Use of small molecule compound s89 in restoring mitochondrial dysfunction

Abstract

The application provides application of a small molecule compound S89 in preparation of a drug for treating diseases related to mitochondrial abnormalities, and belongs to the field of medicines. The small molecule compound S89 can enhance the enzyme activity of mitochondrial MFN1 protein, has the effects of promoting mitochondrial fusion of cells, restoring mitochondrial morphology and function, and can improve axon degeneration of motor neurons of CMT2A patients, and can be used for preparing a drug for treating CMT2A type Charcot-Marie-Tooth disease and a heart ischemia-reperfusion injury syndrome, and can also be used for preparing a drug for inhibiting proliferation of lung cancer cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to application of a small molecule compound S89 in recovery of mitochondrial dysfunction. BACKGROUND

[0002] Mitochondria is a double-membrane-enclosed organelle in eukaryotic cells, which has very important functions, and provides energy for the life activities of cells through oxidative phosphorylation. Mitochondria also participates in a variety of cellular energy metabolism, such as in cardiomyocytes, the state of mitochondria plays an important role in maintaining the energy metabolism and normal maintenance of contraction function of cardiomyocytes. Mitochondria also participates in numerous immune regulation, and the release of mitochondrial DNA can be used as a signal of immune response to activate a series of inflammatory reactions. Furthermore, mitochondria is also closely related to tumors, and the change of activity of key metabolic enzymes in tumor mitochondria will inhibit the level of mitochondrial oxidative phosphorylation. Mitochondria also participates in cell proliferation, apoptosis, aging, calcium signal transduction and other physiological activities. Mitochondria is also a highly dynamic organelle, which is constantly undergoing fusion, fission and movement. In different cells and different life activities, the situation of mitochondrial dynamics is also different, and cells regulate mitochondrial dynamics to meet the energy demand, metabolic demand and other physiological demands in various life activity stages.

[0003] Mitochondrial fusion is divided into two steps, including outer membrane fusion and inner membrane fusion, which are mediated by different proteins but belong to the dynamin superfamily. They all have large GTPase domains. Mitochondrial outer membrane fusion is mainly mediated by Mitofusion1 (MFN1) and Mitofusion2 (MFN2) located in the outer membrane of mitochondria. These two proteins have great similarity in sequence and structure, and have certain complementarity in mediating mitochondrial fusion function. The protein participating in mitochondrial inner membrane fusion is mainly OPA1 located in the inner membrane of mitochondria, which often forms a multimer to promote the fusion of the inner membrane.

[0004] Charcot-Marie-Tooth (CMT) is the most common inherited motor and sensory neuropathy in humans, which has obvious genetic heterogeneity, and patients often suffer from progressive muscle weakness and atrophy in the distal extremities accompanied by sensory impairment, which seriously endangers human health. CMT is mainly divided into demyelination type (including CMT1, CMT3, CMT4 and CMTX1) and axonal type (CMT2). CMT2A is one of the subtypes, in which type 2 is mainly caused by mutations in the outer mitochondrial membrane protein MFN2 gene, and most of them are dominant mutations. The nerve biopsy of CMT2A disease and the neuron axon experiment of CMT2A disease model in mice found that there were a large number of mitochondrial dynamics abnormalities and dysfunction, and it was considered that this was the main cause of the disease. Targeting mitochondria to restore mitochondrial activity and then correcting mitochondrial dynamics abnormalities and dysfunction is expected to provide a new solution for the treatment of neurodegenerative diseases such as CMT2A disease. SUMMARY

[0005] Therefore, the application provides an application of a small molecule compound S89 in preparation of a drug for treating a disease related to mitochondrial dysfunction.

[0006] Preferably, the mitochondria are mitochondria in DRG neurons, motor neurons or tumor cells.

[0007] Preferably, the small molecule compound S89 enhances the enzyme activity of mitochondrial MFN1 protein.

[0008] Preferably, the small molecule compound S89 regulates mitochondrial morphology and promotes mitochondrial fusion.

[0009] Preferably, the small molecule compound S89 promotes the increase of mitochondrial length, mitochondrial area and mitochondrial density.

[0010] Preferably, the small molecule compound S89 increases the movement proportion of mitochondria, improves the abnormal change of membrane potential, and increases the synthesis amount of ATP.

[0011] Preferably, the small molecule compound S89 improves the axon degeneration of motor neurons.

[0012] Preferably, the small molecule compound S89 inhibits the proliferation of tumor cells.

[0013] The application also provides a drug for treating a disease related to mitochondrial dysfunction, which comprises an active ingredient S89 and a pharmaceutically acceptable carrier, and the effective concentration of the S89 is 0.4-20 μM.

[0014] Preferably, the disease includes CMT2A Charcot-Marie-Tooth disease, lung cancer and cardiac ischemia-reperfusion injury syndrome.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] The present application first proves a new use of the small molecule compound S89: the small molecule compound S89 can enhance the enzyme activity of the mitochondrial outer membrane MFN1 protein, can be used for promoting the fusion of mitochondria in normal cells or abnormal cells of mice or humans, regulating mitochondrial parameters, restoring the normal morphology and function of mitochondria, and can be used for preparing a drug for treating diseases caused by mitochondrial dysfunction (CMT2A Charcot-Marie-Tooth disease, lung cancer). BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For the influence of the small molecule compound S89 on the morphology of mitochondria in wild type MEF cells, A is the detection of mitochondrial morphology by immunofluorescence method, and B is a statistical chart of the proportion of different mitochondrial morphologies.

[0018] Figure 2 For the influence of the small molecule compound S89 on the morphology of mitochondria in MFN2 KO MEF cells, A is the detection of mitochondrial morphology by immunofluorescence method, and B is a statistical chart of the proportion of different mitochondrial morphologies.

[0019] Figure 3 For the influence of the small molecule compound S89 on the morphology of mitochondria in wild type hela cells, A is the detection of mitochondrial morphology by immunofluorescence method, and B is a statistical chart of the proportion of different mitochondrial morphologies.

[0020] Figure 4 For the ATP concentration value of cells, A represents wild type MEF cells, and B represents MFN2 KO MEF cells.

[0021] Figure 5 For the relationship between the concentration of the small molecule compound S89 and the degree of mitochondrial fusion in cells.

[0022] Figure 6 For the relationship between the concentration of the small molecule compound S89, the treatment time and the toxicity, A represents the relationship between different concentrations and toxicity, and B represents the relationship between different concentrations and different lengths of time.

[0023] Figure 7 For the morphological changes of mitochondria in cells after elution of the small molecule compound S89.

[0024] Figure 8 For the recovery of mitochondrial activity of the small molecule compound S89, A is the detection of mitochondrial morphology by immunofluorescence method, B is a statistical chart of the proportion of different mitochondrial morphologies, and C is the ATP production level of cells.

[0025] Figure 9 The small molecule compound S89 is related to the enzyme activity of the MFN1 protein.

[0026] Figure 10 The reprogramming plasmid map.

[0027] Figure 11 The small molecule compound S89 has an effect on the axon degeneration of motor neurons of CMT2A patients, A is the growth condition of neurons, and B is the statistical diagram of the axon area / cell body aggregate area.

[0028] Figure 12 The small molecule compound S89 has an effect on the mitochondria in the motor neurons of CMT2A patients, A is the statistical diagram of the mitochondria length, B is the statistical diagram of the mitochondria area, C is the statistical diagram of the mitochondria density, and D is the statistical diagram of the mitochondria movement proportion.

[0029] Figure 13 The small molecule compound S89 has an effect on the mitochondrial membrane potential in the motor neurons of CMT2A patients, A is a fluorescence microscope diagram, and B is a statistical diagram of the mitochondria depolarization proportion.

[0030] Figure 14 The small molecule compound S89 has an effect on the mitochondrial fusion in the axon of DRG neurons.

[0031] Figure 15 The small molecule compound S89 has an inhibitory effect on the proliferation of A549 lung cancer cells. DETAILED DESCRIPTION

[0032] The structural formula of the small molecule compound S89 is as follows:

[0033]

[0034] R1 and R2 are both selected from hydrogen atoms.

[0035] The compound S89 is synthesized by the method disclosed in the US invention patent “15-OXOSPIRAMILACTONE DERIVATIVES, PREPARATION METHODS AND USES THEREOF” (Patent No. US 10,112,918B2).

[0036] It is found that the small molecule compound S89 can enhance the enzyme activity of the MFN1 protein on the outer membrane of the mitochondria. Since the MFN1 protein and the MFN2 protein can jointly mediate the fusion of the outer membrane of the mitochondria, the small molecule compound S89 can effectively promote the fusion of the outer membrane of the mitochondria by enhancing the enzyme activity of the MFN1 protein, and then promote the fusion of the mitochondria.

[0037] The present application finds that the small molecule compound S89 can promote the fusion of mitochondria in the axons of mouse DRG neurons. DRG is the primary sensory center of the body and viscera, and is rich in peripheral nervous system sensory neurons. The small molecule compound S89 can effectively restore the morphology and function of neurons by promoting the fusion of mitochondria in the axons of DRG neurons.

[0038] The present application finds that the small molecule compound S89 can promote the fusion of mitochondria in the axons of mouse DRG neurons. DRG is the primary sensory center of the body and viscera, and is rich in peripheral nervous system sensory neurons. The small molecule compound S89 can effectively restore the morphology and function of neurons by promoting the fusion of mitochondria in the axons of DRG neurons.

[0039] The present application finds that the small molecule compound S89 can restore the normal morphology and function of mitochondria in the motor neurons of CMT2A patients, improve the decrease of mitochondrial membrane potential in the motor neurons of CMT2A patients, and improve the axon degeneration of motor neurons in CMT2A patients, thereby promoting the restoration of the morphology and function of motor neurons in CMT2A patients. This indicates that the small molecule compound S89 can be used as an active ingredient to prepare a drug for treating CMT2A disease.

[0040] Changes in the activity of key metabolic enzymes in tumor cell mitochondria can inhibit the level of mitochondrial oxidative phosphorylation, and mitochondrial abnormalities in tumor cells are closely related to the proliferation of tumor cells. The present application finds that the small molecule compound S89 can effectively inhibit the colony formation of A549 lung cancer cells and reduce the proliferation ability of A549 lung cancer cells by restoring the normal function of mitochondria in A549 lung cancer cells, and can be used as an active ingredient to prepare a drug for inhibiting the growth of A549 lung cancer cells.

[0041] The present application utilizes the restoring effect of the small molecule compound S89 on mitochondrial function to prepare a drug for treating diseases related to mitochondrial dysfunction, which also includes abnormalities caused by mitochondrial DNA mutations. The drug uses the small molecule compound S89 as an active ingredient, and adds a pharmaceutically acceptable carrier; the effective concentration of S89 is 0.4-20 μM, preferably 1-10 μM, and more preferably 2-5 μM.

[0042] The diseases related to mitochondrial dysfunction mentioned in the present application include CMT2A type of peroneal muscular atrophy, lung cancer, and cardiac ischemia-reperfusion injury syndrome.

[0043] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0044] In specific embodiments of the present application:

[0045] The drug solution S89 was prepared with DMSO.

[0046] The cell lines used were: wild type mouse cells presented as MEF, MEF cells with MFN1 gene knockout, MFN2 gene knockout from the laboratory of David C Chan at California Institute of Technology, wild type hela cells from ATCC.

[0047] The polyclonal antibody of TOM20 was from Invitrogen. Mitotracker Orange CMTMRos, Mitotracker Red dye was from Invitrogen. ENZCHEK PHOPHATE ASSAY KIT was from Invitrogen. ATP detection kit was from Biyun Tian. GTP was from Thermo Fisher.

[0048] FBS, DMEM culture medium were from Gibco. hiPSC / hESC culture medium-ncTarget, ncLaminin511 coating protein, hiPSC / hESC high-efficiency cryopreservation solution, ROCK channel inhibitor, EDTA cell passage working solution, Solase single cell digestion solution, 0.25% trypsin digestion solution, hPSC-motor neuron differentiation kit were from Zhiseng Sourcing Company.

[0049] Plasmid pEP4EO2SEN2K, pEP4EO2SET2K, pCEP4-M2L were from Addgene, Bac to Bac baculovirus expression system kit was from Thermo Fisher.

[0050] The cell culture method in the examples is:

[0051] 1) Cell passage: WT, MFN1 KO, MFN2 KO MEF cells and WT, MFN2 KO hela cells are all adherent growth, which are cultured in a constant temperature cell incubator at 37°C with 5% CO2, and the culture medium used is 90% DMEM + 10% serum. When the cells in the culture dish grow to about 90%, remove the culture medium, wash with preheated EDTA for 3 times, then add preheated trypsin and place in the incubator for 3-5 min, then under the microscope, the cells present as crystal beads, add preheated DMEM to terminate digestion, blow the cells with a pipette gun, then move the cells to a 15 mL centrifuge tube, centrifuge at 4°C 1000 rpm for 5 minutes, aspirate the supernatant, suspend the cells with fresh DMEN, according to the required proportion, seed in a new culture dish, shake horizontally and cross, then place in the incubator.

[0052] 2) Cell recovery: Take the cell cryovial out of the liquid nitrogen tank, put it into the 37℃ water bath immediately, shake it gently by hand, take it out when the ice crystal in the cell suspension disappears under naked eye, then centrifuge it at 1000rpm for 5min at 4℃, aspirate the cryopreservation solution, add 1mL fresh DMEM to suspend the cells, then put it into a new culture dish and put it into the incubator.

[0053] 3) Cell cryopreservation: Take the cells out of the incubator, add preheated EDTA and wash three times, then add preheated trypsin and put it into the incubator for 3-5min, add preheated DMEM to terminate the digestion, blow the cells with a pipette after washing, then put it into a 15mL centrifuge tube, centrifuge it at 1000rpm for 5min at 4℃, aspirate the supernatant, add the prepared cryopreservation solution (90% FBS + 5% DMSO + 5% DMEM) to suspend the cells, then aliquot them into cryovials, label them and put them into a cryopreservation box overnight at -80℃, the next day, transfer the cells into a liquid nitrogen tank.

[0054] All the statistical graphs in the examples are drawn by Graphpad Prism 6.

[0055] Example 1

[0056] This example explores the effect of small molecule compound S89 on the morphology of mouse cell mitochondria.

[0057] 1) Wild type and MFN2 gene knockout MEF cells were subcultured into 6-well plates with glass slides in advance, and after the cells were completely spread, they were divided into experimental and control groups for treatment, the experimental group was treated with 2μM S89 (final concentration, prepared with DMSO) for 24h, and the control group was treated with the same concentration of DMSO for 24h.

[0058] 2) After the cell treatment was completed, the culture solution was aspirated, and the cells were washed with 1xPBS twice.

[0059] 3) The prepared 3.7% paraformaldehyde was used to fix the cells in a 37℃ incubator for 20min, then the paraformaldehyde was aspirated, and the cells were washed twice with 1xPBS, then 0.2% Triton X100 was added and 4℃ punched for 15min.

[0060] 4) After punching, Triton X100 was aspirated, washed with 1xPBS three times, and the primary antibody (TOM20) was diluted with 1xPBS to the appropriate concentration (1:200), and added to the cells on the glass slide, and incubated at 4℃ overnight.

[0061] 5) The primary antibody was aspirated, washed with 1xPBS three times, and the required FITC-labeled goat anti-rabbit secondary antibody was diluted with 1xPBS to the appropriate concentration (1:200), and added to the glass slide, and incubated at room temperature for one hour.

[0062] 6) Aspirate the secondary antibody on the glass slide, and wash with 1xPBS for three times, then mount with mounting reagent, seal the glass slide with nail polish, after sealing, you can take it to the fluorescence microscope to take pictures or store it at -20℃, the result of fluorescence microscope is shown in Figures 1-2 .

[0063] As can be seen from Figure 1 , after treating the cells with small molecule compound S89 for 24h, the number of cells with broken mitochondria decreased slightly, and the number of cells with normal mitochondrial morphology increased significantly, indicating that small molecule compound S89 can promote mitochondrial fusion and promote the recovery of normal mitochondrial morphology in wild-type MEF cells.

[0064] As can be seen from Figure 2 , after treating the cells with small molecule compound S89 for 24h, the number of cells with broken mitochondria decreased significantly, and the number of cells with normal mitochondrial morphology increased significantly, indicating that small molecule compound S89 can promote mitochondrial fusion and promote the recovery of normal mitochondrial morphology in MFN2 KO MEF cells.

[0065] Example 2

[0066] This example explores the effect of small molecule compound S89 on the mitochondrial morphology of human cells.

[0067] 1) Wild-type hela cells were passaged into 6-well plates with glass slides in advance, and after the cells were completely spread, they were divided into experimental and control groups for treatment, the experimental group was treated with 2μM S89 for 24h, and the control group was treated with the same concentration of DMSO for 24h.

[0068] 2) After the treatment of the cells was completed, the culture solution was aspirated, and the cells were washed with 1xPBS twice.

[0069] 3) The prepared 3.7% paraformaldehyde was used to fix the cells in a 37℃ incubator for 20min, then the paraformaldehyde was aspirated, and the cells were washed with 1xPBS twice, then 0.2% Triton X100 was added to punch for 15min at 4℃.

[0070] 4) After punching, the Triton X100 was aspirated, and the cells were washed with 1xPBS three times, and the primary antibody (TOM20) was diluted with 1xPBS to an appropriate concentration (1:200), and added to the side of the glass slide with cells, and incubated at 4℃ overnight.

[0071] 5) Aspirate the primary antibody, wash with 1xPBS three times, dilute the secondary antibody of FITC-labeled goat anti-rabbit to the appropriate concentration (1:200) with 1xPBS, and add it to the glass slide, and incubate at room temperature for one hour.

[0072] 6) Aspirate the secondary antibody on the glass slide, and wash it with 1x PBS for three times, then seal it with mounting medium, seal the glass slide with nail polish, and then take it to the fluorescence microscope for imaging or store it at -20℃. The fluorescence microscope imaging results are shown in Figure 3 .

[0073] As can be seen from Figure 3 , after the cells are treated with the small molecule compound S89 for 24 h, the number of cells with broken mitochondria slightly decreases, and the number of cells with intermediate morphology and normal mitochondrial morphology significantly increases, indicating that the small molecule compound S89 can promote the fusion of mitochondria in human cells and promote the recovery of normal morphology of mitochondria in human cells.

[0074] Example 3

[0075] This example explores the effect of the small molecule compound S89 on the generation of ATP in cells.

[0076] 1) Wild-type and MFN2 gene knockout MEF cells are plated in a 6-well plate, and the cells are treated in an experimental group and a control group, respectively. The experimental group is treated with 2 μM S89 for 24 h, and the control group is treated with the same concentration of DMSO for 24 h.

[0077] 2) Aspirate the cell culture solution, and wash it with PBS once. Add 200 μL of cell lysis solution to each well, and lyse the cells on ice for 30 min.

[0078] 3) After lysis, centrifuge at 4℃ 13000 rpm for 15 min, and aspirate the supernatant into a new 1.5 mL centrifuge tube. After adjusting the concentration, it is used for subsequent ATP concentration determination.

[0079] 4) Dissolve the ATP detection reagent on ice, and dilute the ATP standard concentration solution to 0.1 μM, 1 μM, 10 μM, 50 μM, and 100 μM with ATP detection lysis solution to determine the standard curve of ATP concentration.

[0080] 5) Prepare a 96-well plate that is opaque between wells, and add 100 μL of ATP detection working solution to each well as needed. Place it at room temperature for 5 min to consume the background ATP.

[0081] 6) Add 10 μL of sample to each detection well, mix well with a gun, and immediately measure the RLU value (RLU value or CPM can be measured with a luminometer or liquid scintillation instrument) after 3 seconds. According to the standard sample curve, the absolute value of the ATP concentration in the sample is calculated, and the results are shown in Figure 4 .

[0082] As can be seen from Figure 4It can be seen that the ATP concentration of wild type MEF cells and MFN2 KO MEF cells is significantly increased after 24 hours of treatment with 2 μM S89, indicating that small molecule compound S89 can promote the generation of ATP in cells.

[0083] Example 4

[0084] This example investigates the effective concentration of small molecule compound S89.

[0085] 1) The MFN2 gene knockout MEF cells were subcultured into a 6-well plate with a glass slide in advance, and after the cells were completely spread, they were treated with different concentrations of S89 for 24 hours.

[0086] 2) After the cell treatment was completed, the culture solution was aspirated, and the cells were washed with 1xPBS twice.

[0087] 3) The prepared 3.7% paraformaldehyde was used to fix the cells in a 37°C incubator for 20 minutes, then the paraformaldehyde was aspirated, and the cells were washed twice with 1xPBS, then 0.2% Triton X100 was added and 4°C punched for 15 minutes.

[0088] 4) After punching, Triton X100 was aspirated, washed with 1xPBS three times, and the primary antibody (TOM20) was diluted with 1xPBS to the appropriate concentration (1:200), and added to the cell side of the glass slide, and incubated at 4°C overnight.

[0089] 5) The primary antibody was aspirated, washed with 1xPBS three times, and the required FITC-labeled goat anti-rabbit secondary antibody was diluted with 1xPBS to the appropriate concentration (1:200), and added to the glass slide, and incubated at room temperature for one hour.

[0090] 6) The secondary antibody on the glass slide was aspirated, and washed with 1xPBS three times, then mounted with mounting agent, and the glass slide was sealed with nail polish, after sealing, it can be directly taken to the fluorescence microscope for photography or stored at -20°C, the mitochondrial length-width ratio was calculated, and the mitochondrial length-width ratio was used to reflect the degree of mitochondrial fusion, and the experimental results are shown in Figure 5 .

[0091] From Figure 5 it can be seen that the EC 50 value of small molecule compound S89 corresponds to a concentration of 436±85nM, indicating that the effective concentration of small molecule compound S89 for promoting mitochondrial fusion is about 0.4 μM.

[0092] Example 5

[0093] This example investigates the toxicity of small molecule compound S89.

[0094] 1) The MFN2 gene knockout MEF cells were passaged into 6-well plates or 96-well plates with glass slides in advance, and after the cells were completely spread, different concentrations of S89 were used to treat the cells for different time.

[0095] 2) The toxic effects of the drugs were indicated by MTT method and PI staining method, and the results were shown in Figure 6 .

[0096] From Figure 6 it can be seen that after the MFN2 KO MEF cells were treated with different concentrations of S89 for 24 hours, the IC 50 value of S89 was 9.48±0.76 μM; the cell death rate was close to 0 when the MFN2 KO MEF cells were treated with 10 μM concentration for 6 hours, and if the treatment time was increased, the cell death rate increased, and there was a very obvious toxic effect when the cells were treated with 10 μM for 24 hours; the toxic effect appeared when the MFN2 KO MEF cells were treated with 20 μM concentration for 6 hours.

[0097] Example 6

[0098] This example explores the effect of small molecule compound S89 on mitochondrial fusion after elution.

[0099] 1) The MFN2 gene knockout MEF cells were passaged into 6-well plates with glass slides in advance, and after the cells were completely spread, 2 μM S89 was used to treat the cells for 24 hours.

[0100] 2) After the treatment of the cells, MitoTracker red was added for staining for half an hour, and after the staining, fresh culture medium was used to elute S89, and then the cells were directly taken to the fluorescence microscope for photographing, and the results were shown in Figure 7 .

[0101] From Figure 7 it can be seen that after the elution of small molecule compound S89, the effect of mitochondrial fusion gradually disappeared within 15 minutes.

[0102] Example 7

[0103] This example explores the relationship between the effect of small molecule compound S89 on restoring mitochondrial activity and MFN1 protein.

[0104] The MFN1 gene knockout MEF cells were passaged into 6-well plates with glass slides in advance, and after the cells were completely spread, the cells were divided into experimental group and control group, and the cells were treated, the experimental group was treated with 2 μM S89 for 24 hours, and the control group was treated with the same concentration of DMSO for 24 hours, and then the mitochondrial fusion was detected according to the method of immunofluorescence in Example 1, and the ATP production was detected according to the method in Example 3, and the results were shown in Figure 8.

[0105] By Figure 8 It can be seen that the cells with knocked-out MFN1 gene cannot restore normal morphology after treatment with small molecule compound S89, and the ATP concentration is slightly lower than that of the untreated group, indicating that the action of small molecule compound S89 to restore mitochondrial activity requires MFN1 protein.

[0106] Example 8

[0107] This example explores the relationship between small molecule compound S89 and the enzyme activity of MFN1 protein.

[0108] 1. Expression and purification of MFN1 protein in insect system

[0109] The cDNA fragment of full-length MFN1 protein was cloned into the vector pFastBac, and after transformation, blue-white spot screening and gel electrophoresis identification, the recombinant plasmid Bacmid was obtained. The recombinant plasmid was transfected into SF9 cells, and P3 generation high titer virus was collected for subsequent infection.

[0110] 1) SF9 cells were suspended in a 1L conical flask with a density of 1×10 6 / mL. When the cells grew to the logarithmic phase with a density of 2×10 6 / mL, P3 generation virus was added, and the cells were collected after 48-72h of infection.

[0111] 2) The collected cells were poured into a centrifuge cup, centrifuged at 4000 rpm for 10 min, and the supernatant was removed. Then they were washed once with PBS, also at 4000 rpm for 10 min, and the supernatant was removed. The cells were suspended in protein buffer (25mM HEPES, 2.5mM β-ME, 150mM KCL), and protease inhibitors were added, then transferred to a dounce tube for crushing. The crushing efficiency was detected under a microscope to reach 95%.

[0112] 3) Triton-100 was added to a final concentration of 2.5%, mixed well, and then incubated at 4°C for 2h. Then centrifuged at 32000g at 4°C for 1h, and the supernatant was collected. 1-2mL strep beads were added to the supernatant in proportion (1:30), and incubated at 4°C for 1h.

[0113] 4) The incubated strep beads were sequentially passed through the protein purification column, and then the impurities were eluted with 50 times the column volume of 25mM HEPES, 150mM KCL, 0.1% Triton-100, 2.5mM β-ME.

[0114] 5) Elute the protein of interest into 15 mL conical tubes by incubating the column with 20 mL of 25 mM Hepes (pH 7.4), 150 mM KCl, 0.1% Triton-100, 5 mM d-Desthiobiotin elution buffer at 4°C for 20 min, then centrifuge at 4000 rpm for 1 h at 4°C, transfer the collected protein into a centrifuge tube, check the purity and concentration of the protein by SDS-PAGE and Coomassie blue staining, or store at -80°C after immersing in liquid nitrogen.

[0115] 2. Detection of the enzymatic activity of MFN1 protein

[0116] The enzymatic activity of protein GTP was detected using Invitrogen's EnzChek Phosphate Assay Kit. 100 μL of reaction system was added with 20x reaction buffer (1 M Tris-HCl, 20 mM MgCl2pH 7.5, and 2 mM sodium azide), 0.1 U purine nucleoside phosphorylase, 200 mM 2-amino-6-mercapto-7-methylpurine riboside, and 1 μM MFN1 protein, and the remaining volume was supplemented with purified protein buffer (25 mM Hepes (pH 7.4), 150 mM KCl). After incubation with 10 μM small molecule compound S89 at room temperature for 30 min, the system was added to the bottom of a transparent 96-well plate, and a multifunctional enzyme marker was used to set the temperature to 37°C and detect the light absorption value at 360 nm. After the reading and temperature were stable, 0.5 mM GTP was added using a syringe, and the light absorption value at 360 nm was read every 30 s. The time reading when GTP was added was set to 0, and the efficiency of phosphate release was calculated by comparing the standard curve. The detection of the ability of MFN1 to hydrolyze GTP was the enzymatic activity of MFN1, and the results are shown in Figure 9 .

[0117] As can be seen from Figure 9 , the small molecule compound S89 can enhance the enzymatic activity of MFN1 protein.

[0118] Example 9

[0119] This example explores the relationship between the small molecule compound S89 and the axonal degeneration of motor neurons in CMT2A patients.

[0120] Two CMT2A patients with different MFN2 gene mutations (R94W, T105M) and their family normal controls were collected for the experiment.

[0121] 1. Reprogramming of human peripheral blood CD34+

[0122] The reprogramming plasmid contains three plasmids, which contain a total of seven genes: human OCT4, SOX2, NANOOG, LIN28, c-MYC, KLF4, and SV40LT (see [link to plasmid]). Figure 10 Each transfection used 3.0 μg pEP4EO2SEN2K, 3.2 μg pEP4EO2SET2K, and 2.4 μg pCEP4-M2L DNA.

[0123] Prepare 1×10 6 CD34+ cells were transfected with a reprogramming plasmid and then seeded in 6-well plates coated with fibronectin / matrigel containing CD34+ cell expansion medium. Fibronectin and matrigel were used together to promote cell recovery after electroporation. The DMEM / F12 medium used for cell culture was supplemented with N-2, B-27, bFGF, PD0325901, CHIR99021, A-83-01, hLIF, and HA-100. These factors supported cell reprogramming culture from 2 to 11 days after electroporation. iPSCs were then amplified using NuwaCell hPSC Medium, and the total number of iPSC clones was counted on day 17 after electroporation.

[0124] 2. Culture of induced pluripotent stem cells (iPSCs)

[0125] 1) Cell passage: iPSCs were cultured on Nuwacell hPSC medium without a feeder layer using Matrigel as the substrate. Passage was performed using EDTA. When iPSC confluence reached approximately 85%, the cells were washed once with DPBS (calcium- and magnesium-free), followed by incubation at 37°C for 8 minutes with 0.5 mM EDTA (2 mL / well in a 6-well plate). The EDTA solution was then aspirated, and 2 mL of Nuwacell hPSC medium was gradually added to disperse the cell clumps. After a few gentle shakes, most cells detached from the substrate. The digested cell clumps were quickly aliquoted into Matrigel-coated culture plates pre-filled with fresh Nuwacell hPSC medium. To promote cell adhesion and survival, 2.5 μM of the ROCK inhibitor Blebbistatin was added on the first day of passage. Using this method, cells were passaged at a ratio of 1:8 to 1:10, every 3–4 days.

[0126] 2) Cell recovery: First, preheat the water bath to 37°C, place the Matrigel-coated 6-well plate in the incubator about 1 hour in advance to recover to room temperature, take 4 mL of Nova complete medium, add 1 μL of Nuwacell TMBlebbistatin (10 mM) at a ratio of 1:4000, and recover to room temperature. Take out one frozen cell and place it in a 37°C water bath, gently shake it by hand, and thaw it within 1 min. When the ice crystals in the cell suspension completely disappear, take it out and move it to the previously prepared 15 mL centrifuge tube. Use a pipette to take 10 mL of DMEM / F12 and add it drop by drop to the frozen cell suspension. Centrifuge at 160 x g for 5 min, discard the supernatant, add 4 mL of pre-warmed Blebbistatin + Nova medium to mix the cells, and try to avoid blowing. Discard the Matrigel coating liquid in the 6-well plate, mix the cells, and inoculate 2 mL per well into 2 wells in the incubator.

[0127] 3) Cell freezing: When the cell confluence reaches about 85%, it can be harvested for freezing. Take the NuwacellTM hPSC freezing solution in the 4°C refrigerator, pre-warm it to room temperature, and shake it well before use. Discard the hiPSC culture supernatant, add 2 mL per well of DPBS (without calcium and magnesium), shake gently several times, and then discard it. Add 2 mL per well of hPSC subculture working solution, and place the cells in a 37°C incubator. After digestion, gently remove the culture plate and discard the EDTA. Shake the pre-warmed Nuwacell TM hPSC freezing solution, add 1 mL of freezing solution per well, and gently blow and shake it evenly. Then take the cell suspension and add it to a 1.5 / 2 mL freezing tube, label the cell name, passage (P#), date, and operator ID. Place the cells in a gradient program cooling box and place them in a -80°C refrigerator overnight, and then transfer them to a liquid nitrogen tank for long-term storage the next day.

[0128] 3. Inducing pluripotent stem cells (iPSC) to differentiate into motor neurons

[0129] Use Zhongsheng traceable motor neuron differentiation kit, which includes differentiation complete medium A, B, C, D and motor neuron precursor cell freezing solution.

[0130] 1) On the same day (DAY0), when the hPSC reaches 80-90% confluence in the culture dish, remove the medium, add 2 mL per well of DPBS (without calcium and magnesium), shake gently and discard.

[0131] 2) Add 1 mL per well of pre-warmed Solase digestion solution to completely cover the bottom of the well, and incubate in the cell culture incubator for 5-7 min. Gently shake the well plate to completely detach the cells from the matrix.

[0132] 3) Transfer the cell suspension into a 1.5 mL centrifuge tube and centrifuge for 10-15 seconds in a hand-held centrifuge.

[0133] 4) Aspirate the supernatant and resuspend the cells in 1 mL of human motor neuron differentiation complete medium A. Gently pipette the cells to disperse them as single cells as much as possible and count the cells.

[0134] 5) Seed 3-5 x 10 5 cells / well into a 6-well plate coated with Matrigel and add 2 mL of human motor neuron differentiation complete medium A (containing 10 μΜ / mL of Blebbistatin) per well.

[0135] 6) Incubate in the cell culture incubator. After 22-24 hours, aspirate the cell culture medium and add 2 mL of human motor neuron differentiation complete medium A per well. Continue incubation in the cell culture incubator and change the medium every day.

[0136] 7) On the fourth day (DAY 4), aspirate the medium and add 2 mL of human motor neuron differentiation complete medium B per well. Continue incubation in the cell culture incubator and change the medium every day (DAY 4-9).

[0137] 8) On DAY 9, aspirate the medium and add 2 mL / well of DPBS (without calcium and magnesium), gently shake and aspirate. Add 1 mL / well of pre-warmed Solase digestion solution to completely cover the bottom of the well, and incubate in the cell culture incubator for 8-12 minutes. Gently shake the plate to completely detach the cells.

[0138] 9) Transfer the cell suspension into a 1.5 mL centrifuge tube and centrifuge for 10-15 seconds in a hand-held centrifuge.

[0139] 10) Aspirate the supernatant and resuspend the cells in 1 mL of differentiation complete medium C. Gently pipette the cells to disperse them as single cells as much as possible and count the cells. The motor neuron precursor cells obtained here can be frozen by adding 1 mL of motor neuron precursor cell freezing solution to freeze the cells at a cell number of 2 x 10 6 cells / tube.

[0140] 11) Seed at a density of 1-3 x 10 5 cells / well into a 12-well plate coated with Matrigel and add 1 mL / well of differentiation complete medium C (containing 10 μΜ / mL of Blebbistatin). Incubate in the cell culture incubator. After 22-24 hours, aspirate the cell culture medium and add 1 mL / well of differentiation complete medium C. Continue incubation in the cell culture incubator and change the medium every day (DAY 9-12).

[0141] 12) On Day 12, aspirate the cell culture medium and add 1 mL / well of complete differentiation medium D. Treat the cells in the experimental and control groups separately. The experimental group received 0.5 μM S89 cells, while the control group received the same concentration of DMSO. Place the cells in an incubator and change the medium every other day. Mature motor neurons can be obtained by Days 30–35. Observe the growth of motor neurons under a bright-field microscope. Figure 11 .

[0142] Depend on Figure 11 It can be seen that after treatment with the small molecule compound S89, the axonal state of neurons was restored and the ratio of axonal area to cell body aggregate area was significantly increased, indicating that the addition of the small molecule compound S89 can improve the axonal degeneration of motor neurons in CMT2A patients.

[0143] Example 10

[0144] This embodiment investigates the effects of the small molecule compound S89 on mitochondrial abnormalities in motor neurons of CMT2A patients.

[0145] 1. Packaging of the mitodendra virus

[0146] 1) Transfer 293T cells to a 10cm culture dish in advance. When the cells reach about 70% confluency, transfect them with lipo3000. The solution is 10μg mitodendra plasmid, 7.5μg packaging plasmid 1 (PSPAX), and 2.5μg packaging plasmid 2 (PMD2.G). Change the medium after 6 hours.

[0147] 2) After 24 hours, collect the first supernatant into a 50mL centrifuge tube, wrap it with aluminum foil and place it at 4℃. After 60 hours, collect the second supernatant and mix it with the first supernatant.

[0148] 3) Centrifuge the collected virus solution at 2000 rpm for 5 minutes at 4°C. Filter the supernatant through a 0.22 μm filter into a new 50 mL centrifuge tube. Centrifuge the filtered virus solution at 34000 rpm for 3 hours at 4°C. Discard the supernatant, suspend the precipitate in 60 μL of PBS containing 0.1% BSA, and then aliquot into PCR tubes and store at -80°C.

[0149] 2. Experimental Procedure

[0150] Motor neurons differentiated from induced pluripotent stem cells (iPSCs) were treated with the small molecule compound S89 according to the method in Example 9. The morphology, size, and density of mitochondria were observed using the immunofluorescence method described in Example 1 (mitochondrial length, area, and density were statistically analyzed using ImageJ software). On day 25 of differentiation, mitochondria were labeled with mitodendra virus infection, and mitochondrial movement was captured in real-time on live cells. Mitochondrial parameters and movement status are shown below. Figure 12 .

[0151] Depend on Figure 12 It has been shown that the small molecule compound S89 can increase mitochondrial length and surface area, as well as mitochondrial density and the proportion of mitochondrial motility. This indicates that the small molecule compound S89 can improve mitochondrial abnormalities in motor neurons of CMT2A patients.

[0152] Example 11

[0153] This embodiment investigates the effect of the small molecule compound S89 on the mitochondrial membrane potential in motor neurons of CMT2A patients.

[0154] Motor neurons differentiated from induced pluripotent stem cells (iPSCs) were treated with the small molecule compound S89 according to the method in Example 9, stained with MitoTracker Orange CMTMRos for 30 min, washed off, and then labeled with TOM20 antibody using the immunofluorescence method in Example 1. The samples were then observed under a fluorescence microscope, and the mitochondrial depolarization ratio was calculated. The results are shown in [Figure 1]. Figure 13 .

[0155] Depend on Figure 13 It can be seen that the mitochondrial depolarization ratio was significantly reduced after treatment with the small molecule compound S89, indicating that the small molecule compound S89 can improve the reduction of mitochondrial membrane potential in motor neurons of CMT2A patients.

[0156] Example 12

[0157] This embodiment investigates the fusion effect of the small molecule compound S89 on mitochondria in DRG neurons.

[0158] 1. DRG neuron isolation steps:

[0159] 1) Prepare sterile slides: Soak slides in strong acid overnight, wash thoroughly with deionized water, and store in 75% ethanol. Before isolating DRG neurons, treat slides with 25 μg / mL Poly-L-Ornithine (diluted with PBS) and incubate overnight.

[0160] 2) Use Ca-free 2+ / Mg 2+ PBS wash the glass slides for 1h each time, wash once with ddH2O, and finally coat with 5μg / mL Laminin for 1h before use;

[0161] 3) Anesthetize the mouse by intraperitoneal injection of tribromoethanol (20mg / mL) (180mg / kg), sterilize the mouse with 75% alcohol, cut the skin along the midline of the spine and expose the whole spinal column region, cut the bone on both sides of the spinal canal until the whole spinal column can be removed, trim off the excess bone if necessary. Cut off the whole spinal column, remove the excess muscle and other tissues. Place the spinal column in a 60mm petri dish containing 5mL of Ca 2+ / Mg 2+ HBSS, try to operate on ice;

[0162] 4) Cut the spinal column along the midline, remove the spinal cord to expose the DRG neurons, carefully pull out the DRG ball from the base with a sterile ophthalmic forceps, try to avoid mechanical damage to the ball, transfer the DRG ball to a Ca 2+ / Mg 2+ HBSS solution, from the back to the abdomen until all DRG ganglia are removed;

[0163] 5) Trim off the excess axon bundles and blood vessels with microscissors, transfer the DRG ball to the collagenase / dispase solution prepared in advance, digest at 37°C for 30min, then place it on a rotator to digest at room temperature for 30min;

[0164] 6) After the digestion is completed, centrifuge at 1000g at 4°C for 2min, remove the supernatant, resuspend the cell pellet with 1mL of the culture medium prepared in advance for 10-15 times (slowly and gently, try to maintain a uniform speed), then filter the single cells with a 70μm cell screen, and remove the tissues that are not digested sufficiently at the same time. Centrifuge the filtered cell suspension at 200g at 4°C for 3min;

[0165] 7) Remove the supernatant, resuspend the cell pellet and count, then seed it into a culture dish, replace the medium after the cells adhere to the dish for several hours. When the glial cells start to proliferate after 3 days or so, add cytosine arabinoside to inhibit the proliferation of the glial cells.

[0166] 2. Experimental procedure

[0167] Isolate and culture the DRG neurons of mice (8 weeks, male) in vitro, and treat them with 0.5μM S89 for 24h. Label the mitochondria in the axons of the DRG neurons with TOM20 antibody according to the method of immunofluorescence in Example 1, observe the slices under a fluorescence microscope after they are prepared, and the results are shown in Figure 14 .

[0168] FromFigure 14 It can be known that the small molecule compound S89 can promote the fusion of mitochondria in DRG neurons.

[0169] Embodiment 13

[0170] This embodiment explores the inhibitory effect of the small molecule compound S89 on the growth of A549 lung cancer cells.

[0171] 1. Experimental steps

[0172] (1) Cell counting

[0173] 1) The adherent cells were digested into a non-adherent state under the action of EDTA and trypsin, and suspended with cell culture medium;

[0174] 2) Part of the cell suspension was taken out and diluted according to a certain proportion;

[0175] 3) 10 μL of the diluted cell suspension was taken and added dropwise into the small chamber of the blood cell counting plate, and the number of cells in four chambers was counted under a microscope; the cell density in the original solution was calculated according to the formula, with the unit of cells per milliliter:

[0176] C = n / 4 x a x 10 4

[0177] N is the number of cells in four chambers, and a is the dilution multiple;

[0178] 4) The cell stock solution was diluted according to the concentration, and the final concentration was 1 cell per microliter, and 100 microliters were inoculated into one hole of a six-well plate (three duplicate holes).

[0179] (2) Every other day, the culture medium containing different concentrations of S89 was replaced with the original culture medium.

[0180] (3) After 9 days of S89 addition, the culture medium was washed away, 1 milliliter of R250 staining solution was added to each well, and it was placed at room temperature for 10 min, then the R250 staining solution was removed, anhydrous ethanol was added dropwise for a few seconds to wash off the floating color, the six-well plate was placed in clean water to terminate the staining. Dry, take pictures, and count the number of colonies. The results are shown in Figure 15 .

[0181] From Figure 15 it can be known that the number of A549 cells in the group added with the small molecule compound S89 is significantly reduced, indicating that the small molecule compound S89 can inhibit the proliferation of A549 lung cancer cells.

[0182] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. The application of the small molecule compound S89 in the preparation of drugs for treating diseases related to mitochondrial dysfunction, characterized in that, The mitochondrial dysfunction-related disease is CMT2A type peroneal muscular atrophy.

2. The application according to claim 1, characterized in that, The mitochondria are those of DRG neurons or motor neurons.

3. The application according to claim 1, characterized in that, The small molecule compound S89 enhances the enzymatic activity of the mitochondrial MFN1 protein.

4. The application according to claim 1, characterized in that, The small molecule compound S89 regulates mitochondrial morphology and promotes mitochondrial fusion.

5. The application according to claim 1 or 4, characterized in that, The small molecule compound S89 promotes an increase in mitochondrial length, mitochondrial area, and mitochondrial density.

6. The application according to claim 1, characterized in that, The small molecule compound S89 increases the proportion of mitochondrial motility, improves abnormal changes in membrane potential, and increases ATP synthesis.

7. The application according to claim 6, characterized in that, The small molecule compound S89 improves the axonal degeneration of motor neurons.

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