Mitochondrial autophagy enhanced chimeric molecule based on E3 protein ubiquitin ligase ITCH and application thereof

Abstract

The invention discloses a mitochondrial autophagy enhanced chimeric molecule based on E3 protein ubiquitin ligase ITCH and application of the mitochondrial autophagy enhanced chimeric molecule, and belongs to the field of medicinal chemistry. The structure of the chimeric molecule is I-L-O ', I represents a ligand combined with ITCH, L represents a connecting chain, and O' represents a ligand combined with mitochondrial outer membrane protein. The mitochondrial outer membrane protein comprises a translocation protein and a voltage-dependent anion channel protein. The chimeric molecule can effectively enhance mitochondrial autophagy and repair mitochondrial damage under low concentration (10-100 nanomolar concentration) so as to enhance mitochondrial functions, and verification is carried out in various cells and animal bodies, so that a blank in the field is filled.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a mitochondrial autophagy-enhancing chimeric molecule based on the E3 protein ubiquitin ligase ITCH and its applications. Background Technology

[0002] Mitochondria are among the most important organelles in cells. They provide energy molecules (ATP) to cells through oxidative phosphorylation and play a key role in many cell biological processes such as cell metabolism, growth, and apoptosis.

[0003] During the process of performing their functions, mitochondria constantly produce free radicals, which can damage them. Factors such as aging and stress can exacerbate mitochondrial damage, leading to mitochondrial dysfunction.

[0004] Mitochondrial dysfunction contributes to the development of many major human diseases, such as Alzheimer's disease, Parkinson's disease, ALS, cardiovascular disease, obesity, diabetes, fibrosis of the liver, kidneys and lungs, gout, and cancer.

[0005] Mitophagy is a normal cellular mechanism for clearing damaged mitochondria. In this process, damaged mitochondria are selectively encapsulated into autophagosomes, then fused with lysosomes and degraded by hydrolases. Mitophagy is a cellular protective mechanism that removes excess or dysfunctional mitochondria, maintaining a healthy number of mitochondria to balance cellular homeostasis. Increasing evidence suggests that mitophagy plays a crucial role in maintaining the health of the mitochondrial network, and abnormalities and deficiencies in mitophagy are closely related to mitochondrial dysfunction. Enhancing cellular mitophagy levels may offer new methods and strategies for treating these diseases.

[0006] Several natural product molecules and synthetic small molecules, such as Uroli thin-A, UMI-77, Kaempferol, and Rhapontigenin, have been reported to induce mitophagy, enhance the cell's ability to clear damaged mitochondria, and have shown some therapeutic effects in animal disease models such as Alzheimer's disease. However, these molecules do not have high specificity in regulating mitophagy; while affecting mitophagy, they may also affect other signaling pathways or biological processes. Furthermore, the concentrations required to induce mitophagy are all at the micromolar level or higher, limiting their potential as new drugs. Small molecules that can specifically enhance cellular mitophagy at low concentrations represent a pressing but unmet market need.

[0007] Protein degradation targeting chimeras (PROTACs) are a novel targeted protein degradation technology. Traditional PROTAC molecules function through the proteasome pathway. Due to the limited pore size of the proteasome, it can only degrade smaller targets such as protein molecules, but cannot degrade organelles, including mitochondria. Therefore, traditional PROTAC molecules cannot achieve the goal of enhancing mitophagy.

[0008] Several chimeric small molecules developed based on PROTAC, such as ATTEC, AUTAC, and AUTOTAC, have been reported to degrade target proteins via autophagy rather than the proteasome. ATTEC and AUTOTAC can degrade target proteins and lipid droplets, but there are no reports of them degrading damaged mitochondria.

[0009] In a 2019 paper published in Molecular Cell (Takahashi, D.; Moriyama, J.; Nakamura, T.; Miki, E.; Takahashi, E.; Sato, A.; Akaike, T.; Itto-Nakama, K.; Arimoto, H. AUTACs: Cargo-Specific Degraders Using Selective Autophagy. Mol. Cell 2019, 76(5), 797, DOI: 10.1016 / j.molcel.2019.09.009), Japanese scientists designed and synthesized a chimeric small molecule, AUTAC4, which can degrade damaged mitochondria. AUTAC4 induces K63 ubiquitination in mitochondria, but the mechanism is unclear. It is unknown whether it works by recruiting a specific E3 ligase. In addition, AUTAC4 requires a concentration of 10 micromoles to degrade damaged mitochondria in cells, and its effectiveness in animals has not been tested. Its clinical application prospects are questionable.

[0010] Mitophagy in cells currently exhibits two known modes: ubiquitination-dependent and ubiquitination-independent. Among these, Pink1 / Parkin-mediated ubiquitination-dependent mitophagy is the most thoroughly studied. In healthy cells without mitochondrial damage, Pink1, acting as a "sentinel," rapidly enters the mitochondria after reaching the outer mitochondrial membrane and is degraded, unable to remain there. However, when mitochondria are damaged, their membrane potential is lost. Pink1 then remains on the outer mitochondrial membrane, phosphorylating pre-existing ubiquitin molecules (Ub) near the membrane, and subsequently binding to Parkin, leading to Parkin's phosphorylation. Phosphorylation and activation of Parkin cause K63 ubiquitination of many proteins on the outer mitochondrial membrane. Through the selective binding of autophagy receptor proteins (currently known to include p62, NBR1, OPTN, NDP52, and TAX1BP1, etc.) to both ubiquitin and LC3 proteins, autophagosomes are recruited to the damaged mitochondria, driving downstream processes of mitophagy.

[0011] Besides Pink1 / Parkin, there are other reports of E3 ligase-mediated ubiquitination of mitochondrial outer membrane proteins driving mitophagy. For example, the mitochondrial E3 ligase MARCH5 directly interacts with FUNDC1, mediating its ubiquitination at lysine 119, and subsequently degrading FUNDC1 to regulate hypoxia-induced mitophagy.

[0012] Therefore, an obvious idea is to design a PROTAC molecule that binds to a specific mitochondrial outer membrane protein at one end, and at the other end recruits a specific E3 ligase to the mitochondrial outer membrane target protein, mimicking the action of activated Parkin to forcibly induce ubiquitination of the mitochondrial outer membrane protein and enhance mitophagy. However, to date, no PROTAC molecule has been developed that can effectively degrade damaged mitochondria. In fact, Japanese scientists attempted this idea in a 2019 paper published in *Molecular Cell*, synthesizing a chimeric molecule that binds to CRBN (one of the most commonly used E3 ligases in PROTAC molecules) at one end and to the exogenously expressed mitochondrial outer membrane protein mito-EGFP-HT at the other end. This molecule successfully brought CRBN to the mitochondrial outer membrane and induced K48 ubiquitination of mito-EGFP-HT, but failed to degrade damaged mitochondria.

[0013] The key technological bottleneck for enhancing chimeric small molecules in mitophagy lies in E3 ligases; finding an E3 ligase that can effectively mimic the activated Parkin is crucial. The human body has nearly 600 E3 ligases, offering immense potential.

[0014] However, when screening and validating new E3 ligases and designing PROTACs, it is necessary to comprehensively consider multiple dimensions such as the PPI of E3 ligases, structural availability, functional necessity, and cellular location to ensure the effectiveness and specificity of PROTACs.

[0015] In summary, while the use of PROTAC-based chimeric small molecules to enhance mitophagy by recruiting specific E3 ligases to mitochondria to drive the ubiquitination of mitochondrial outer membrane proteins (especially K63 ubiquitination) is theoretically feasible, there are no successful cases to date. Summary of the Invention

[0016] In order to overcome at least one of the shortcomings of the prior art, one of the objectives of the present invention is to provide a chimeric molecule that enhances mitophagy.

[0017] A second objective of the present invention is to provide a pharmaceutical composition comprising the above-described chimeric molecule that enhances mitophagy.

[0018] A third objective of this invention is to provide the use of the above-mentioned chimeric molecules and pharmaceutical compositions that enhance mitophagy in the preparation of medicaments for the prevention or treatment of diseases caused by or related to mitochondrial dysfunction.

[0019] The fourth objective of this invention is to provide a treatment method.

[0020] The technical solution adopted in this application is:

[0021] In a first aspect, a chimeric molecule that enhances mitophagy is provided, the structure of which is shown in Formula 1:

[0022] IL-O'

[0023] Formula 1

[0024] Wherein, I represents the ligand that binds to the E3 protein ubiquitin ligase ITCH, L represents the linker chain, and O' represents the ligand that binds to mitochondrial outer membrane proteins; the mitochondrial outer membrane proteins include translocator proteins (TSPO) and voltage-dependent anion channel proteins (VDAC).

[0025] The structural formula of I includes:

[0026]

[0027] L represents an alkoxy group chain, including: -(CH2CH2O) a -、-(CH2CH2CH2O) b - where a and b are natural numbers greater than or equal to 1.

[0028] In some embodiments, the ligand that binds to the mitochondrial outer membrane protein has any of the following structural formulas:

[0029]

[0030]

[0031] Where X is a halogen.

[0032] In some embodiments, X is selected from at least one of fluorine, chlorine, bromine, and iodine.

[0033] In some embodiments, the structure of the chimeric molecule is shown in Formula 2:

[0034]

[0035] Where n is a natural number from 1 to 10.

[0036] In some preferred embodiments, n is a natural number from 1 to 5.

[0037] In some embodiments, the chimeric molecule has any one of the following structural formulas:

[0038]

[0039] In a second aspect, a pharmaceutical composition is provided, comprising the chimeric molecule for enhancing mitophagy as described in any of the preceding claims, and a pharmaceutically acceptable carrier.

[0040] In some embodiments, the pharmaceutically acceptable carrier is any one or more of buffers, emulsifiers, suspending agents, stabilizers, preservatives, excipients, fillers, coagulants, blending agents, surfactants, dispersants, and defoamers.

[0041] Thirdly, the use of the chimeric molecules and pharmaceutical compositions described above that enhance mitophagy in the preparation of medicaments for the prevention and / or treatment of diseases caused by or related to mitochondrial dysfunction is provided.

[0042] In some embodiments, the mitochondrial dysfunction leading to or related to diseases include neurodegenerative diseases, cardiovascular diseases, metabolic diseases, tumors, tissue fibrosis, autoimmune diseases, inflammatory diseases, or diseases related to viral infections.

[0043] The neurodegenerative diseases mentioned include Alzheimer's disease, Parkinson's disease, Alzheimer's disease, and ALS;

[0044] The metabolic diseases mentioned include obesity, diabetes, and gout;

[0045] The tissue fibrosis includes liver fibrosis, kidney fibrosis, and pulmonary fibrosis.

[0046] Fourthly, a treatment method is provided. The treatment method comprises administering the above-described chimeric molecule or pharmaceutical composition that enhances mitophagy to a patient who has a disease caused by or related to mitochondrial dysfunction.

[0047] The beneficial effects of this application are:

[0048] This application discloses a PROTAC-based chimeric molecule (Mitophagy-Enhancing Chimera, or MEC) that can effectively enhance mitophagy at low concentrations (10-100 nanomolar concentrations), repair mitochondrial damage, and thus enhance mitochondrial function. It has been validated in various cells and in animals, thus filling a major gap in this field.

[0049] The chimeric molecule of this application can be used to prevent and treat a variety of diseases related to mitochondrial dysfunction, and has extremely wide application value. Attached Figure Description

[0050] Figure 1 (a) is a schematic diagram of the structure of the MEC chimeric molecule of this application; (b) is a schematic diagram of the mechanism of action of the MEC chimeric molecule of this application; (c) is a basic structural diagram of ITCH (human) of this application.

[0051] Figure 2 This is the hydrogen NMR spectrum of MEC4.

[0052] Figure 3 This is the proton NMR spectrum of MEC9.

[0053] Figure 4 This is the proton NMR spectrum of MEC11.

[0054] Figure 5 This demonstrates the repair effects of different MEC molecules on mitochondria. (a) and (b) show the repair effects of different MEC molecules on mitochondria in HeLa cells damaged by CCCP; where (a) is flow cytometry data stained with the JC-1 probe, and (b) is the staining map of the mitosox probe. (c) and (d) show the repair effects of different MEC molecules on mitochondria in HeLa cells damaged by CCCP. 1-42The restorative effects of different MEC molecules on mitochondria in damaged SH-SY5Y cells; where (c) is a TMRE staining map in the SH-SY5Y cell line, and (d) is a statistical graph of TMRE staining in the SH-SY5Y cell line.

[0055] Figure 6 Shown in Aβ 1-42 The restorative effects of different MEC molecules on mitochondria in damaged BV2 cells. (a) shows the TMRE staining pattern in the BV2 cell line, and (b) shows the statistical graph of TMRE staining in the BV2 cell line.

[0056] Figure 7 The effect of MEC4 small molecules on the restoration of mitochondrial membrane potential in CCCP-damaged HEK293T cells under two conditions.

[0057] Figure 8 The effects of MEC4 on the restoration of mitochondrial membrane potential (TMRE and JC-1) in CCCP-damaged HeLa cells are shown. (a) is a staining map of TMRE, and (b) is a flow cytometry plot of JC-1 staining.

[0058] Figure 9 Shown in Aβ 1-42 MEC4 plays a role in restoring mitochondrial membrane potential in induced-damage BV2 cells and has a "hook effect".

[0059] Figure 10 Shown in Aβ 1-42 The restorative effect of MEC4 on mitochondrial membrane potential in induced-damage primary neurons.

[0060] Figure 11 MEC4 was shown to reduce mitochondrial ROS production in CCCP-damaged Hela cells.

[0061] Figure 12 This demonstrates the effect of MEC4 on cell viability in CCCP-damaged HeLa cells.

[0062] Figure 13 The effect of MEC4 on ATP restoration is shown in HeLa cells damaged by CCCP.

[0063] Figure 14 MEC4 is shown to degrade mitochondrial membrane proteins in CCCP-damaged Hela cells.

[0064] Figure 15 It showed inhibitory effects on cytokines IL-6 and TNF-α in LPS-induced neuroinflammatory BV2 cells.

[0065] Figure 16 The mitochondrial membrane potential of MEC4 small molecules is shown in undamaged HeLa cells.

[0066] Figure 17 The MEC4 molecule was shown to have no effect on cell viability in undamaged HeLa cells.

[0067] Figure 18 The MEC4 small molecule was shown to have no effect on cell viability in undamaged BV2, U87, and PC12 cells.

[0068] Figure 19 MEC4 small molecules do not degrade mitochondrial inner membrane protein TIM23 and mitochondrial outer membrane protein TSPO in undamaged HeLa cells.

[0069] Figure 20 The study showed that MEC4 degradation of mitochondrial proteins in CCCP-damaged HeLa cells was dependent on the autophagy pathway rather than the proteasome pathway. (a) shows the effects of autophagy inhibitors CQ, wortmannin, and bafA1 on MEC4 degradation-damaged mitochondria, and (b) shows the effect of the proteasome inhibitor MG132 on MEC4 degradation-damaged mitochondria.

[0070] Figure 21 The effect of MEC4 small molecules on repairing mitochondrial membrane potential in HeLa cells with CCCP damage can be blocked by autophagy inhibitors. (a) shows the effect of autophagy inhibitors CQ, wortmanin, bafA1 and proteasome inhibitor MG132 on MEC4 repairing damaged mitochondria (JC-1 staining), and (b) shows the flow cytometry results of JC-1 probe staining.

[0071] Figure 22 MEC4 was shown to further enhance CCCP-induced autophagy.

[0072] Figure 23 The results showed that MEC4 lost its role in repairing damaged mitochondria in ATG5 knockout HeLa cells, including its ability to affect mitochondrial membrane potential, mitochondrial ROS, and ATP production. (a) shows the immunoblotting verification of the ATG5 knockout cell line; (b) shows the effect of ATG5 knockout on MEC4's ability to restore membrane potential; (c) shows the effect of ATG5 knockout on MEC4's ability to reduce mitochondrial ROS; (d) shows the effect of ATG5 knockout on MEC4's effect on cell viability; and (e) shows the effect of ATG5 knockout on MEC4's ability to produce ATP.

[0073] Figure 24MitoKeima fluorescence changes showed that MEC4 did not induce mitophagy in undamaged HeLa cells, but enhanced mitophagy in CCCP-damaged HeLa cells.

[0074] Figure 25 Using the Mtphagy Dye method, we showed that MEC4 does not induce mitophagy in undamaged HeLa cells, but enhances mitophagy in CCCP-damaged HeLa cells.

[0075] Figure 26 The Thermal Shift Assay showed that MEC4 has the ability to interact with TSPO and ITCH proteins in cells.

[0076] Figure 27 Thermal Shift Assay showed that MEC4 interacts with TSPO and ITCH proteins in cells, while control molecules AK and PLGO do not.

[0077] Figure 28 PLA method was used to show that MEC4 recruits ITCH and TSPO to interact in HeLa cells.

[0078] Figure 29 Endogenous immunoprecipitation experiments showed that ITCH and TSPO had a significant interaction in BV2 cells under the influence of MEC4.

[0079] Figure 30 The method of extracting mitochondria showed that MEC4 enhances mitochondrial K63 ubiquitination in HeLa cells.

[0080] Figure 31 PLA method was used to show that MEC4 enhances mitochondrial K63 ubiquitination in HeLa cells.

[0081] Figure 32 The results showed that MEC4 lost its role in repairing damaged mitochondria in ITCH knockout HeLa cells, including its effects on mitochondrial membrane potential, mitochondrial ROS, ATP production capacity, mitophagy, and cell viability. (a) shows the immunoblotting validation of the ITCH knockout cell line; (b) shows the effect of MEC4 on restoring mitochondrial membrane potential in the ITCH knockout cell line; (c) shows the effect of MEC4 on reducing mitochondrial ROS in the ITCH knockout cell line; (d) shows the effect of MEC4 on cell viability in the ITCH knockout cell line; and (e) shows the effect of MEC4 on ATP production capacity in the ITCH knockout cell line.

[0082] Figure 33The results showed that MEC4 lost its role in repairing damaged mitochondria in TSPO knockout HeLa cells, including its effects on mitochondrial membrane potential, mitochondrial ROS, ATP production capacity, mitophagy, and cell viability. (a) shows the immunoblotting validation of the TSPO knockout cell line; (b) shows the effect of MEC4 on restoring mitochondrial membrane potential in the TSPO knockout cell line; (c) shows the effect of MEC4 on reducing mitochondrial ROS in the TSPO knockout cell line; (d) shows the effect of MEC4 on cell viability in the TSPO knockout cell line; and (e) shows the effect of MEC4 on ATP production capacity in the TSPO knockout cell line.

[0083] Figure 34 The results showed that knocking down P62 in HeLa cells resulted in the loss of MEC4's ability to repair damaged mitochondria, demonstrating that P62 is a key selective autophagy receptor protein for MEC4 to enhance mitophagy.

[0084] Figure 35 The results showed that knocking down FUNDC1 resulted in the loss of MEC4's function in restoring mitochondrial membrane potential, demonstrating that repairing damaged mitochondria depends on FUNDC1.

[0085] Figure 36 Immunoprecipitation results showed that MEC4 treatment in BV2 cells led to the interaction of TSPO with P62 and FUNDC1.

[0086] Figure 37 The transcriptional levels of genes promoting mitochondrial synthesis are shown in CCCP-induced damaged HEK293T and HeLa cells. (a) shows the transcriptional level of MEC4 promoting TFAM in HEK293T cells, (b) shows the transcriptional level of MEC4 promoting TFAM in HeLa cells, and (c) shows the transcriptional level of MEC4 promoting PGC1-α in HeLa cells.

[0087] Figure 38 It showed that it promotes the expression of proteins related to mitochondrial synthesis in CCCP-induced damaged HEK293T cells.

[0088] Figure 39 Behavioral experiments showed that mice treated with MEC4 exhibited normal autonomous exploration activities and no significant differences in anxiety or depression.

[0089] Figure 40 (a) shows the activity trajectory and dwell time on the platform of WT mice and Alzheimer's mice before and after MEC4 treatment and no treatment. (b) shows the statistics of the number of times the mice crossed the platform in the water maze model and the dwell time on the platform before and after MEC4 treatment.

[0090] Figure 41Behavioral experiments showed that MEC4 improved cognitive memory in AD mice.

[0091] Figure 42 ELISA results showed that MEC4 treatment effectively reduced the accumulation of Aβ in the hippocampus, including insoluble Aβ protein. 1-40 ELISA analysis and soluble protein Aβ 1-42 ELISA analysis.

[0092] Figure 43 Immunofluorescence showed that MEC4 enhanced microglia's phagocytosis of Aβ and slowed down Aβ deposition in AD model mice.

[0093] Figure 44 (a) shows the electron microscopy results in AD model mice, which show that MEC4 promotes the clearance of damaged mitochondria. (b) is a statistical graph of the electron microscopy results, showing that MEC4 treatment improves the morphology of mitochondria and clears damaged mitochondria.

[0094] Figure 45 (a) Immunohistochemistry showing that MEC4 improves neuronal cell damage in AD model mice; (b) Nissl body staining showing that MEC4 increases Nissl bodies in AD model mice.

[0095] Figure 46 Unilateral administration of MEC4 small molecules using a stereotaxic device showed that MEC4 restored the mitochondrial membrane potential in AD mice.

[0096] Figure 47 Unilateral administration of MEC4 small molecules via stereotaxic imaging showed that MEC4 enhanced TSPO ubiquitination. Detailed Implementation

[0097] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0098] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the description of this application, unless otherwise stated, terms such as "multiple / a variety" mean two / more than one.

[0099]

Terminology Explanation

[0100] Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.

[0101] As used in this article, when referring to specific numerical values, it means that the value can vary within a range of no more than 5%.

[0102] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed.

[0103] In this application, small molecules refer to molecules with a molecular weight of less than 2000.

[0104] This application provides a chimeric molecule that enhances mitophagy.

[0105] The MEC (Mitophagy-Enhancing Chimera) chimeric molecule disclosed in this application, also known as a MEC small molecule, consists of three parts: a molecule that can bind to the E3 ubiquitin ligase ITCH protein (ITCH Binder), a molecule that can bind to the specific protein TSPO on the outer mitochondrial membrane (OMM(TSPO)binder), and an intermediate linker molecule, as illustrated in the diagram below. Figure 1 (a) It should be noted that, Figure 1 The diagram in (a) is only for differentiation and does not define the molecular structure of each part.

[0106] A chimeric molecule that enhances mitophagy, the structure of which is shown in Formula 1:

[0107] IL-O' (Formula 1)

[0108] Where I represents the ligand that binds to ITCH, L represents the linker chain, and O' represents the ligand for the outer mitochondrial membrane protein.

[0109] The operational mode of MEC is as follows: Figure 1As shown in (b), through the ability of this chimeric molecule to simultaneously bind to ITCH and mitochondrial outer membrane proteins, MEC recruits ITCH to the mitochondrial outer membrane. The E3 ligase activity of ITCH leads to K63 ubiquitination of mitochondrial outer membrane proteins (including TSPO, VDAC, or other mitochondrial outer membrane proteins). Under the premise of K63 ubiquitination of mitochondrial outer membrane proteins induced by MEC4, these proteins are recruited in large numbers to the damaged mitochondrial outer membrane. Subsequently, they cooperate with the selective autophagy receptor protein P62 to recruit autophagosomes. The gradually extending autophagosomes encapsulate the damaged mitochondria, fuse with lysosomes, and then degrade the damaged mitochondria through the action of lysosomal hydrolases. After the outer membrane protein ubiquitination process is completed, the MEC molecule (or the MEC molecule combined with ITCH) can detach from the TSPO target protein and bind to the next TSPO molecule in the next mitochondrial cell, repeating the process and cyclically degrading more damaged mitochondria, thus producing the PROTAC-signature "catalytic" mode effect.

[0110] Figure 1 (c) is a basic structural diagram of ITCH (human) in this application.

[0111] The products used in the embodiments include:

[0112] CCCP (Carbonyl Cyanide m-Chlorophenylhydrazone), structural formula is CCCP is an inhibitor of oxidative phosphorylation (OXPHOS) and a mitochondrial proton carrier uncoupling agent. CCCP inhibits the activation of STING and its downstream signaling molecules TBK1 and IRF3.

[0113] Aβ 1-42 This represents a 1 to 42 amino acid segment of amyloid-beta.

[0114] MG132 (Z-Leu-Leu-Leu-al) is a widely used proteasome inhibitor that effectively blocks the proteolytic activity of the 26S proteasome complex. It is a potent, reversible, and cell-permeable proteasome inhibitor. MG132 is also an autophagy activator, affecting the cellular autophagy process.

[0115] Bafilomycin A1 (BAF-1) is a macrolide antibiotic, a specific and reversible V-ATPase inhibitor, and a late-stage autophagy inhibitor. It blocks the fusion of autophagosomes and lysosomes and inhibits acidification and protein degradation in lysosomes.

[0116] Chloroquine (CQ) is an autophagy inhibitor that disrupts / interrupts autophagosome-lysosome fusion in its initial stages and enhances the antiproliferative effects of chemotherapeutic drugs. CQ and its derivative, Hydroxychloroquine, are the only autophagy inhibitors approved by the FDA for clinical trials. When used as adjunctive options in anticancer clinical trials, they can enhance the body's sensitization to chemotherapy.

[0117] WORT: Wortmannin. Wortmannin (SL-2052) is a potent, irreversible, selective PI3K inhibitor that blocks autophagy and effectively inhibits Polo-like kinase 1 (PlK1) and Plk3.

[0118] In the description of this application, it should be noted that other conditions not specifically specified in the embodiments are performed under conventional conditions. Reagents or instruments whose manufacturers are not specified are all commercially available products, and experiments are typically conducted under conventional conditions or according to the conditions recommended by the manufacturer.

[0119] The present application will be further described below with reference to the embodiments.

[0120] Example 1: Preparation of MEC molecules

[0121] (1) Synthesis of intermediate AK-PEG-JQ1

[0122] Reaction process

[0123]

[0124] Specifically, the following steps are included:

[0125]

[0126] Step 1: Sulfuric acid (H₂SO₄, 3 mL) was added to a mixture of 2,6-dimethoxybenzene-1,4-diol 1 (1 g, 5.9 mmol, 1.0 eq.) and ethyl 3-oxobutyrate 2 (1.15 g, 8.8 mmol, 1.5 eq.), and stirred at 25 °C for 1 hour. The reaction was monitored by LCMS. After the reaction was complete, the mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (EtOAc, 3 times × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, petroleum ether: ethyl acetate = 5:1) to give 6-hydroxy-5,7-dimethoxy-4-methylchroman-2-one 3 (AK-EG2-TSPO-int-2) (380 mg, yield 27%) as a yellow oil. M / z: [M+H] + =237.2.

[0127]

[0128] Step 2: N,N-dimethylformamide (DMF, 5 ml) was added to a mixture of 6-hydroxy-5,7-dimethoxy-4-methylthrom-2-one 3 (380 mg, 1.61 mmol, 1.0 eq.), tert-butyl(2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate 4 (688 mg, 1.93 mmol, 1.2 eq.), and potassium carbonate (K₂CO₃, 445 mg, 3.22 mmol, 2.0 eq.), and the mixture was stirred at 80 °C for 2 hours. The reaction was monitored by LCMS. After the reaction was complete, the mixture was quenched with pure water (30 ml) and extracted with ethyl acetate (EtOAc, 3 times × 30 ml). The combined organic layers were washed with saturated brine (50 ml), dried over anhydrous sodium sulfate (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, petroleum ether:ethyl acetate = 5:1) to give tert-butyl N-[1-(5,7-dimethoxy-4-methyl-2-oxochroman-6-yl)-1,4,7,10-tetraoxadodecane-12-yl]carbamate 5 (380 mg, yield 46%) as a yellow oil. M / z: [M+H] + =512.5.

[0129]

[0130] Step 3: Trifluoroacetic acid (TFA, 1 ml) was added to a solution of tert-butyl N-[1-(5,7-dimethoxy-4-methyl-2-oxochroman-6-yl)-1,4,7,10-tetraoxadododecane-12-yl]carbamate 5 (350 mg, 0.68 mmol, 1.0 eq.) in dichloromethane (CH2Cl2, 3 ml). The mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the mixture was cooled with saturated sodium bicarbonate aqueous solution (aq.sat.NaHCO3, 30 ml) and extracted with dichloromethane (CH2Cl2, 3 times × 20 ml). The combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure to give 6-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)-5,7-dimethoxy-4-methylchroman-2-one 6 (240 mg, yield 85%) as a yellow oil. M / z: [M+H] + =412.4.

[0131]

[0132] Step 4: HATU (50 mg, 0.12 mmol, 1.0 eq.) was added to a solution of 6-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)-5,7-dimethoxy-4-methylsomn-2-one 6 (50 mg, 0.12 mmol, 1.0 eq.), (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazacycloheptane-6-yl)acetic acid 7 (48.7 mg, 0.12 mmol, 1.0 eq.), and diisopropylethylamine (DIPEA, 47 mg, 0.36 mmol, 3.0 eq.) in dichloromethane (CH2Cl2, 5 mL). The mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LCMS. After the reaction was complete, the mixture was cooled with pure water (20 ml) and extracted with dichloromethane (CH2Cl2, 3 times × 20 ml). The combined organic layers were dried over anhydrous sodium sulfate (Na2SO4) and concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (prep-HPLC, containing 0.05% NH3·H2O) to give a white solid (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazacycloheptane-6-yl)-N-(2-(2-(2-(2-((5,7-dimethoxy-4-methyl-2-oxo-2H-chroman-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)acetamide, named AK-PEG-JQ1 (28.0 mg, yield 28%). M / z: [M+H] + =794.2.

[0133] (2) Synthesis of intermediate AK-EG2-TSPO-int1

[0134] Reaction process

[0135]

[0136] Specifically, the following steps are included:

[0137]

[0138] Step 1: Oxaloyl chloride (2.31 g, 18.2 mmol, 3.5 eq.) was added to a solution of 2-phenyl-1H-indole 1 (1 g, 5.2 mmol, 1.0 eq.) in tetrahydrofuran (THF, 20 mL) at 0 °C. The mixture was stirred at 25 °C for 16 h under nitrogen protection. The reaction was monitored by LCMS. After the reaction was complete, methanol (CH3OH, 1 mL) was added dropwise to the mixture at 0 °C. The mixture was then concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, petroleum ether: ethyl acetate = 3:1) to give methyl 2-oxo-2-(2-phenyl-1H-indole-3-yl)acetate 2 (1.2 g, 83% yield) as a brown solid. M / z: [M+H] + =280.0.

[0139]

[0140] Step 2: Lithium hydroxide monohydrate (LiOH·H2O, 300 mg, 7.16 mmol, 4.0 eq.) was added to a solution of methyl 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetate 2 (500 mg, 1.79 mmol, 1.0 eq.) in methanol (CH3OH, 30 mL) and water (H2O, 6 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was added to 1 N hydrochloric acid (HCl, 20 mL) and extracted with ethyl acetate (EtOAc, 30 mL × 3). The combined organic layers were washed with brine (brine, 100 mL), dried over anhydrous sodium sulfate (Na2SO4), and filtered. The filtrate was concentrated under reduced pressure to give 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetic acid 3 (AK-EG2-TSPO-int-1) (470 mg, 85% yield) as a yellow solid. M / z: [M+H] + =266.0.

[0141] (3) Synthesis of MEC 4 (AK-EG4-TSPO)

[0142] Reaction process

[0143]

[0144] Specifically, the following steps are included:

[0145] Compound 1: See the synthesis of AK-PEG-JQ1.

[0146] Compound 2: See the synthesis of AK-EG2-TSPO-int1.

[0147] Step 1: HATU (296 mg, 0.787 mmol, 1.2 eq.) was added to a solution of 6-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)-5,7-dimethoxy-4-methylsomn-2-one 1 (270 mg, 0.656 mmol, 1.0 eq.), oxo(2-phenyl-1H-indol-3-yl)acetic acid 2 (174 mg, 0.656 mmol, 1.0 eq.), and diisopropylethylamine (DIPEA, 252 mg, 1.968 mmol, 3.0 eq.) in dichloromethane (DCM, 20 mL). The mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LCMS. After the reaction was complete, the mixture was cooled with pure water (H2O, 40 mL) and extracted with dichloromethane (CH2Cl2, 3 × 40 mL). The combined organic layers were dried over anhydrous sodium sulfate (Na2SO4) and concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (prep-HPLC, containing 0.05% NH3·H2O) to give a white solid N-[1-(5,7-dimethoxy-4-methyl-2-oxochroman-6-yl)-1,4,7,10-tetraoxadododecane-12-yl]-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (i.e., MEC4, AK-EG4-TSPO) (31 mg, yield 7.2%). M / z: [M+H]+=659.2. 1HNMR (400MHz, DMSO-d6) δ12.35 (s, 1H), 8.50 (t, J = 5.6Hz, 1H), 8.07 (d, J = 7.2Hz, 1H ),7.56(dd,J=6.8,3.2Hz,2H),7.48(dd,J=7.2,2.8Hz,3H),7.30-7.19(m,2H),6.89(s,1H),6.12(s,1H) ,4.04-3.97(m,2H),3.91(s,3H),3.87(s,3H),3.71-3.65(m,2H),3.60-3.56(m,2H),3.56-3.53(m,2H),3.53-3.49(m,2H),3.49-3.44(m,2H),3.33(s,3H),3.23(t,J=6.2Hz,2H),2.91(dd,J=12.0,6.0Hz,2H). The proton NMR spectrum of MEC4 is shown below. Figure 2 .

[0148] (4) Synthesis of MEC 9 (AK-EG2-TSPO)

[0149] Reaction process

[0150]

[0151] Specifically, the following steps are included:

[0152] Compound 5: See the synthesis of AK-EG2-TSPO-int1

[0153]

[0154] Step 1: Potassium carbonate (72 mg, 0.50 mmol, 2.0 eq.) was added to a solution of 6-hydroxy-5,7-dimethoxy-4-methylsomn-2-one 1 (60 mg, 0.25 mmol, 1.0 eq.) and [2-(2-bromoethoxy)ethyl]aminotert-butylcarbamate 2 (82 mg, 0.30 mmol, 1.2 eq.) in N,N-dimethylformamide (DMF, 2 mL), and the mixture was treated at 80 °C for 2 h. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was diluted with ethyl acetate (EtOAc, 20 mL) and washed with brine (brine, 20 mL). The organic layer was dried over anhydrous sodium sulfate (Na₂SO₄) and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, petroleum ether: ethyl acetate = 3:1) to give a yellow oily tert-butyl (2-{2-[(5,7-dimethoxy-4-methyl-2-oxochroman-6-yl)oxy]ethoxy}ethyl)carbamate 6 (74 mg, yield 66%). M / z: [M+H]+ = 424.0.

[0155]

[0156] Step 2: Trifluoroacetic acid (TFA, 1 ml) was added to a solution of tert-butyl(2-{2-[(5,7-dimethoxy-4-methyl-2-oxochroman-6-yl)oxy]ethoxy}ethyl)carbamate 3 (90 mg, 0.212 mmol, 1.0 eq.) in dichloromethane (CH2Cl2, 3 ml). The mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the reaction mixture was cooled with a saturated aqueous solution of sodium bicarbonate (aq.sat.NaHCO3, 30 ml) and extracted with dichloromethane (CH2Cl2, 3 times × 20 ml). The combined organic layers were dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was concentrated under reduced pressure to give a yellow oily substance 6-[2-(2-aminoethoxy)ethoxy]-5,7-dimethoxy-4-methylchroman-2-one 4 (65 mg, 90% yield). M / z: [M+H]+=324.0.

[0157]

[0158] Step 3: HATU (82 mg, 0.22 mmol, 1.2 eq.) was added to a solution of 6-[2-(2-aminoethoxy)ethoxy]-5,7-dimethoxy-4-methylchroman-2-one 4 (60 mg, 0.19 mmol, 1.0 eq.), oxo(2-phenyl-1H-indol-3-yl)acetic acid 5 (59 mg, 0.22 mmol, 1.2 eq.), and diisopropylethylamine (DIPEA, 73 mg, 0.56 mmol, 3.0 eq.) in dichloromethane (CH2Cl2, 5 mL). The mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was cooled with pure water (H2O, 20 mL) and extracted with dichloromethane (CH2Cl2, 3 times × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate (Na2SO4) and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (prep-HPLC, containing 0.05% NH3) to give a yellow solid N-(2-{2-[(5,7-dimethoxy-4-methyl-2-oxochroman-6-yl)oxy]ethoxy}ethyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (i.e. MEC9, AK-EG2-TSPO) (29.2 mg, yield 26.7%). M / z: [M+H]+=571.2.1H NMR (400MHz, DMSO-d6) δ12.35 (s, 1H), 8.50 (t, J = 5.6Hz, 1H), 8.08 (d, J = 7. 2Hz,1H),7.59-7.55(m,2H),7.51-7.45(m,4H),7.29-7.20(m,2H),6.89(s, 1H),6.12(d,J=1.2Hz,1H),4.03-3.99(m,2H),3.90(s,3H),3.87(s,3H),3 .68-3.63(m,2H),3.29(s,2H),2.95(dd,J=12.0,6.0Hz,2H),2.49(s,3H). The proton NMR spectrum of MEC9 is shown below. Figure 3 .

[0159] (5) Synthesis of MEC 11 (AK2-TSPO)

[0160] Reaction process

[0161]

[0162] Specifically, the following steps are included:

[0163] Compound 5: See the synthesis of AK-EG2-TSPO-int1

[0164]

[0165] Step 1: Under nitrogen protection, 6-hydroxy-5,7-dimethoxy-4-methyl-2H-chroman-2-one 1 (340 mg, 1.44 mmol, 1.0 eq.) was stirred in N,N-dimethylformamide (DMF, 5 mL), followed by the addition of potassium carbonate (497 mg, 3.60 mmol, 2.5 eq.) and tert-butyl(2-iodoethyl)carbamate 2 (470 mg, 1.73 mmol, 1.2 eq.). The reaction mixture was stirred at 60 °C for 16 hours. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was added to water (10 mL) and extracted with ethyl acetate (EtOAc, 10 mL × 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give a colorless oily tert-butyl (2-((5,7-dimethoxy-4-methyl-2-oxo-2H-chroman-6-yl)oxy)ethyl)carbamate 3 (86 mg, yield 15%). M / z: [M+H]+ = 380.2.

[0166]

[0167] Step 2: Under nitrogen atmosphere, a solution of tert-butyl(2-((5,7-dimethoxy-4-methyl-2-oxo-2H-chroman-6-yl)oxy)ethyl)carbamate 3 (86 mg, 0.23 mmol, 1.0 eq.) in dichloromethane (CH2Cl2, 2 ml) was stirred, and then hydrochloric acid / 1,4-dioxane (HCl / dioxane, 5 ml) was added. The reaction mixture was stirred at 25 °C for 2 hours. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to give a white solid 6-(2-aminoethoxy)-5,7-dimethoxy-4-methyl-2H-chroman-2-one 4 (70 mg, 98% yield). M / z: [M+H]+ = 280.1.

[0168]

[0169] Step 3: In a solution of 5-oxo-2-(2-phenyl-1H-indol-3-yl)acetic acid 5 (50 mg, 0.19 mmol, 1.0 eq.) in dichloromethane (CH2Cl2, 5 mL), HATU (86 mg, 0.22 mmol, 1.2 eq.), DIEA (37 mg, 0.28 mmol, 1.5 eq.), and 6-(2-aminoethoxy)-5,7-dimethoxy-4-methyl-2H-chroman-2-one 4 (70 mg, 0.22 mmol, 1.2 eq.) were added. The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LCMS. After the reaction was complete, the reaction mixture was added to water (5 mL) and extracted with dichloromethane (5 mL × 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate (Na2SO4), and filtered. The filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative high performance liquid chromatography (prep-HPLC, containing 0.1% FA) to give a yellow solid N-(2-((5,7-dimethoxy-4-methyl-2-oxo-2H-chroman-6-yl)oxy)ethyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (i.e. MEC11, AK2-TSPO) (27.35 mg, yield 27%). M / z: [M+H]+=527.2. ¹H NMR (400MHz, DMSO-d6) δ 12.40 (s, ¹H), 8.54 (t, J=5.6Hz, ¹H), 8.13–8.07 (m, ¹H), 7.57–7.53 (m, 2H), 7.50–7.43 (m, 4H), 7.30–7.21 (m, 2H), 6.91 (s, ¹H), 6.13 (d, J=1.2Hz, ¹H), 3.09–3.85 (m, 6H), 3.71 (t, J=6.4Hz, 2H), 3.12 (q, J=6.4Hz, 2H), 2.50 (s, 3H). The ¹H NMR spectrum of MEC11 is shown below. Figure 4 .

[0170] Example 2: Comparison of the ability of different MEC molecules to repair mitochondrial damage (restore mitochondrial membrane potential) in HeLa cells

[0171] In this embodiment, the MEC molecules synthesized in this application and their ability to repair CCCP-induced mitochondrial damage (restore mitochondrial membrane potential) in HeLa cells at a concentration of 10 nanomolar (10 nM) are scored in Table 1. The values ​​are: +++ (strong), ++ (medium), + (weak), - (no).

[0172] The specific steps included: mitochondrial membrane potential was detected using the JC-1 mitochondrial membrane potential detection kit. HeLa cells (cervical cancer cell line) were aliquoted into 24-well plates at a density of 50,000 per well. Different MECs and AUTAC4 were co-treated with 20 μmol of carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 3 hours, followed by staining according to the JC-1 kit instructions. After staining for 30 minutes, the cells were washed with PBS, and finally, images were captured using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). Image analysis was performed using the software included with the Nikon camera. The results are shown in Table 1.

[0173] Table 1. Mitochondrial damage repair capacity of MEC molecules

[0174]

[0175] As shown in Table 1, when ITCH is used as the targeted E3 ligase and EG is used as the linker molecule, the length of the linker molecule significantly affects the repair effect of MEC molecules on damaged mitochondria. EG4 is the best linker, followed by EG2. When there is no linker molecule, the repair ability of MEC molecules is the weakest, but there is still some repair ability.

[0176] Example 3: Comparison of the effects of different MEC molecules on restoring damaged mitochondria

[0177] Changes in mitochondrial membrane potential and mitochondrial ROS are the gold standard for distinguishing between healthy and damaged mitochondria.

[0178] (1) Mitochondrial membrane potential

[0179] The levels of CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) or Aβ in HeLa, SH-SY5Y, and BV2 cells were evaluated using both JC-1 and TMRE methods. 1-42 The effects of different MEC small molecules on the recovery of mitochondrial membrane potential after mitochondrial damage caused by (human β-amyloid protein 1-42) are shown in the following results. Figure 5-6 As shown.

[0180] Specifically, the steps include: staining with the JC-1 probe according to the method in Example 2. After staining with tetramethylrhodamine ethyl ester (TMRE), flow cytometry cells were collected and analyzed using a flow cytometer (BD FACSAria). TMThe detection was performed using a III-cell sorter. Tetramethylrhodamine ethyl ester (TMRE) dye is a membrane-permeable cationic fluorescent probe that specifically recognizes mitochondrial membrane potential, thereby attaching to mitochondria and producing bright fluorescence. At certain concentrations, rhodamine dyes have low toxicity to cells and are therefore widely used to detect mitochondria in animal cells, plant cells, and microorganisms. Cell preparation was consistent with the JC-1 staining method. TMRE was prepared into a 5 mmol stock solution according to the reagent manufacturer's instructions, and the cells were co-incubated with the solution for 20 minutes in the dark. The cells were washed once with PBS, and finally, fluorescence changes between different wells were photographed using a fluorescence microscope (Nikon TI fluorescencemicroscopy; Nikon, TI-DH, Japan). The images were analyzed using the software included with the Nikon camera.

[0181] Figure 5 (a) Figure 5 (c) and (d) Figure 6 Results (a) and (b) show that at 10 nanomolar, MEC4 has the strongest repair effect on damaged mitochondrial membrane potential, MEC9 is in the middle, and MEC11 has the weakest repair ability, but it still has a certain ability to restore membrane potential compared with CCCP.

[0182] (2) Mitochondrial ROS-MitoSOX Red

[0183] MitoSOX Red superoxide indicator is a novel fluorescent dye specifically targeting mitochondria in living cells. Mitosox superoxide oxidizes MitoSOX reagent, producing bright red fluorescence. The effect of MEC (microorganisms) incubation on mitochondrial ROS in CCCP-damaged HeLa cells was assessed using Mitosox probes. The specific steps included: HeLa cells were seeded at a density of 50,000 per well in 24-well plates and cultured overnight. Afterward, different MEC molecules were treated for 3 hours, followed by staining with MitoSOX Red (superoxide indicator) in the dark for 20 minutes according to the manufacturer's instructions. The cells were washed once with PBS and photographed using a fluorescence microscope.

[0184] like Figure 5 As shown in (b), the results indicate that in damaged HeLa cells, MEC4 had the strongest effect in reducing mitochondrial ROS, followed by MEC9, while MEC11 had a relatively weaker ability to reduce ROS compared to MEC4 and MEC9.

[0185] Example 4: The role of MEC4 in repairing damaged mitochondria in various cell types.

[0186] 1. MEC4 repairs mitochondrial damage, enhances mitochondrial function, and inhibits cell death in various damaged cells.

[0187] 1.1 Mitochondrial membrane potential

[0188] (1) HEK293T cells

[0189] The TMRE probe was used to assess the changes in mitochondrial membrane potential in human embryonic kidney cells (HEK293T) after 30 min of pretreatment with CCCP (20 μM) and 30 min of cotreatment with different concentrations of MEC4 small molecules.

[0190] The specific steps include: seeding HEK293T cells at a density of 100,000 per well in a 24-well plate, culturing overnight, or pretreating with CCCP for 30 minutes followed by MEC4 treatment for 6 hours, or co-incubating different concentrations of MEC4 with CCCP for 6 hours, and then performing TMRE staining according to the HeLa cell procedure in Example 3. The results are as follows... Figure 7 As shown, under both treatments, MEC4 can restore mitochondrial membrane potential well at 10 nM and 100 nM, and the restoration of mitochondrial membrane potential has a "hook effect".

[0191] (2) HeLa cells

[0192] The effects of different concentrations of MEC4 on mitochondrial membrane potential in CCCP-damaged HeLa cells were evaluated using both JC-1 and TMRE methods. The specific steps included: Mitochondrial membrane potential was detected using the JC-1 mitochondrial membrane potential assay kit. HeLa cells (cervical cancer cell line) were aliquoted into 24-well plates at a density of 50,000 per well. After co-treatment with CCCP (20 μM) and different concentrations of MEC4 for 3 hours, staining was performed according to the JC-1 kit instructions. After 30 minutes of staining, the cells were washed with PBS, and finally, JC-1 staining was analyzed using flow cytometry. TMRE detection was performed using HEK293T cells. Results are as follows: Figure 8 As shown in (a) and (b), MEC4 can restore mitochondrial membrane potential well at both 10 and 100 nM, and the restoration of mitochondrial membrane potential has a "hook effect".

[0193] (3) BV2 cells (mouse microglia)

[0194] The TMRE probe was used to stain mitochondrial membrane potential to evaluate the effects of different concentrations of MEC4 on co-treated Aβ in BV2 cells. 1-42 The repair effect of (10 μM) treatment on mitochondrial damage caused by 24 h specifically includes the following steps: referring to the above method, replace CCCP with Aβ. 1-42 (10μM) was used to stain and photograph BV2 cells after the reaction.

[0195] like Figure 9 The results all show that MEC4 can restore the electrical potential of damaged mitochondrial membranes, with the best effect at 100 nM and a "hook effect".

[0196] (4) Primary neurons

[0197] Preparation of primary neurons: Pregnant mice at day 18 (e18) of embryonic development (newborn mice at p0-p1 can be used for mouse cell culture, anesthetized on ice) were anesthetized with ether or isoflurane gas mixture. The fetus was quickly removed via cesarean section on ice, and its head was cut off and placed in ice-cold culture medium. Skin and skull tissue were removed, and the brain, connected to the cerebellum, was removed and placed in another small dish containing ice-cold culture medium. The dish was placed on ice, and the brain was divided in half, severing at the ventral base where it connects to the cerebellum to facilitate exposure of the hippocampus. The meninges were removed, and the hippocampus was cut out with small scissors. Due to the large number of cortical cells, the cortical tissue near the cerebellum could be removed at this stage. The tissue was minced with small scissors. The culture medium containing the tissue fragments was transferred to a 5 μL centrifuge tube. After standing for a period of time until the tissue fragments settled to the bottom, the supernatant was aspirated (it does not need to be completely aspirated). In another 5ml centrifuge tube, mix trypsin, DNase, and digestion solution at a volume ratio of 1:1:3. Filter the mixture and add it to the tissue block. Digest at 37°C for 7-8 minutes, not exceeding 15 minutes (digestion time depends on enzyme concentration), mixing every 2 minutes. Meanwhile, prepare a 1:4 mixture of trypsin inhibitor (TI) and dissection medium in a 5ml centrifuge tube (or use 5% FBS + Neurobasal + B27). Add a certain amount of the 5% FBS + Neurobasal + B27 mixture to a pre-coated dish. Remove the digested tissue block from 37°C, centrifuge, or allow it to settle naturally. Aspirate any excess liquid, add the mixture to stop digestion, and incubate at 37°C for 2-3 minutes. The following steps must be performed on ice. After incubation, allow the tissue block to settle to the bottom or centrifuge, aspirate the supernatant, then add DNase and agitate. Centrifuge for 5-6 minutes and aspirate the supernatant. After adding 2-3 ml of culture medium, use a pipette or tube to disperse the tissue fragments. After culturing for 24 hours until the cells adhere, replace the medium with complete culture medium (DMEM / F12 + 2% B27) and culture for 3 days, then observe the neuronal growth.

[0198] The cells were cultured in cytarabine medium (final concentration 2.5 μg / ml, half-replaced) for 3 days to inhibit the growth of glial cells and other cells, obtaining pure cultured primary nerve cells. The whole culture medium was changed every 3 days, with half-replaced each time.

[0199] The mitochondrial membrane potential was detected using JC-1 to evaluate the effects of different concentrations of MEC4 on Aβ. 1-42 The effect of (10 μM) treatment on mitochondrial membrane potential in primary neurons was investigated, specifically including the following steps: Neuronal cells were cultured using a special culture medium. After the cells adhered to the plate, they were digested and divided into 24-well plates at 100,000 cells per well. After overnight incubation, the cells were divided into 6 treatment groups, with each group consisting of an untreated control group (Cont) and a treatment group treated with 10 μM Aβ alone. 1-42 For the 6-hour treatment group, Aβ was used first. 1-42 The combined group (Aβ) was treated with 10, 100 nM, 1 μM, and 10 μM MEC4 for 4 hours after 6 hours of treatment. 1-42 +MEC4), after treating the cells according to the above method, the cells were stained with the JC-1 kit according to the instructions. After staining for 30 minutes, the cells were washed with PBS, and finally the mitochondrial membrane potential of each group of cells was photographed using a fluorescence microscope. Figure 10 As shown, the results indicate that MEC4 has a restorative effect on mitochondrial membrane potential in primary neurons damaged by Aβ1-42, with the best effect observed at 100 nM.

[0200] 1.2 Mitosox ROS

[0201] Mitosox probes were used to stain mitochondrial ROS in HeLa cells. ROS levels were assessed after 3 hours of co-incubation with 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM MEC4 small molecules in 20 μM CCCP. The specific steps included: HeLa cell seeding and small molecule treatment according to the treatment in Example 2, followed by staining according to the MitoSOX instructions. The probes were incubated with the cells for 20 minutes in the dark, washed once with PBS, and photographed under a fluorescence microscope.

[0202] like Figure 11 As shown, the results indicate that MEC4 small molecules inhibit mitochondrial ROS production in HeLa cells in a concentration-dependent manner, with the best effect at 10 nM and 100 nM, while the effect decreases at 10 μM, suggesting that MEC4 small molecules have a "hook" effect in HeLa cells.

[0203] 1.3 Cell proliferation

[0204] The cell viability of MEC4 at concentrations ranging from 1 nM to 1 μM in CCCP-induced damaged HeLa cells after 24 h of incubation was detected using the CCK8 method. The specific steps included: HeLa cells were seeded at a density of 10,000 per well in 96-well plates and cultured overnight. After incubation, the cells were co-incubated with 50 μmol of CCCP and different concentrations of MEC4 for 24 h. Then, 10 μL of CCK8 reagent was added to each well, and the cells were cultured at 37 °C, 5% CO2, and 90% humidity for 2 h. The absorbance was measured at 450 nm using a multi-functional microplate reader (BioTek Synergy H1).

[0205] like Figure 12 As shown, the results indicate that CCCP can significantly induce cell death. MEC4 small molecules inhibit CCCP-induced cell death in a gradient range of 1-100 nM, while having little effect at 1 μM, reflecting the "hook" effect of MEC4 and its potential toxicity at high concentrations.

[0206] 1.4 ATP production capacity

[0207] The effect of different concentrations of MEC4 on mitochondrial ATP production capacity in CCCP-damaged HeLa cells was evaluated using the following steps: HeLa cells were seeded at a density of 50,000 per well in 24-well plates and cultured overnight. Afterward, the cells were co-incubated with 50 μmol CCCP and different concentrations of MEC4 for 24 hours. The culture supernatant was discarded, and 500 μL of ATP lysis buffer was added to each well. Protein quantification and luminescent detection were performed using a multi-functional microplate reader (BioTek Synergy H1) according to the ATP assay kit (Shanghai Beyotime Biotechnology Co., Ltd. S0026). ATP quantification was performed based on the results. Figure 13 As shown, CCCP can significantly reduce ATP production, while MEC4 molecules significantly restore ATP production capacity at 10 and 100 nM, but have no significant effect at 1 μM.

[0208] 1.5 Degradation of mitochondrial inner membrane protein TIM23 and mitochondrial outer membrane protein TSPO

[0209] Western blotting was used to evaluate the changes in mitochondrial inner and outer membrane protein levels after 6 hours of treatment with different MEC4 molecules in CCCP-damaged HeLa cells. The specific steps included: HeLa cells were aliquoted into 24-well plates at 50,000 cells per well and allowed to adhere overnight. Cells were then treated in three ways: an untreated control group, a control group treated with 20 μM CCCP for 30 minutes followed by washing and replacement with fresh culture medium, and a control group treated with CCCP for 30 minutes followed by treatment with different concentration gradients (1, 10, 100, 1000 nM) of MEC4 for 6 hours. After removing the cell supernatant, 80 μL of sample buffer was added to each well, and cell lysates were collected, boiled at 100°C, and then analyzed by Western blotting to detect the expression of relevant proteins. Figure 14 As shown, the results indicate that treatment with 10 and 100 nM MEC4 small molecules after CCCP treatment can degrade mitochondrial membrane proteins (inner membrane protein TIM23 and outer membrane protein TSPO).

[0210] 1.6 Cytokine Release

[0211] Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the secretion of cytokines IL-6 and TNF-α by MEC4 in lipopolysaccharide (LPS)-treated BV2 cells.

[0212] The specific steps include: Cells were treated in the following groups: untreated cells (Cont), cells treated with 1 μg / ml lipopolysaccharide (LPS) alone (LPS), and cells treated with LPS for 3 hours followed by treatment with different concentrations of MEC4 for 6 hours (LPS+MEC4). Cell culture supernatants were then collected, and the levels of IL-6 and TNF-α in the supernatants were detected according to the instructions of the mouse IL-6 and TNF-α ELISA kit. Finally, absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader, and data analysis was performed using ELISA Calc software.

[0213] The results are as follows Figure 15 As shown, different concentrations of MEC4 in BV2 cells can inhibit LPS-induced release of IL-6 and TNF-α.

[0214] 2. MEC4 small molecules themselves do not cause mitochondrial damage and are non-cytotoxic.

[0215] 2.1 Mitochondrial membrane potential

[0216] Mitochondrial membrane potential was stained in HeLa cells using a TMRE probe. MEC4 and HeLa cells were co-incubated for 6 hours with 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM of MEC4, respectively. The specific experimental procedures were as described in Example 2. The results are as follows: Figure 16 As shown, in undamaged HeLa cells, different concentrations of MEC4 had no significant effect on the membrane potential of HeLa cells.

[0217] 2.2 Cell proliferation

[0218] (1) HeLa cells

[0219] The cell viability of MEC4 at concentrations ranging from 1 nM to 10 μM in undamaged HeLa cells after 24 h of incubation was detected using the CCK8 method. The specific steps included: HeLa cells were seeded at a density of 10,000 per well in 96-well plates and cultured overnight. After incubation, the cells were co-incubated with different concentrations of MEC4 for 24 h. Then, 10 μL of CCK8 reagent was added to each well, and the cells were cultured at 37 °C, 5% CO2, and 90% humidity for 2 h. The absorbance was measured at 450 nm using a multi-functional microplate reader (BioTek Synergy H1).

[0220] like Figure 17 As shown, the results indicate that MEC4 does not affect cell viability in the concentration range of 1 nM to 10 μM, and only at 10 μM does MEC4 exhibit certain cytotoxic effects.

[0221] (2) Microglia (BV2) and neurons (U87, PC12)

[0222] The cell viability of MEC4 at concentrations ranging from 1 nM to 10 μM in undamaged BV2, PC12, and U87 cells after 24 h of incubation was detected using the CCK8 assay. The specific steps included: cells were seeded at a density of 10,000 per well in 96-well plates and cultured overnight. After incubation, the cells were co-incubated with different concentrations of MEC4 for 24 h. Then, 10 μL of CCK8 reagent was added to each well, and the cells were cultured at 37 °C, 5% CO2, and 90% humidity for 2 h. The absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader.

[0223] like Figure 18 As shown, different concentrations of MEC4 from 1 nM to 10 nM did not affect the cell viability of BV2, U87, and PC12 cells.

[0224] 2.3 MEC4 does not degrade TSPO or induce autophagy in HeLa cells without mitochondrial damage.

[0225] Western blotting was used to evaluate the changes in mitochondrial inner and outer membrane proteins and autophagy levels after 6 hours of treatment with MEC4 in undamaged HeLa cells. The specific steps included: HeLa cells were aliquoted into 24-well plates at a density of 50,000 per well and allowed to adhere overnight. After the cells adhered, they were treated with different concentrations of MEC4 for 6 hours. After removing the cell supernatant, 80 μL Sample Buffer was added to each well, and the cell lysates were collected. The samples were boiled at 100°C and then the expression of relevant proteins was detected by Western blotting.

[0226] The results are as follows Figure 19 As shown, different concentrations of MEC4 molecules do not degrade mitochondrial membrane proteins (inner membrane protein TIM23 and outer membrane protein TSPO) in cells without mitochondrial damage.

[0227] Example 5: MEC4 small molecules repair mitochondrial damage in HeLa cells by enhancing mitophagy.

[0228] 1. MEC4's role in repairing damaged mitochondria is independent of the proteasome pathway but depends on the autophagy pathway.

[0229] 1.1 Immunoblotting

[0230] Western blotting was used to evaluate the effects of the proteasome inhibitor MG132, autophagy inhibitor BAF-1, WORT, and CQ on the outer mitochondrial membrane protein TSPO and the inner mitochondrial membrane protein TIM23 under CCCP induction in HeLa cells. Cells were treated as described above, and cell lysates were collected for Western blotting detection of TSPO and TIM23.

[0231] The results are as follows Figure 20 As shown, (a) shows the effect of autophagy inhibitors CQ, wortmanin, and bafA1 on MEC4 degradation-damaged mitochondria, and (b) shows the effect of proteasome inhibitor MG132 on MEC4 degradation-damaged mitochondria.

[0232] Figure 20 The results showed that in CCCP-damaged HeLa, MG132 could not block the effect of MEC4 on degrading mitochondrial membrane proteins, while autophagy inhibitors could block MEC4-induced membrane protein degradation, indicating that the role of MEC4 in repairing damaged mitochondria depends on the autophagy pathway rather than the proteasome pathway.

[0233] 1.2 Mitochondrial membrane potential

[0234] The JC-1 probe was used to stain mitochondrial membrane potential to evaluate the role of MEC4 in restoring mitochondrial membrane potential damaged by CCCP. The specific steps included: HeLa cells were treated according to the method described in Example 2. After co-incubating with MEC4, CCCP, autophagy inhibitor BafA1, proteasome inhibitor MG132, and CQ for 3 hours, staining was performed according to the JC-1 instruction manual. Subsequently, fluorescence microscopy and flow cytometry analysis were performed.

[0235] See results Figure 21 (a) shows the effects of autophagy inhibitors CQ, wortmanin, bafA1, and proteasome inhibitor MG132 on MEC4 repair of damaged mitochondria (JC-1 staining), and (b) shows the flow cytometry results of JC-1 probe staining.

[0236] like Figure 21 As shown in (a) and (b), the results show that the autophagy inhibitor BafA1 significantly inhibited the role of MEC4 small molecules in repairing mitochondrial membrane potential in CCCP-damaged HeLa cells, while the proteasome inhibitor MG132 could not, demonstrating that the role of MEC4 small molecules in restoring mitochondrial membrane potential depends on autophagy rather than the proteasome pathway.

[0237] 2. MEC4 further enhances CCCP-induced autophagy.

[0238] HeLa cells were treated according to the method in Example 4, 1.5, except that different antibodies were incubated, specifically LC3 antibody, overnight at 4°C.

[0239] like Figure 22 As shown, Western blot results indicated that 10 nM and 100 nM MEC4 in HeLa cells further enhanced the LC3 type I to type II conversion induced by 30 minutes of CCCP treatment, suggesting that MEC1 further enhanced CCCP-induced autophagy.

[0240] 3. Assess whether the role of MEC4 in repairing damaged mitochondria depends on autophagy.

[0241] To further evaluate the role of MEC4 in repairing damaged mitochondria, which depends on autophagy, we constructed HeLa cells with ATG5 (a gene essential for autophagy) knocked out, such as... Figure 23 As shown in (a), Western blotting confirmed that complete knockout of the ATG5 gene resulted in the absence of ATG5 protein production.

[0242] (1) JC-1 and Mitosox probes were used to stain mitochondrial membrane potential and mitochondrial ROS, respectively, to evaluate the role of MEC4 in repairing CCCP-damaged mitochondria after ATG5 knockout. The specific steps included: HeLa cells were treated according to the method described in Example 2; after co-incubation with MEC4 and CCCP for 3 hours, staining was performed according to the instructions for JC-1 and Mitosox. Figure 23 As shown in (b) and (c), the results show that CCCP significantly damaged mitochondria in ATG5 knockout cells, leading to a decrease in mitochondrial membrane potential and an increase in mitochondrial reactive oxygen species levels, while MEC4 had no restorative effect, proving that the role of MEC4 in repairing mitochondrial damage depends on autophagy.

[0243] (2) The effect of MEC4 on cell viability and mitochondrial ATP production capacity after CCCP damage in ATG5 knockout HeLa cells was evaluated. The specific steps included: ATG5 knockout HeLa cells were seeded at a density of 50,000 per well in 24-well plates and cultured overnight. Afterward, the cells were co-incubated with 50 μM CCCP and 10 nM MEC4 for 24 hours. Cell viability was determined according to the experimental procedure in 1.3 "Cell Proliferation" of Example 4, while mitochondrial ATP production capacity was determined according to the procedure in 1.4 "ATP Production Capacity" of Example 4. In short, after discarding the culture supernatant, 500 μL of ATP detection lysis buffer was added to each well. Protein quantification and luminescence detection were performed using a multi-functional microplate reader (BioTek Synergy H1) according to the method of the ATP detection kit (Shanghai Beyotime Biotechnology Co., Ltd. S0026). ATP quantification was performed based on the detection results.

[0244] The results are as follows Figure 23 As shown in (d) and (e), CCCP significantly reduced ATP levels in ATG5 knockout HeLa cells, while MEC4 molecules could not restore ATP production capacity at 1-1000 nM and had no further promoting effect on cell proliferation, further demonstrating that the effect of MEC4 molecules in restoring ATP production capacity depends on autophagy.

[0245] 4. MEC4 enhances mitophagy in damaged cells.

[0246] Keima proteins exhibit different fluorescence signals in acidic and neutral pH environments. Therefore, Keima proteins located in mitochondria (also referred to as mitoKeima) can visualize the degree of mitochondrial autophagy by showing mitochondria entering lysosomes via the autophagy pathway.

[0247] MitoKeima was stably expressed in HeLa cells overexpressing parkin. Mitophagy was induced by CCCP treatment, and changes in fluorescence signals excited by the 440nm channel (neutral pH) and 586nm channel (acidic pH) were observed. The specific steps included: HeLa cells overexpressing Parkin and mitoKeima proteins were seeded at a density of 50,000 per well in 24-well plates. The cells were treated with CCCP for 3 hours or treated with 10 nM MEC4 for 6 hours without treatment. No staining was required. The changes in fluorescence under the 440nm and 586nm channels were observed directly under a fluorescence microscope, and the results were analyzed using software.

[0248] like Figure 24 As shown, the results indicate that the addition of MEC4 after CCCP treatment enhanced the red fluorescence in the 586nm channel, suggesting that MEC4 enhances CCCP-induced mitophagy.

[0249] Furthermore, Mtphagy Dye, through chemical binding, is fixed to mitochondria within cells and emits weak fluorescence. When mitochondria undergo autophagy, damaged mitochondria fuse with lysosomes, causing the pH to drop and become acidic, at which point Mtphagy Dye produces stronger fluorescence. In HeLa cells overexpressing GFP-parkin, after incubation with the Mtphagy Dye probe for 1 hour, the probe was washed away, followed by CCCP pretreatment for 30 minutes, and finally, 10 nM MEC4 molecules were added. The specific steps included: HeLa cells overexpressing GFP-parkin were seeded at a density of 50,000 per well in 24-well plates and cultured overnight. The Mtphagy Dye probe was then loaded into the cells and incubated for 1 hour, washed away, and then treated with 10 nM MEC4 molecules for 6 hours. No further staining was required. Changes in red fluorescence were observed under a fluorescence microscope and analyzed using software.

[0250] like Figure 25 As shown, the results indicate that MEC4 does not induce mitophagy in undamaged HeLa cells, but enhances mitophagy levels in a dose-dependent manner in CCCP-damaged HeLa cells.

[0251] Example 6: Verification of the mechanism by which MEC4 small molecules repair damaged mitochondria by enhancing mitophagy

[0252] 1. MEC4 combines TSPO and ITCH respectively.

[0253] CETSA (Cellular Thermal Shift Assay) is an experiment to detect the efficiency of intracellular drug binding to target proteins. Its principle is that target proteins often have a protective effect when binding to drug molecules. Specifically, as temperature increases, proteins denature and aggregate, which can be removed by centrifugation. However, when proteins bind to drugs, the amount of denatured protein decreases at the same temperature.

[0254] Specifically, the following steps are included: Based on this principle, the extract of HeLa cells is divided into 5 equal parts, and MEC4 small molecules of different concentrations are incubated with the cell extract on ice for 1 hour, then incubated at 55°C for 5 minutes, followed by centrifugation at 20,000g for 30 minutes, and the supernatant is collected for immunoblotting detection.

[0255] like Figure 26 As shown, the results indicate that MEC4 effectively protects against the degradation of ITCH and TSPO at 10 nM, demonstrating that MEC4 has the ability to interact with TSPO and ITCH proteins in cells.

[0256] The same method is as follows Figure 27 As shown, the addition of MEC4 small molecules protected the degradation of TSPO and ITCH proteins in cells, while the control molecules AK and PLGO at both ends had no interaction ability.

[0257] 2. The interaction between TSPO and ITCH is induced by MEC4 small molecule treatment.

[0258] 2.1 Adjacency Linkage (PLA)

[0259] In HeLa cells induced with mitochondrial damage by treatment with 10 μM CCCP for 30 min, treatment with 10 nM MEC4 for 60 min was performed separately. Figure 28 As shown, the adjacent linkage technique (PLA) revealed that MEC4 treatment alone resulted in the interaction between ITCH and TSPO. In HeLa cells damaged by CCCP, MEC4 treatment further enhanced the interaction between ITCH and TSPO.

[0260] 2.2 Co-immunoprecipitation technique (CO-IP)

[0261] Simultaneously, Aβ was used in BV2 cells via endogenous co-immunoprecipitation (CO-IP). 1-42 Mitochondrial damage was induced by treatment with 10 nM MEC4 for 3 h. Proteins were collected, immunoprecipitated with TSPO antibody, and then analyzed for ITCH. The results are as follows: Figure 29 This shows that in Aβ 1-42 The protein expression of ITCH in the +MEC4 group was significantly higher than that in the Aβ group. 1-42The results showed that, under the influence of MEC4, ITCH and TSPO had a significant interaction.

[0262] 3. MEC4 enhances mitochondrial K63 ubiquitination.

[0263] 3.1 Isolation of mitochondria and cytoplasm

[0264] Mitochondria were extracted by separating and extracting mitochondria from HeLa cells. The cells were treated with 10 nM MEC4 for 3 h, 10 μM CCCP for 30 min, or 10 μM CCCP for 30 min, washed, and then treated with 10 nM MEC4 for 3 h. The cell pellet was then collected and the mitochondria were extracted.

[0265] like Figure 30 As shown, the results indicate that mitochondrial K63 ubiquitination was significantly enhanced after treatment with MEC4 alone or CCCP alone, while mitochondrial K63 ubiquitination was further enhanced after treatment with MEC4 and CCCP together, indicating that MEC4 enhances mitochondrial K63 ubiquitination.

[0266] 3.2 Adjacency Linkage (PLA)

[0267] Using adjacent-linking (PLA) technology, HeLa cells induced with mitochondrial damage by treatment with 10 μM CCP for 30 min were then treated with 10 nM MEC4 for 30 min. Figure 31 As shown in the PLA, TSPO and K63 interaction was present 30 min after MEC4 treatment alone, while mitochondrial K63 ubiquitination was further enhanced after combined treatment with MEC4 and CCCP.

[0268] 4. MEC4's role in repairing CCCP-damaged mitochondria depends on ITCH.

[0269] ITCH knockout HeLa cells were constructed, and Western blotting (WB) confirmed that ITCH protein was not expressed in ITCH knockout HeLa cells. Figure 32 As shown in (a).

[0270] WT HeLa and ITCH- / -HeLa cells were divided into 24-well plates at a density of 50,000 cells per well. The cells were treated with 20 μM CCCP for 3 h to induce mitochondrial damage, and with 20 M CCCP and 10 nM MEC4 for 3 h. The mitochondrial membrane potential was then stained with JC-1 and photographed using a fluorescence microscope.

[0271] like Figure 32As shown in (b), the results indicate that MEC4 can restore mitochondrial membrane potential damaged by CCCP in WT HeLa cells, while MEC4 cannot repair the loss of mitochondrial membrane potential caused by CCCP damage in ITCH- / -HeLa cells. Figure 32 The assessments of mitochondrial ROS, cell proliferation capacity, and ATP production capacity in (c), (d), and (e) yielded the same conclusion: MEC4 small molecules have the function of repairing damaged mitochondria in WT Hela cells, but this function is lost in MEC4 small molecule ITCH- / -Hela cells.

[0272] 5. MEC4's role in repairing CCCP-damaged mitochondria depends on TSPO.

[0273] TSPO knockout HeLa cells were constructed, and Western blotting showed that TSPO protein was not expressed in TSPO knockout HeLa cells. Figure 33 As shown in (a).

[0274] Mitochondrial damage was induced in WT HeLa and TSPO- / -HeLa cells by treatment with 20 μM CCCP for 3 h and by co-treatment with 20 μM CCCP and 10 nM MEC4 for 3 h. The mitochondrial membrane potential was detected using the JC-I method, which included the following steps: 50,000 WTHela and TSPO- / -HeLa cells were divided into 24-well plates per well. After cell attachment, each cell line was treated with the drugs as described above. After treatment, the mitochondrial membrane potential was stained with JC-I and then photographed using a fluorescence microscope.

[0275] like Figure 33 As shown in (b), the results indicate that MEC4 can effectively restore the mitochondrial membrane potential damaged by CCCP in WT HeLa, but not in TSPO- / -HeLa, demonstrating that the role of MEC4 in repairing damaged mitochondria is TSPO-dependent, which is consistent with expectations.

[0276] Mitosox probes were used to stain mitochondrial ROS to evaluate the role of MEC4 in repairing CCCP-damaged mitochondria after TSPO knockout. The specific steps included: HeLa cells were treated as described above, and after co-incubating MEC4 and CCCP for 3 hours, staining was performed according to the Mitosox instructions. CCK8, ATP production, and mitoKeima were investigated using the methods described earlier.

[0277] like Figure 33As shown in (c), (d), and (e), the results indicate that MEC4 effectively inhibits ROS generated by CCCP treatment in WT HeLa cells but not in TSPO- / -HeLa cells. MEC4 also fails to restore cell viability and ATP production capacity in CCCP-damaged TSPO- / -HeLa cells.

[0278] 6. MEC4's role in repairing CCCP-damaged mitochondria depends on p62.

[0279] The specific steps include: dividing WTHela cells into 24-well plates at 50,000 cells per well, transferring P62 knockdown siRNA into WT Hela using lipo3000, and treating each well for 48 hours as follows: untreated control group (Cont), cells treated with 20 μM CCCP for 3 hours, and cells treated with 20 μM CCCP simultaneously with 1 nM, 10 nM, 100 nM, and 1 μM MEC4 for 3 hours, respectively, followed by mitochondrial membrane potential staining.

[0280] like Figure 34 The results showed that knocking down P62 in HeLa cells resulted in the loss of MEC4's ability to repair damaged mitochondria, demonstrating that P62 is a key selective autophagy receptor protein for MEC4 to enhance mitophagy.

[0281] 7. MEC4's role in repairing CCCP-damaged mitochondria depends on FUNDC1.

[0282] In HeLa cells, FUNDC1, NBR1, NDP52, OPA1, and BNIP3L were knocked down. Specifically, WT HeLa cells were divided into 24-well plates at a density of 50,000 cells per well. The knockdown siRNAs for FUNDC1, NBR1, NDP52, OPA1, and BNIP3L were transferred to WT HeLa cells using a Lipo3000 transfection agent. After 48 hours, each well was treated as follows: untreated control (Cont), cells treated with 20 μM CCCP for 3 hours, and cells treated with both 20 μM CCCP and 10 nM MEC4 for 3 hours. Mitochondrial membrane potential staining was then performed, followed by fluorescence intensity analysis using multicolor flow cytometry. Results are as follows: Figure 35 The results showed that after knockdown, MEC4 lost its function of restoring mitochondrial membrane potential, demonstrating that the repair of damaged mitochondria depends on FUNDC1.

[0283] An immunoprecipitation experiment was conducted in BV2 cells using Aβ. 1-42 Mitochondrial damage was induced by treatment with 10 nM MEC4, and the antibodies used for incubation included P62, FUNDC1, NBR1, NDP52, OPA1, and BNIP3L. Figure 36Immunoprecipitation results showed that p62 and FUNDC1 were present in MEC4 in conjunction with Aβ. 1-42 The expression of TSPO was significantly increased under co-treatment, indicating that MEC4 treatment in BV2 cells led to the interaction of TSPO with P62 and FUNDC1.

[0284] Example 7: MEC4 small molecules repair damaged mitochondria and promote mitochondrial regeneration

[0285] 1. RT-PCR

[0286] PCG1-α and TFAM are both important genes related to mitochondrial synthesis. In HEK293T and HeLa cells treated with CCCP and MEC4, the expression of these genes after 24 hours of treatment was detected by RT-PCR. Results are as follows: Figure 37 As shown, in CCCP-induced damaged HEK293T and HeLa cells, CCCP treatment significantly reduced the mRNA levels of PGC1-α and TFAM. After the addition of MEC4, the mRNA levels of PCG1-α and TFAM increased compared with the CCCP group, indicating that MEC4 promotes the expression of mitochondrial synthesis-related genes at 24h.

[0287] 2. Western Blot

[0288] PCG1-α and TFAM are both important mitochondrial synthesis-related proteins. In HEK293T cells treated with CCCP and MEC4, the expression of these proteins at 1h, 6h, and 24h was detected by Western blotting. Results are as follows: Figure 38 As shown, in CCCP-induced damaged HEK293T cells, the expression of PCG1-α and TFAM proteins was increased after 24 h of MEC4 addition compared with the CCCP group, indicating that MEC4 promotes the expression of mitochondrial synthesis-related proteins after 24 h.

[0289] Example 8: Progress of MEC4 small molecule intervention in Alzheimer's mice by enhancing mitophagy

[0290] 1. Behavioral experiments

[0291] 1) In Alzheimer's disease models, the open field test is a commonly used behavioral experiment to assess the experimental animals' ability to engage in spontaneous activities, exploratory behavior, and adaptability to new environments. Choosing suitable Alzheimer's disease model animals, such as FAD... 4TTransgenic mice are used, and it is essential to ensure that the animals acclimatize to the experimental environment for a period of time before the experiment, generally 1-2 weeks, to reduce the impact of stress on the experimental results. Before the experiment, the animals should be fasted but allowed water for a period of time, usually 4-6 hours, to avoid the influence of food on their activity; however, the fasting period should not be too long to avoid affecting the animals' health. The open space box should be placed in a quiet, well-lit, and temperature-appropriate laboratory (generally 20-25℃). Before the experiment begins, the open space box needs to be cleaned and disinfected to remove any residual odors and contaminants and avoid interfering with the animals' behavior. The animals are gently placed in the center of the open space box, and the video equipment is turned on to record the animals' activities in the open space. The experiment typically lasts 5-15 minutes, and the specific time can be adjusted according to the species of experimental animals and the experimental purpose. During the experiment, the experimenters should avoid making noise or sudden movements to avoid startling the animals.

[0292] Observation indicators: ① Total activity distance: The total activity distance of the animals during the experiment was calculated by analyzing their movement trajectories in the open field. The total activity distance of animals in the Alzheimer's disease model may be significantly reduced, reflecting a decline in their activity capacity. ② Activity time: The time the animals spent in an active state in the open field was recorded. Animals in the Alzheimer's disease model may exhibit a shortened activity time and an increased time spent at rest. ③ Central area dwell time and number of entries: The open field box was divided into a central area and a peripheral area, and the dwell time and number of entries into the central area were analyzed. Normal animals usually exhibit some exploratory behavior in the central area, while animals in the Alzheimer's disease model may reduce their exploration of the central area due to anxiety, fear, or cognitive impairment, resulting in a decrease in both dwell time and number of entries into the central area. ④ Movement speed: The average movement speed of the animals was calculated based on their activity distance and time. The movement speed of animals in the Alzheimer's disease model may be reduced.

[0293] The results are as follows Figure 39 As shown, compared with AD model mice, MEC4-treated mice exhibited normal autonomous exploration activities and no significant differences in anxiety and depression.

[0294] 2) In Alzheimer's disease models, the Morris water maze is a commonly used behavioral experiment, mainly consisting of two parts: a navigation test and a spatial exploration test. ① Navigation test: This tests the animal's learning ability and spatial memory acquisition ability regarding the location of a hidden platform. A circular or square pool is used. For mice, the pool diameter is typically around 80-150 cm; for rats, the diameter is generally around 120-200 cm. The pool is filled with water, the depth of which should ensure that the animal cannot stand and rest by touching the bottom, but also not too deep to increase the risk of drowning. The water temperature is maintained at around 22-25℃ to simulate a more suitable living environment for the animal. A hidden platform is placed underwater in one quadrant of the pool (usually a fixed quadrant). The diameter of the platform depends on the size of the animal; for mice, the platform diameter is generally 5-8 cm, and for rats, it is 10-15 cm. The animal is placed in the pool facing the pool wall from different entry points. Each entry point can represent a different starting position, such as east, west, south, or north. Record the time it takes for the animal to find the underwater platform, i.e., the escape latency period. If the animal fails to find the platform within a specified time (e.g., 60-120 seconds), the experimenter guides it to the platform and allows it to stay there for a period of time (e.g., 10-30 seconds) before conducting the next experiment. Training is conducted multiple times daily, such as 4-6 times per day for mice, for several consecutive days (usually 4-7 days), to observe whether the time it takes to find the platform gradually shortens with increasing training frequency, reflecting an improvement in its learning ability. ② Spatial exploration experiment: This tests the animal's ability to retain the memory of previously learned platform locations and its spatial exploration behavior. This is conducted one day after the completion of the navigational positioning experiment, at which time the hidden platform in the pool is removed. The animal is placed in the water from the quadrant opposite to the original platform's location, and the swimming trajectory, number of times it crosses the original platform location, time spent in the original platform quadrant, total swimming distance, and average swimming speed are recorded within a certain time period (e.g., 60-120 seconds). Normal animals will exhibit more exploratory behavior in the quadrant where the original platform is located, while Alzheimer's disease model animals may show memory loss of the original platform location and reduced exploratory behavior in the original platform quadrant.

[0295] The results are as follows Figure 40 As shown in (a) and (b), the water maze test showed that AD model mice had significantly reduced learning and memory abilities compared to wild-type mice. However, after MEC4 treatment, the learning and memory abilities of AD model mice were significantly improved.

[0296] 3) In a mouse model of Alzheimer's disease, the Y-maze spontaneous alternation behavioral experiment is primarily used to assess the mice's spatial working memory. The Y-maze spontaneous alternation experiment is based on the animal's natural instinct to explore new environments. Normal mice have an instinct to explore new areas and tend to enter maze arms that have been explored less frequently in the Y-maze. However, Alzheimer's mice, due to impaired brain function, may have deficits in spatial working memory, exhibiting a reduction in spontaneous alternation behavior. A Y-maze typically consists of three arms of equal length, with an angle of 120 degrees between each arm. The arm size can be selected according to the size of the mouse; generally, a mouse's Y-maze arm is 28 cm long, 5 cm wide, and 10 cm high. The maze should ideally be light-colored and opaque so that the mouse can clearly distinguish between the different arms. Different geometric shapes can be attached to the arms as visual markers to aid the mouse's spatial orientation. Suitable Alzheimer's model mice should be selected, ensuring they are in good health and free from other diseases or injuries that could affect behavior. Before the experiment, allow the mice to acclimatize to the experimental environment. For example, place the mice in the experimental room beforehand for a period of time to allow them to adapt, reducing stress caused by unfamiliar surroundings. Prepare video equipment to record the mice's behavior in the maze; use 75% alcohol and paper towels to remove odors from the maze after each mouse's experiment to prevent the scent of the previous mouse from affecting the behavior of the next mouse. Place the mice in the Y-maze and allow them to explore freely for 5-10 minutes to familiarize themselves with the maze environment. Do not record data during this stage. Place the mice into the maze from a fixed arm (starting arm) and allow them to explore freely for a period of time, generally 5-10 minutes (the specific time can be determined based on experimental needs and preliminary experimental results). Record the order and number of times the mice enter each arm.

[0297] The results are as follows Figure 41 The results showed that AD model mice had significantly lower cognitive and memory abilities compared to WT wild-type mice, and MEC4 intervention improved the problem of poor cognitive and memory abilities in AD model mice.

[0298] 2. Enzyme-linked immunosorbent assay (ELISA)

[0299] Sacrificing mice and extracting hippocampal tissue. After weighing the tissue, add an appropriate amount of protein extraction reagent and homogenize the tissue thoroughly using a homogenizer or ultrasonic homogenizer to fully release the protein into the extraction solution. Centrifuge the homogenate at a low temperature (e.g., 4°C) at an appropriate speed (e.g., 10,000-15,000 rpm) for a period of time (usually 10-30 minutes) to remove tissue fragments and other impurities. Collect the supernatant, which is the extracted hippocampal tissue protein solution. Protein concentration can be determined using protein quantification methods such as the BCA method for subsequent calculation of Aβ content. (The text then abruptly shifts to a seemingly unrelated topic about mouse Aβ levels.) 1-40 and Aβ 1-42The Aβ content in the supernatant was detected according to the ELISA kit instructions. Finally, the absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader, and the data were analyzed using Elsa Calc software.

[0300] The results are as follows Figure 42 The results showed that MEC4 treatment significantly reduced the content of Aβ in the hippocampus.

[0301] 3. Immunofluorescence

[0302] Mouse hippocampal tissue sections were prepared by sacrificing mice. These sections can be prepared via perfusion fixation (4% paraformaldehyde), cryosectioning, or paraffin sectioning. The section thickness is generally 4-20 μm. The sections were placed in a humidified chamber, and an appropriate amount of blocking solution was added. The sections were incubated at room temperature for a certain time (e.g., 1-2 hours) to block non-specific binding sites on the sections. A diluted primary antibody solution was added, ensuring the sections were completely covered. The dilution ratio of the primary antibody was determined according to the antibody's instructions and preliminary experimental results. A diluted secondary antibody solution was added and incubated at room temperature for a certain time (e.g., 1-2 hours) to allow the secondary antibody to specifically bind to the primary antibody. The dilution ratio of the secondary antibody was also determined according to the instructions and preliminary experimental results. To observe the cell nuclei, a nuclear staining agent, such as DAPI (4',6-diamidinyl-2-phenylindole), could be added after secondary antibody incubation and incubated at room temperature for a few minutes to stain the cell nuclei. The sections were then observed and photographed using a confocal microscope.

[0303] The results are as follows Figure 43 The results showed that MEC4 treatment significantly activated microglia in the hippocampus of mice, which then engulfed Aβ aggregates, ultimately leading to the clearance of Aβ in AD model mice and thus improving the phenotype of the AD model.

[0304] 4. Transmission electron microscopy (TEM)

[0305] For the observation of mitochondria, it is usually necessary to fix, dehydrate, and embed the cells or tissues, and then cut them into thin sections using an ultramicrotome. In this case, mice are sacrificed, and the hippocampus is carefully removed and fixed in electron microscopy fixative.

[0306] The results are as follows Figure 44 (a) and (b) show that in the hippocampal tissue, normal mice have normal mitochondrial morphology, are not shrunken, and have neatly distributed cristae. In contrast, AD model mice have significantly shrunken and smaller mitochondria, and the cristae are difficult to distinguish. After treatment with MEC4, the mitochondrial morphology was restored, and the cristae distribution was visible. Statistical graphs show that MEC4 restored both the size and number of mitochondria.

[0307] 5. Immunohistochemistry

[0308] Mouse hippocampal tissue samples were fixed, dehydrated, and embedded to prepare paraffin sections. Utilizing the principle of specific antigen-antibody binding in immunology, a chemical substance from the tissue or cell is first extracted as an antigen or hapten. This antigen is then used to immunize the animal to obtain a specific antibody, which is then used to detect similar antigenic substances in the tissue or cell. Since antigen-antibody complexes are colorless, histochemical methods must be used to visualize the antigen-antibody binding site, thereby enabling qualitative, localization, or quantitative studies of unknown antigens in the tissue or cell.

[0309] The results are as follows Figure 45 As shown in (a), WT represents the wild-type mouse group, PBS represents AD model mice injected with PBS, and MEC represents AD model mice injected with MEC4.

[0310] Figure 45 (a) The results showed that neurons were lost in AD model mice, while MEC4 treatment significantly increased neuronal expression.

[0311] 6. Nissl body staining

[0312] Nissl staining is named after the German psychiatrist and neuropathologist Franz Nissl. This staining method is primarily used to stain Nissl bodies in the cytoplasm of neurons in paraffin or frozen sections. It utilizes the property of basic dyes to bind to nucleic acids within the cell. Commonly used basic dyes include tar violet (also known as cresol violet or khaki violet), thionine, methylene blue, and toluidine blue. These dyes can bind to RNA or DNA, thus staining Nissl bodies containing a large amount of nucleic acid. Large and numerous Nissl bodies usually indicate strong protein synthesis in nerve cells; conversely, when nerve cells are damaged, the number of Nissl bodies decreases or even disappears. Therefore, Nissl staining can be used to assess the health and functional status of neurons.

[0313] The results are as follows Figure 45 As shown in (b), WT represents the wild-type mouse group, PBS represents AD model mice injected with PBS, and MEC represents AD model mice injected with MEC4.

[0314] Figure 45 (b) Nissl staining results showed that the number of Nissl bodies was significantly reduced in the AD model, while the number of Nissl bodies was significantly increased after MEC4 intervention.

[0315] Example 9: Exploring the in vivo mechanism of MEC4 small molecules intervening in Alzheimer's mice by enhancing mitophagy.

[0316] 1) Using a stereotaxic instrument, WT and AD model mice were administered unilaterally in the CA1 region. The left side received 500 nmol of PBS, and the right side received 1 μg / 500 nmol of MEC4. After 8 h of treatment, the brain tissue was completely removed after cardiac perfusion with PBS. The brain tissue was frozen and then sectioned. Fresh sections were stained with MT-1 (mitochondrial membrane potential) probe, fixed, and then stained with DAPI for nuclear staining. After mounting, the slides were scanned using a scanner.

[0317] The results are as follows Figure 46 The results showed that the membrane potential of the AD model was significantly lower than that of the WT mouse, but the membrane potential was restored after unilateral administration of MEC4 molecules.

[0318] 2) The above frozen sections were subjected to PLA experiments, and the results are as follows: Figure 47 The results showed that unilateral administration of MEC4 induced K63 ubiquitination of TSPO in both WT and AD model mice, further validating the mechanism of action of MEC4 in vivo.

[0319] The advantages of MEC4 (which recruits ITCH as an E3 ligase) compared to MEC1 (which recruits MAP3K1 as an E3 ligase) in this application are:

[0320] (1) The optimal concentration of MEC4 is lower (10 nM, while MEC1 requires 100 nM).

[0321] (2) MEC4 has a smaller molecular weight than MEC1, higher solubility, and better drug-like properties.

[0322] (3) MEC4 has a better ability to penetrate the blood-brain barrier than MEC1, and has great potential for treating neurological diseases.

[0323] (4) The small molecule AK-087 that binds to ITCH at one end of MEC4 has the ability to enhance the activity of ITCH E3 ligase (while the small molecule IKAM-1 that binds to MAP3K1 does not have this ability), making it more suitable for the functional requirements of MEC.

[0324] (5) The molecular weight of ITCH (103 kDa) is much smaller than that of MAP3K1 (196 kDa), and unlike MAP3K1, it does not possess both E3 ligase and protein kinase activities. Therefore, the impact of "hijacking" ITCH on cells is likely less than that of "hijacking" MAP3K1, and theoretically, its biosafety is better. This is because MAP3K1, in addition to being a serine / threonine kinase, also has E3 ubiquitin ligase activity, which complicates its role in cells. The choice of ITCH in this application can greatly reduce this risk.

[0325] (6) The expression levels of ITCH and MAP3K1 vary greatly in different tissues and cells, and each has its most suitable application scenario.

[0326] The chimeric molecule described in this application, as an innovative biomedical tool, has demonstrated extremely broad application value in the prevention and treatment of various diseases associated with mitochondrial dysfunction. Mitochondrial dysfunction is the pathological basis of many diseases, including neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), cardiovascular diseases, metabolic diseases (such as diabetes), muscle diseases, and certain genetic diseases. Through its unique structure and mechanism of action, this chimeric molecule can effectively intervene in key processes such as mitochondrial energy metabolism, oxidative stress response, and apoptosis, thus providing new ideas and methods for the treatment of these diseases. Its high efficiency and safety demonstrated in preclinical studies indicate its enormous potential in the future medical field, and it is expected to make significant contributions to improving patients' quality of life and extending their lifespan.

[0327] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A chimeric molecule that enhances mitophagy, characterized in that, The structure of the chimeric molecule is shown in Formula 1: IL-O' Formula 1 Wherein, I represents the ligand that binds to the E3 protein ubiquitin ligase ITCH, L represents the linker chain, and O' represents the ligand that binds to mitochondrial outer membrane proteins; the mitochondrial outer membrane proteins include translocator proteins (TSPO) and voltage-dependent anion channel proteins (VDAC). The structural formula of I includes: L represents an alkoxy group chain, including: -(CH2CH2O) a -、-(CH2CH2CH2O) b - where a and b are natural numbers greater than or equal to 1.

2. The chimeric molecule according to claim 1, characterized in that, The ligand that binds to the mitochondrial outer membrane protein has any of the following structural formulas: Where X is a halogen.

3. The chimeric molecule according to claim 2, characterized in that, X is selected from at least one of fluorine, chlorine, bromine, and iodine.

4. The chimeric molecule according to any one of claims 1-3, characterized in that, The structural formula of the chimeric molecule is shown in Formula 2: Where n is a natural number from 1 to 10.

5. The chimeric molecule according to claim 4, characterized in that, n is a natural number between 1 and 5.

6. The chimeric molecule according to any one of claims 1-5, characterized in that, The chimeric molecule has any one of the following structural formulas:

7. A pharmaceutical composition, characterized in that, The invention includes the chimeric molecule that enhances mitophagy as described in any one of claims 1-6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carrier is any one or more of the following: buffer, emulsifier, suspending agent, stabilizer, preservative, excipient, filler, coagulant, blending agent, surfactant, dispersant, and defoamer.

9. The use of the chimeric molecule that enhances mitophagy according to any one of claims 1-6, and the pharmaceutical composition according to any one of claims 7-8, in the preparation of a medicament for the prevention and / or treatment of diseases caused by or related to mitochondrial dysfunction.

10. The application according to claim 9, characterized in that, The diseases caused by or related to mitochondrial dysfunction include neurodegenerative diseases, cardiovascular diseases, metabolic diseases, tumors, tissue fibrosis, autoimmune diseases, inflammatory diseases, or diseases related to viral infections. The neurodegenerative diseases mentioned include Alzheimer's disease, Parkinson's disease, Alzheimer's disease, and ALS; The metabolic diseases mentioned include obesity, diabetes, and gout; The tissue fibrosis includes liver fibrosis, kidney fibrosis, and pulmonary fibrosis.

Images