yellow, NTRK2 is shown in purple, and the binding affinity score is -9.6 kcal / mol. Figure 4

[0031] The binding sites of pimozide and risperidone have significant spatial similarity.

[0032] Example 3 To explore the potential regulatory effect of drugs on the formation of the TMEM132E-TrkB complex, the three screened psychoactive compounds pimozide, reboroise, and risperidone were used to treat HEK293T cells overexpressing TMEM132E-HA and Flag-TrkB.

[0033] (1) The prepared pLVX-IRES-TMEM132E-Puro lentivirus and blank vector control pLVX-IRES-Puro were used to infect human embryonic epithelial cells HEK293T, and Puro puromycin screening was performed to construct a TMEM132E-HA stable high-expression HEK293T cell strain.

[0034] The Flag-TrkB plasmid was transfected into the HEK293T cells stably expressing TMEM132E-HA, and after the cells were cultured for 12 hours, 5 μM pimozide, reboroise, and risperidone were added for 12 hours of drug treatment, and co-immunoprecipitation (CoIP) experiments were carried out, with immunoglobulin G (IgG) as a negative control.

[0035] ​The process of co-immunoprecipitation (CoIP) experiment includes: collecting cells, ultrasonically lysing cells using 1 mL of IP lysis solution, taking 100 μL of supernatant after centrifugation as an Input positive control, adding 450 μL of protein to magnetic beads and 3 μL of HA-tag antibody, adding 450 μL of protein to magnetic beads and 3 μL of IgG antibody, binding at 4°C for 4 hours, washing magnetic beads, adding SDS denaturation, and then performing Western Blotting experiment.

[0036] The results, as shown in Figure 5 , show that pimozide, rebimastat and risperidone can significantly enhance the interaction between TMEM132E and TrkB.

[0037] (2) The HeLa cells overexpressing TMEM132E-HA were treated with the three drugs respectively, and TrkB and TMEM132E-HA were stained, and the colocalization of TMEM132E-HA (red fluorescence) and TrkB (green fluorescence) was observed by confocal microscopy, and the scale = 50 μm. The results of immunohistochemical fluorescence staining are shown in Figure 6 .

[0038] The results show that in the HeLa cells overexpressing TMEM132E-HA, compared with the group without adding drugs, the treatment of the three drugs can increase the colocalization ratio and fluorescence intensity of TMEM132E-HA and TrkB. Similarly, in the HEK293 cells overexpressing TMEM132E-HA, the treatment of the three drugs also increases the colocalization of TMEM132E-HA and TrkB on the cell membrane.

[0039] Example 4 The HEK293T cells were treated with the three psychoactive compounds pimozide, rebimastat and risperidone respectively, and the phosphorylation level of the key molecule ERK in the TrkB downstream signaling pathway was detected.

[0040] The prepared pLVX-IRES-TMEM132E-Puro lentivirus and blank vector control pLVX-IRES-Puro were used to infect human embryonic epithelial cells HEK293T, and Puro puromycin screening was performed to construct a HEK293T cell strain stably overexpressing TMEM132E-HA.

[0041] HEK293T cells stably expressing empty vector or TMEM132E-HA were treated with 5 μM drugs, respectively. In the pimozide experimental group, there were empty vector without drug group, empty vector with pimozide treatment group, overexpressed TMEM132E-HA without drug group, and overexpressed TMEM132E-HA with pimozide treatment group. In the rebotegrin experimental group, there were empty vector without drug group, empty vector with rebotegrin treatment group, overexpressed TMEM132E-HA without drug group, and overexpressed TMEM132E-HA with rebotegrin treatment group. In the risperidone experimental group, there were empty vector without drug group, empty vector with risperidone treatment group, overexpressed TMEM132E-HA without drug group, and overexpressed TMEM132E-HA with risperidone treatment group.

[0042] The phosphorylation level of the key signal molecule ERK1 / 2 downstream of TrkB (p-ERK) was detected by Western blotting, and statistical significance was determined by two-way ANOVA combined with Tukey's multiple comparison test, ; the data are expressed as mean ± standard error. The protein band and protein band gray value quantitative analysis of Western blotting data of each group are shown in Figure 7 .

[0043] The results show that in the pimozide and rebotegrin experimental groups, the ERK phosphorylation level of the empty vector cells decreases after the addition of pimozide or rebotegrin treatment, and in the risperidone experimental group, the ERK phosphorylation level of the empty vector cells increases after the addition of risperidone treatment. In the three drug experimental groups, compared with the empty vector without drug group, the ERK phosphorylation level of the overexpressed TMEM132E-HA without drug group increases. In the three experimental groups, compared with the empty vector with drug treatment group and the overexpressed TMEM132E-HA without drug group, the three drugs can increase the phosphorylation level of extracellular signal-regulated kinase (ERK) in the overexpressed TMEM132E-HA with drug treatment group.

[0044] In summary, pimozide, rebotegrin, and risperidone can promote the formation of the TMEM132E-TrkB complex and activate the downstream signal cascade. This finding determines a new therapeutic target for the three compounds and suggests that their combined application with antidepressants has potential synergistic effects.

[0045] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements will not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Application of a depression treatment target TMEM132E in drug repositioning for depression. 2.The application of the depressive treatment target TMEM132Ein the reutilization of depressive drugs according to claim 1, characterized in that: The drug is a drug targeting the interaction between TMEM132E and TrkB. 3.The application of the depressive treatment target TMEM132Ein the reutilization of depressive drugs according to claim 1, characterized in that: The drug is pimozide, rebotiepine or risperidone.

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