Chimeric molecule for enhancing mitophagy and use thereof

Abstract

A chimeric molecule for enhancing mitophagy, which, at a low concentration, can effectively enhance mitophagy, repair mitochondrial damage, and thus enhance mitochondrial functions, and has been verified in various cells and in animals. The chimeric molecule has wide application values in the prevention or treatment of diseases of mitochondrial dysfunction.

Description

Chimeric molecules for enhancing mitochondrial autophagy and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202311601281.1 filed with the Chinese Patent Office on November 27, 2023, entitled “Chimeric molecules for enhancing mitochondrial autophagy and their applications,” the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the field of medicinal chemistry, and in particular to a chimeric molecule for enhancing mitochondrial autophagy and an application thereof. Background Art

[0004] Mitochondria are one of the most important organelles in cells. They provide energy molecules (ATP) to cells through oxidative phosphorylation and play a key role in multiple cellular biological processes such as cell metabolism, growth, and apoptosis.

[0005] Mitochondria continuously produce free radicals in the process of performing their functions, causing damage to the mitochondria. Factors such as aging and stress aggravate mitochondrial damage and lead to mitochondrial dysfunction.

[0006] Mitochondrial dysfunction promotes the occurrence and development of many major human diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cardiovascular disease, obesity, diabetes, fibrosis of liver, kidney and lung tissues, gout, cancer, etc.

[0007] Mitochondrial autophagy is a normal cellular mechanism for clearing damaged mitochondria. During this process, damaged mitochondria are selectively encapsulated and transported into autophagosomes, where they fuse with lysosomes and are degraded by hydrolases. Mitochondrial autophagy is a cellular protective mechanism that clears excess or dysfunctional mitochondria and maintains the number of healthy mitochondria to balance cellular homeostasis. Accumulating evidence indicates that mitophagy plays an important role in maintaining the health of the mitochondrial network. Abnormal and insufficient mitophagy is closely associated with mitochondrial dysfunction. Enhancing mitophagy levels in cells is expected to provide new approaches and strategies for the treatment of these diseases.

[0008] Several natural product molecules and synthetic small molecules, such as Urolithin-A, UMI-77, Kaempferol, and Rhapontigenin, have been reported to induce mitophagy, enhancing cells' ability to clear damaged mitochondria and demonstrating therapeutic efficacy in animal models of diseases such as Alzheimer's disease. However, these molecules are not highly specific in regulating mitophagy, and while influencing mitophagy, they are likely to affect other signaling pathways or biological processes. Furthermore, the concentrations required to induce mitophagy are typically micromolar or higher, limiting their potential for new drug development. Small molecules that can specifically enhance mitophagy at low concentrations represent an urgent but unmet market need.

[0009] Proteolysis targeting chimeras (PROTACs) are a novel targeted protein degradation technology. Traditional PROTAC molecules work through the proteasome pathway. Due to the limited pore size of the proteasome, they can only degrade smaller targets such as proteins, but cannot degrade organelles including mitochondria. Therefore, traditional PROTAC molecules cannot achieve the purpose of enhancing mitophagy levels.

[0010] Several chimeric small molecules developed based on PROTACs have been reported to utilize the autophagy pathway rather than the proteasome to degrade target proteins, such as ATTEC, AUTAC, and AUTOTAC. ATTEC and AUTOTAC can degrade target proteins and lipid droplets, but there have been no reports of their ability to degrade damaged mitochondria.

[0011] In a paper published in Molecular Cell in 2019 (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 AUTAC4 chimeric small molecules that can degrade damaged mitochondria. AUTAC4 causes K63 ubiquitination in mitochondria, but the mechanism is unclear, and whether it works by recruiting a specific E3 ligase is unknown. In addition, AUTAC4 requires a concentration of 10 micromolar to degrade damaged mitochondria in cells, and its effectiveness in animals has not yet been tested, leaving its clinical application prospects in doubt.

[0012] Mitophagy is currently understood to occur in two modes: ubiquitination-dependent and ubiquitination-independent. The 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 and is degraded upon reaching the outer mitochondrial membrane, preventing it from residing there. However, when mitochondria are damaged, their membrane potential is lost, and Pink1 is retained in the outer mitochondrial membrane. It phosphorylates pre-existing ubiquitin molecules (Ub) near the outer mitochondrial membrane and then binds to Parkin, leading to its phosphorylation. Phosphorylated and activated Parkin induces K63 ubiquitination of numerous proteins on the outer mitochondrial membrane. Through selective autophagy receptors (currently known to include p62, NBR1, OPTN, NDP52, and TAX1BP1), Pink1 simultaneously binds to ubiquitin and LC3, recruiting autophagosomes to damaged mitochondria, driving the downstream processes of mitophagy.

[0013] In addition to Pink1 / Parkin, there are also reports of other 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.

[0014] Therefore, an obvious idea is to design a PROTAC molecule that binds to a mitochondrial outer membrane protein at one end, and its other end recruits a specific E3 ligase to the mitochondrial outer membrane target protein to simulate the effect of Parkin after activation, forcibly leading to the ubiquitination of mitochondrial outer membrane proteins and enhancing mitochondrial autophagy. However, so far, no PROTAC molecule that can effectively degrade damaged mitochondria has been developed. In fact, Japanese scientists attempted this idea in a paper published in Molecular Cell in 2019, synthesizing a chimeric molecule that binds CRBN (one of the most commonly used E3 ligases for PROTAC molecules) at one end and 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 triggered K48 ubiquitination of mito-EGFP-HT, but was unable to degrade damaged mitochondria.

[0015] The technical bottleneck for chimeric small molecules that could potentially enhance mitophagy lies in the E3 ligase. Finding an E3 ligase that can effectively mimic Parkin after activation is crucial. The human body has nearly 600 E3 ligases, offering enormous potential.

[0016] In summary, based on PROTAC design, chimeric small molecules that recruit specific E3 ligases to mitochondria, drive ubiquitination of mitochondrial outer membrane proteins (especially K63 ubiquitination) and thus enhance mitochondrial autophagy are theoretically feasible, but there have been no successful cases yet.

[0017] Summary of the Invention

[0018] In order to overcome the problems existing in the prior art, the present invention provides the following technical solutions:

[0019] The first aspect of the present invention provides a chimeric molecule for enhancing mitochondrial autophagy, the structure of the chimeric molecule is shown in Formula 1: MLO Formula 1

[0020] Among them, M represents the ligand of MAP3K1, L represents the linker chain, and O represents the ligand of the mitochondrial outer membrane protein;

[0021] The structural formula of M includes:

[0022] L is an alkoxy chain, including: -(CH2CH2O) a -、-(CH2CH2CH2O) b -, where a and b are natural numbers greater than or equal to 1.

[0023] Preferably, the mitochondrial outer membrane proteins include TSPO and VDAC.

[0024] Preferably, the structural formula of O includes: Wherein, X is a halogen.

[0025] Preferably, X is selected from at least one of fluorine, chlorine, bromine and iodine.

[0026] Preferably, the chimeric molecule has a structural formula as shown in Formula 2 or Formula 3:

[0027] Where n is a natural number from 1 to 10; X 1 is a halogen;

[0028] Where m is a natural number from 1 to 10; X 2 、X 3 、X 4 All are halogens.

[0029] More preferably, n is a natural number of 1-5.

[0030] More preferably, m is a natural number of 1-5.

[0031] Preferably, the chimeric molecule is selected from:

[0032] The second aspect of the present invention provides a pharmaceutical composition comprising the chimeric molecule for enhancing mitochondrial autophagy as described above, and optional pharmaceutical excipients.

[0033] The third aspect of the present invention provides the use of the chimeric molecule and pharmaceutical composition for enhancing mitochondrial autophagy described above in the preparation of a drug for preventing or treating diseases caused by mitochondrial dysfunction.

[0034] Preferably, the diseases of mitochondrial dysfunction include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cardiovascular disease, obesity, diabetes, cancer, fibrosis of liver, kidney, lung and other tissues, gout, autoimmune or inflammatory diseases, and viral infections.

[0035] The beneficial effects of the present invention include at least:

[0036] This invention discloses for the first time a PROTAC-based chimeric molecule (Mitophagy-Enhancing Chimera, or MEC) that effectively enhances mitophagy at low concentrations (10 to 100 nanomolar), repairing mitochondrial damage and thus enhancing mitochondrial function. This discovery has been validated in a variety of cell types and in animals, filling a significant gap in this field. The chimeric molecule of this invention has broad application value in preventing or treating diseases associated with mitochondrial dysfunction.

[0037] The features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 shows a schematic structural diagram of the MEC chimeric molecule of the present invention.

[0039] FIG2 is a schematic diagram showing the mechanism of action of the MEC chimeric molecule of the present invention.

[0040] FIG3 shows the basic structure of MAP3K1 (human) of the present invention.

[0041] FIG4 shows the restorative effect of different MEC small molecules on mitochondrial membrane potential in CCCP-damaged HeLa cells.

[0042] FIG5 shows the restorative effect of MEC1 and MEC7 chimeric molecules on mitochondrial membrane potential in Aβ-damaged SN4741 cells.

[0043] FIG6 shows the restorative effect of several MEC small molecules on mitochondrial membrane potential in CCCP-damaged SH-SY5Y cells detected by TMRE method.

[0044] FIG7 shows the restoration function of MEC1 and MEC7 on mitochondrial membrane potential in CCCP-damaged SH-SY5Y cells detected by JC-1.

[0045] FIG8 shows the restorative effects of different MEC small molecules and AUTAC4 on mitochondrial membrane potential in angiotensin II-injured vascular smooth muscle cells.

[0046] FIG9 shows that MEC1 small molecules do not affect mitochondrial membrane potential in intact HeLa cells.

[0047] FIG10 shows that MEC1 small molecules do not affect mitochondrial membrane potential in intact SH-SY5Y cells.

[0048] FIG11 shows that MEC1 small molecules do not affect mitochondrial ROS in uninjured HeLa cells.

[0049] FIG12 shows that MEC1 small molecules do not affect ATP production capacity in intact HeLa cells.

[0050] FIG13 shows that MEC1 small molecules do not affect cell viability in intact HeLa cells.

[0051] FIG14 shows the dose-dependency and “Hook” effect of MEC1 small molecule in restoring mitochondrial membrane potential in CCCP-damaged HeLa cells.

[0052] FIG15 shows that MEC1 small molecules restore mitochondrial membrane potential in CCCP- or OA-damaged SN4741 cells in a dose-dependent manner.

[0053] FIG16 shows that MEC1 small molecule restores mitochondrial membrane potential in CCCP-injured SH-SY5Y cells in a dose-dependent manner.

[0054] FIG17 shows the differential effects of MEC1 on mitochondrial membrane potential in uninjured and CCCP-injured AML-12 cells.

[0055] FIG18 shows the dose-dependency and “Hook” effect of MEC1 small molecules in restoring mitochondrial membrane potential in CCCP-injured HEK293T cells.

[0056] FIG19 shows that MEC1 small molecule reduces global ROS and mitochondrial ROS levels in CCCP-injured HeLa cells in a dose-dependent manner.

[0057] FIG20 shows that MEC1 small molecule reduces global ROS levels in CCCP-injured SH-SY5Y cells in a dose-dependent manner.

[0058] FIG21 shows the effect of MEC1 small molecule on reducing mitochondrial ROS levels in Aβ-damaged SN4741 cells.

[0059] FIG22 shows that MEC1 small molecules restore the ability of CCCP-injured HeLa cells to produce ATP in a dose-dependent manner.

[0060] FIG23 shows the effect of MEC1 small molecule on enhancing cell viability in CCCP-injured HeLa cells in a dose-dependent manner and the “Hook” effect.

[0061] FIG24 shows that MEC1 small molecules do not degrade mitochondrial inner and outer membrane proteins nor change autophagy levels in intact HeLa cells.

[0062] FIG25 shows that MEC1 small molecules degrade mitochondrial inner and outer membrane proteins via autophagy rather than proteasome pathway in CCCP-damaged HeLa cells.

[0063] FIG26 shows that MEC1 did not affect mitochondrial protein levels in uninjured SH-SY5Y cells, but degraded mitochondrial inner and outer membrane proteins in CCCP-injured cells in a concentration-dependent manner.

[0064] FIG27 shows that MEC1 further enhances CCCP-induced autophagy in HeLa cells.

[0065] FIG28 shows that the effects of MEC1 small molecules on restoring mitochondrial membrane potential and reducing mitochondrial ROS levels in CCCP-injured Hela cells can be blocked by autophagy inhibitors.

[0066] FIG29 shows that MEC1 loses its function of repairing damaged mitochondria in ATG5 knockout Hela cells, including its effects on mitochondrial membrane potential, global ROS, mitochondrial ROS and ATP production capacity.

[0067] FIG30 shows that MEC1 does not induce mitophagy in uninjured Hela cells, but enhances mitophagy in CCCP-injured Hela cells, as shown by colocalization of mitochondrial and autophagosome fluorescence.

[0068] FIG31 shows that the level of mitophagy in CCCP-injured Hela cells is enhanced in a dose-dependent manner as shown by changes in MitoKeima fluorescence.

[0069] FIG32 shows that MEC1 does not induce mitophagy in uninjured Hela cells using the Mitophagy Dye method, but enhances the mitophagy level in CCCP-injured Hela cells in a dose-dependent manner and exhibits a "Hook" effect.

[0070] FIG33 shows that MEC1 has the ability to interact with TSPO protein in cells through Thermal Shift Assay.

[0071] Figure 34 shows that MEC1 has the ability to interact with Flag-MAP3K1 expressed in cells and exogenously expressed and purified His-MAP3K1-CTD protein through Thermal Shift Assay.

[0072] FIG35 shows that MEC1 can simultaneously interact with expressed and purified GST-TSPO and His-MAP3K1-CTD to form a tripartite complex.

[0073] FIG36 shows the temporal effect of MEC1 recruiting MAP3K1 to TSPO in HeLa cells using PLA.

[0074] FIG37 shows that MEC1 enhances K63 ubiquitination of mitochondria in HeLa cells using mitochondrial extraction.

[0075] FIG38 shows that MEC1 enhances K63 ubiquitination of TSPO and VDAC by exporting HA-tagged ubiquitin and co-immunoprecipitation (co-IP).

[0076] FIG39 shows that MAP3K1 protein is not expressed in MAP3K1 knockout Hela cells.

[0077] FIG40 shows that MEC1 loses its function of repairing damaged mitochondria in MAP3K1 knockout Hela cells, including the loss of its effects on mitochondrial membrane potential, global ROS, mitochondrial ROS and cell viability.

[0078] FIG41 shows that the function of MEC1 in repairing damaged mitochondria is restored after exogenous expression of Flag-tagged MAP3K1 protein in MAP3K1 knockout Hela cells.

[0079] FIG42 shows that the effect of MEC1 on enhancing mitochondrial K63 ubiquitination is lost in MAP3K1 knockout HeLa cells.

[0080] FIG43 shows that the function of MEC1 in degrading TSPO and TIM23 is lost in MAP3K1-knockout HeLa cells under CCCP injury.

[0081] FIG44 shows that there is no TSPO protein expression in TSPO knockout Hela cells.

[0082] FIG45 shows that the effect of MEC1 in restoring damaged mitochondrial membrane potential is lost in TSPO knockout HeLa cells, but the effect of MEC7 is not affected.

[0083] FIG46 shows that MEC1 has no effect on restoring the ATP production capacity of damaged mitochondria and reducing the overall ROS level in TSPO knockout HeLa cells.

[0084] FIG47 shows the exogenous expression of Myc-tagged TSPO protein in TSPO knockout Hela cells.

[0085] Figure 48 shows that the function of MEC1 in repairing damaged mitochondria was restored after exogenous expression of Myc-tagged TSPO protein in TSPO knockout Hela cells.

[0086] Figure 49 shows that knockdown of NBR1 in HeLa cells leads to the loss of the function of MEC1 in repairing damaged mitochondria, demonstrating that NBR1 is a key selective autophagy receptor protein for MEC1-enhanced mitochondrial autophagy.

[0087] Figure 50 shows that Nur77 is efficiently recruited to the mitochondrial outer membrane by MEC1 only when mitochondria are damaged, and Nur77 knockdown leads to the loss of MEC1's function in repairing damaged mitochondria, proving that Nur77 is the key factor that determines MEC1's selective degradation of damaged mitochondria but not normal mitochondria.

[0088] FIG51 shows that the MEC1 small molecule itself does not affect the cell viability of macrophages.

[0089] FIG52 shows that MEC1 small molecule itself does not induce macrophage cell death by detecting LDH release.

[0090] FIG53 shows that the MEC1 small molecule itself has no effect on the basal autophagy level of macrophages.

[0091] FIG54 shows that MEC1 increases mitophagy levels in NLRP3 inflammasome-activated macrophages.

[0092] FIG55 shows that MEC1 inhibits IL-1β release triggered by NLRP3 inflammasome activation in wild-type macrophages in a concentration-dependent manner, but does not affect TNF-α release that is unrelated to NLRP3 inflammasome activation.

[0093] FIG56 shows that MEC1 inhibits LDH release and Gasdermin D cleavage induced by NLRP3 inflammasome activation in wild-type macrophages in a concentration-dependent manner.

[0094] FIG57 shows that MEC1 inhibits IL-1β release, mitochondrial ROS production, and Gasdermin D cleavage induced by NLRP3 inflammasome activation in CAPS mutant macrophages in a concentration-dependent manner.

[0095] FIG58 shows that nigericin-induced NLRP3 inflammasome activation leads to loss of mitochondrial membrane potential in macrophages, while MEC1 restores mitochondrial membrane potential.

[0096] FIG59 shows that nigericin-induced NLRP3 inflammasome activation leads to a significant increase in ROS and mitochondrial ROS levels in macrophages, while MEC1 concentration-dependently reduces the overall ROS and mitochondrial ROS levels in cells.

[0097] FIG60 shows that MEC1 repairs lysosomal damage caused by NLRP3 inflammasome activation in a concentration-dependent manner.

[0098] Figure 61 shows that the effects of MEC1 in inhibiting IL-1β release and degradation of mitochondrial inner and outer membrane proteins in NLRP3 inflammasome-activated macrophages can be blocked by autophagy inhibitors.

[0099] FIG62 shows that the effects of MEC1 on inhibiting IL-1β release and degradation of mitochondrial inner and outer membrane proteins in NLRP3 inflammasome-activated macrophages cannot be blocked by proteasome inhibitors.

[0100] FIG63 shows that the inhibitory effect of MEC1 on the increase in mitochondrial ROS levels and overall ROS levels caused by NLRP3 inflammasome activation can be blocked by autophagy inhibitors.

[0101] FIG64 shows that MEC1 significantly inhibits NLRP3 inflammasome activation in MSU and Alum mouse models.

[0102] FIG65 shows that MEC1 significantly inhibits acute liver injury induced by NLRP3 inflammasome activation in the LPS+D-Gal mouse model.

[0103] FIG66 shows that MEC1 significantly slows down the weight gain of mice in a high-fat-fed obesity model.

[0104] FIG67 shows that high-fat feeding leads to a significant decrease in the oxygen consumption and carbon dioxide production capacity of mice, while MEC1 administration restores the oxygen consumption and carbon dioxide production capacity of high-fat fed mice to a considerable extent.

[0105] FIG68 shows that glucose tolerance of high-fat-fed mice is impaired, while MEC1 administration significantly improves glucose tolerance of high-fat-fed mice.

[0106] FIG69 shows that MEC1 administration significantly reduced the weight of abdominal white fat, epididymal white fat, and brown fat in high-fat-fed mice.

[0107] FIG70 shows that high-fat feeding leads to elevated serum alanine aminotransferase, aspartate aminotransferase, cholesterol, triglyceride, and LDL-C levels in mice, whereas MEC1 administration significantly reduces these indicators.

[0108] Figure 71 shows that vacuoles were found in the liver tissue cells and lipid droplets in the adipose tissue of mice fed a high-fat diet, while vacuoles in the liver cells decreased and lipid droplets in the adipose tissue became smaller after MEC1 administration.

[0109] Figure 72 shows that mitochondrial morphology was abnormal in liver cells and adipocytes of high-fat-fed mice, while mitochondrial morphology was restored after MEC1 treatment.

[0110] Figure 73 shows that the expression levels of lipid synthesis-related genes in high-fat-fed mice were significantly increased by RNA sequencing and gene set enrichment score analysis, while the expression of these genes was downregulated after MEC1 administration.

[0111] Figure 74 shows the expression of mitochondrial autophagy-related genes in the transcriptome sequencing results. A considerable number of mitochondrial autophagy-related genes were upregulated after MEC1 administration. DETAILED DESCRIPTION

[0112] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is 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 present invention are only exemplary descriptions of the specific embodiments of the present invention, intended to be used for explaining the present invention, and do not constitute a limitation of the present invention.

[0113] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and a separate point value, and the separate point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. In the description of this application, unless otherwise stated, the meaning of similar terms such as "multiple / multiple" means two or more.

[0114]

Terminology

[0115] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0116] As used herein, when a specific numerical value is mentioned, it is intended that the numerical value may vary within a range of no more than 5%.

[0117] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed.

[0118] In the present invention, small molecules refer to molecules with a molecular weight of 2000 or less.

[0119] A first aspect of the present invention provides a chimeric molecule that enhances mitophagy.

[0120] The MEC (Mitophagy-Enhancing Chimera) chimeric molecule disclosed in the present invention, also known as a MEC small molecule, consists of three parts: a small molecule that binds to the E3 ligase MAP3K1 (MAP3K1 ligand), a small molecule that binds to a mitochondrial outer membrane protein (OMM ligand), and an intermediate linker molecule (Linker). A schematic diagram is shown in Figure 1. It should be noted that the diagram in Figure 1 is only for distinction and does not limit the molecular structure of each part.

[0121] The structural formula of the chimeric molecule of the present invention is shown in Formula 1: MLO Formula 1

[0122] Among them, M represents the ligand of MAP3K1, L represents the connecting chain, and O represents the ligand of mitochondrial outer membrane protein.

[0123] The mode of action of MEC is shown in Figure 2. Leveraging the chimeric molecule's ability to simultaneously bind to MAP3K1 and mitochondrial outer membrane proteins, MEC recruits MAP3K1 to the mitochondrial outer membrane. Through MAP3K1's E3 ligase activity, MEC leads to K63 ubiquitination of mitochondrial outer membrane proteins (including TSPO, VDAC, and other mitochondrial outer membrane proteins). If mitochondria are intact and membrane potential is normal, Nur77 (an orphan receptor protein primarily localized in the nucleus) is retained in the nucleus and unable to reach the mitochondria. In this situation, K63 ubiquitination of mitochondrial outer membrane proteins does not trigger subsequent mitophagy. However, mitochondrial damage induces Nur77 to export from the nucleus. Under the premise of MEC1-induced K63 ubiquitination of mitochondrial outer membrane proteins, Nur77 is recruited to the damaged mitochondrial outer membrane in large quantities. It then works in conjunction with the selective autophagy receptor protein NBR1 to recruit autophagosomes. The gradually expanding autophagosomes encapsulate the damaged mitochondria, fuse with lysosomes, and then complete the degradation of the damaged mitochondria by the action of hydrolytic enzymes within the lysosomes. After the ubiquitination process of the outer membrane protein is completed, the MEC molecule (or the combination of the MEC molecule and MAP3K1) can dissociate from the TSPO target protein and bind to the TSPO molecule of the next mitochondria to repeat the action, cyclically degrading more damaged mitochondria, thereby producing the PROTAC signature "catalytic" mode effect.

[0124] The MEC molecules disclosed in the present invention have two major characteristics that are crucial. First, although MEC produces K63 ubiquitination on the outer membrane of both damaged and non-damaged mitochondria through the action of MAP3K1, only damaged mitochondria will recruit autophagosomes and lead to degradation, while non-damaged mitochondria will not recruit autophagosomes and will not be degraded. In addition, consistent with the above results, MEC only increases the level of mitophagy in cells with damaged mitochondria, clears damaged mitochondria, and thus repairs mitochondrial function, but does not change the level of mitophagy in cells with undamaged mitochondria and has no degradation effect on normal mitochondria. When human cells are damaged by mitochondria, they will initiate a naturally occurring mitophagy process (such as the widely studied Pink1 / Parkin-mediated mitophagy), and MEC enhances this process by increasing K63 ubiquitination on the outer membrane of damaged mitochondria. As we all know, autophagy (including mitophagy) is a double-edged sword. It may produce beneficial biological effects in the prevention or treatment of diseases, but it may also bring biosafety risks. The two features of increasing the level of mitophagy only in cells with damaged mitochondria and selectively clearing damaged mitochondria give MEC molecules a high degree of biosafety, which greatly contributes to the clinical application of MEC molecules.

[0125] Two other considerations in the design of MEC molecules are also critical. On the one hand, the selected E3 ligase must be able to quickly and efficiently cause ubiquitination of the target protein itself or other mitochondrial outer membrane proteins adjacent to it after being recruited to the target protein on the outer mitochondrial membrane, and preferably K63 ubiquitination, thereby driving subsequent mitophagy mediated by selective autophagy receptor proteins. On the other hand, the purpose of the present invention is to degrade mitochondria rather than target proteins. After being recruited to the target protein on the outer mitochondrial membrane by the MEC molecule, the E3 ligase preferably does not cause the target protein to be rapidly degraded by the proteasome, thereby having enough time to catalyze the ubiquitination of mitochondrial outer membrane proteins including the target protein. In other words, the ideal MEC molecule of the present invention should have the ability to efficiently bind to the target protein and the E3 ligase to form a tripartite, which must be possessed by an excellent PROTAC molecule, and the recruited E3 ligase must be in an activated state to have the ability to catalyze ubiquitination of adjacent proteins, but has defects in driving proteasome-mediated target protein degradation (thus being an atypical PROTAC molecule).

[0126] The key to MEC technology, and also its bottleneck, is identifying the right E3 ligase that can effectively drive mitophagy. Currently, there are no effective predictive tools or methods, requiring experimental verification. Of the 600 E3 ligases found in human cells, a significant number lack suitable binding small molecules for MEC construction, making experimental testing challenging.

[0127] The main breakthrough of the present invention is the creative discovery of a suitable E3 ligase, namely MAP3K1, which can lead to K63 ubiquitination of mitochondrial outer membrane proteins and drive ubiquitination-dependent mitophagy, thereby degrading and damaging mitochondria. The three E3 ligases tested in the present invention (CRBN, APC CDC20 Among the E3 ligases, MAP3K1 and MAP3K2 (P<0.05), only MAP3K1 has the effect of enhancing mitophagy, while the other two E3 ligases are ineffective.

[0128] The basic structure of E3 ligase MAP3K1 is shown in Figure 3. MAP3K1 not only has a ubiquitin ligase domain (433-492), but also has a protein kinase domain (1243-1508), which is its biggest difference compared with many other E3 ligases.

[0129] A key aspect of the present invention is the use of a suitable small molecule that binds to MAP3K1 (corresponding to the M in the structural formula, i.e., a ligand for MAP3K1). Ideally, the small molecule should be highly specific (binding only to MAP3K1 and not to other cellular proteins), possess a high binding constant (relatively high binding constant to MAP3K1), and not inhibit the E3 ligase activity of MAP3K1.

[0130] Small molecules capable of binding to MAP3K1 include IKAM-1, SM1-71, etc., among which IKAM-1 (1-(3-bromo-4-fluorophenyl)-3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)u rea)) is preferred:

[0131] IKAM-1 is an inhibitor of MAP3K1 kinase function. It directly interacts with the C-terminal kinase domain of the MAP3K1 protein, thereby recruiting MAP3K1 to the outer mitochondrial membrane to exert its E3 ligase activity. However, the ability to bind to the MAP3K1 kinase domain or inhibit MAP3K1 kinase activity is not a requirement for the practice of the present invention and should not be construed as a limitation. Small molecules that bind to other MAP3K1 sites, as long as they do not inhibit MAP3K1's E3 ligase activity, are also suitable for the practice of the present invention.

[0132] Accordingly, the structural formula of M includes:

[0133] In some preferred embodiments, X is selected from at least one of fluorine, chlorine, bromine, and iodine.

[0134] The role of linker molecules in MEC is crucial, and the optimization methods and strategies commonly used in PROTAC technology can be used to optimize the linker molecules of MEC.

[0135] In the present invention, alkenyl alcohols are used as linker molecules. Preferably, ethylene glycol (EG) is used as the linker molecule, including EG2, EG4, etc., preferably EG4.

[0136] Accordingly, L in the chimeric molecule is an alkoxy group, including: -(CH2CH2O) a -、-(CH2CH2CH2O) b -, wherein a and b are respectively natural numbers greater than or equal to 1. In some preferred embodiments, a is a natural number from 1 to 10. In some preferred embodiments, b is a natural number from 1 to 10. In some more preferred embodiments, a is a natural number from 1 to 5. In some more preferred embodiments, b is a natural number from 1 to 5.

[0137] The mitochondrial outer membrane proteins of the present invention can be selected from a wide range. Any mitochondrial outer membrane protein that meets the following two conditions can be a target protein for the implementation of the present invention: specific expression in the mitochondrial outer membrane (not expressed elsewhere in the cell) and high abundance in the mitochondrial outer membrane. Preferred mitochondrial outer membrane proteins of the present invention include TSPO and VDAC. Other mitochondrial outer membrane proteins such as TOM20, Miro1, MFN 1 / 2, and Fis1 can also meet the requirements of the present invention.

[0138] In some preferred embodiments, the mitochondrial outer membrane proteins include TSPO and VDAC.

[0139] Correspondingly, in the chimeric molecule, the structure of O can be selected within a wide range, and any structure that has the ability to bind to mitochondrial outer membrane proteins (including TSPO and VDAC) can be used in the practice of the present invention.

[0140] In some preferred embodiments, the ideal small molecule that binds to TSPO or VDAC has a high degree of specificity (binding only to TSPO or VDAC without binding to other proteins in the cell) and has a medium to high binding constant, but the higher the binding ability, the better. In fact, on the premise that the small molecule can efficiently produce MAP3K1-mediated K63 ubiquitination of TSPO or VDAC (and other adjacent mitochondrial outer membrane proteins) after binding to TSPO or VDAC, the relatively low binding ability may have an advantage, making it easier for the MEC small molecule carrying MAP3K1 to fall off the target protein after completing the ubiquitination of the mitochondrial outer membrane protein, and then bind to a new TSPO or VDAC molecule to drive the next round of ubiquitination, thereby realizing the characteristic "catalytic" function of the PROTAC molecule. In addition, the ideal MEC small molecule should not affect the normal biological function of TSPO or VDAC at an effective concentration for enhancing mitochondrial autophagy.

[0141] A variety of small molecules that can bind to TSPO have been reported, including natural TSPO ligands such as cholesterol and porphyrin compounds, various synthetic molecules such as 2-phenylindole-3-glyoxyamides, PK-11195, benzodiazepines, Compounds (such as Ro5-4864 and AHN-086), imidazopyridine compounds (such as alpidem), indole derivatives (such as FGiN-1-27 and SSr180575), pyrrolobenzoxazepines, phenoxyphenyl acetamide derivatives (such as DAA1106 and Pbr28), Isoquinoline carboxamide and Quinoline carboxamide, etc., can be used in the practice of the present invention, among which 2-phenylindole-3-glyoxylamide is preferred:

[0142] Similar to TSPO, a variety of small molecules can be used to bind to VDAC, including VBIT-4, VBIT-12, NSC 15364, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), etc., among which VBIT-4 is preferred:

[0143] Preferably, the structural formula of O includes: Wherein, X is a halogen.

[0144] In some preferred embodiments, X is selected from at least one of fluorine, chlorine, bromine, and iodine.

[0145] In some preferred embodiments, the chimeric molecule of the present invention has a structural formula as shown in Formula 2 or Formula 3:

[0146] Where n is a natural number from 1 to 10; X 1 is a halogen;

[0147] In some more preferred embodiments, X 1 At least one selected from fluorine, chlorine, bromine and iodine. 1 Most preferred is bromine.

[0148] Where m is a natural number from 1 to 10; X 2 、X 3 、X 4 All are halogens.

[0149] In some more preferred embodiments, X 2 、X 3 、X 4 At least one selected from fluorine, chlorine, bromine and iodine.

[0150] In some further preferred embodiments, X 2 is bromine, X 3 is chlorine, X 4 For fluorine.

[0151] In some more preferred embodiments, n is a natural number of 1-5.

[0152] In some more preferred embodiments, m is a natural number of 1-5.

[0153] In some preferred embodiments, the chimeric molecule is selected from the group consisting of:

[0154] It should be noted that the various materials and reagents used in the following preparation methods are commonly used materials and reagents in this field and can be obtained commercially.

[0155] The second aspect of the present invention provides a pharmaceutical composition comprising the chimeric molecule for enhancing mitochondrial autophagy as described above, and optional pharmaceutical excipients.

[0156] In the present invention, "pharmaceutical excipients" include but are not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by relevant government regulatory authorities as acceptable for use by humans or animals such as livestock.

[0157] The MEC molecules or pharmaceutical compositions described in the present invention can drive mitochondrial autophagy to remove damaged mitochondria and thus repair mitochondrial function in a variety of human and animal cells, and are expected to be used for the prevention and treatment of a variety of major diseases related to mitochondrial autophagy dysfunction, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cardiovascular disease, obesity, diabetes, cancer, fibrosis of liver, kidney, lung and other tissues, gout, autoimmune or inflammatory diseases, viral infections, etc.

[0158] In the present invention, "pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to animals (including humans and livestock, etc.). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. The term "pharmaceutical" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compound of the present invention and is relatively non-toxic, that is, the substance can be administered to an individual without causing adverse biological reactions or interacting in an adverse manner with any component contained in the composition.

[0159] The MEC molecules of the present invention can be prepared into various pharmaceutical dosage forms and administered through various modes such as oral administration, injection, transdermal administration, inhalation, and mucosal administration.

[0160] As used herein, the terms "effective amount," "therapeutically effective amount," or "effectively administered amount" refer to an amount of at least one agent or compound sufficient to provide some relief to some degree from one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. For example, a therapeutically effective amount is the amount of a composition comprising a compound disclosed herein that provides a clinically significant relief of symptoms. Techniques such as dose escalation studies can be used to determine the effective amount appropriate for any individual case.

[0161] The MEC molecules of the present invention repair damaged mitochondria in a concentration-dependent manner. In cell-based experiments, effective concentrations range from 100 picomolar to 10 micromolar, with a preferred concentration range of 1 nanomolar to 1 micromolar, and an optimal range of 10 to 200 nanomolar. In animal experiments, effective dosages range from 0.2 μg / kg (animal body weight) to 20 mg / kg, with a preferred concentration range of 2 μg / kg to 5 mg / kg, and an optimal range of 20 μg / kg to 2 mg / kg.

[0162] Similar to many PROTACs used to degrade target proteins, the effect of MEC molecules on repairing damaged mitochondria exhibits a "Hook" effect, that is, the effect of repairing damaged mitochondria in the low concentration range increases with the increase of MEC molecule concentration, but after reaching a certain concentration value (this value is 1 micromolar for MEC1 molecules in cell experiments), the effect of repairing damaged mitochondria instead decreases with the increase of MEC molecule concentration. A reasonable explanation is that MEC molecules need to bind to MAP3K1 and TSPO at the same time to form a tripartite in order to drive mitophagy. The number of MAP3K1 and TSPO protein molecules in cells is limited. When the concentration of MEC molecules is low, the number of MAP3K1 and TSPO molecules is excessive, and most MEC molecules can bind to MAP3K1 and TSPO at the same time. At this time, the increase in MEC molecule concentration will lead to a linear increase in the effect of driving mitophagy. However, when the MEC concentration reaches a certain value, the number of MEC molecules is excessive, resulting in the formation of duplexes that are only bound to MAP3K1 or only bound to TSPO and cannot drive mitophagy, and reducing the possibility of triplet formation. The greater the excess of MEC molecules, the lower the probability of forming an effective triplet. This result suggests that the dosage of MEC molecules will be an important issue in practical applications, and it is necessary to ensure that the dosage of MEC molecules is below the concentration required to produce the "Hook" effect.

[0163] The third aspect of the present invention provides the use of the chimeric molecule and pharmaceutical composition for enhancing mitochondrial autophagy described above in the preparation of a drug for preventing or treating diseases caused by mitochondrial autophagy dysfunction.

[0164] In some preferred embodiments, the diseases caused by mitochondrial autophagy dysfunction include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cardiovascular disease, obesity, diabetes, cancer, fibrosis of liver, kidney, lung and other tissues, gout, autoimmune or inflammatory diseases, and viral infections.

[0165] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0166] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Please note that these are intended to explain rather than limit the present invention.

[0167] Example 1 Preparation of MEC molecules

[0168] (1) Synthesis of intermediate JQ1-PEG4-MKK

[0169] Reaction process

[0170] The specific steps include:

[0171] Step 1: At 0 ° C, NaH (60% in oil) (0.74 g, 18.4 mmol, 2.0 eq.) was added to a solution of 2-bromo-4-nitrophenol 1 (2.0 g, 9.2 mmol, 1.0 eq.) in THF (20 mL) in several portions. The mixture was stirred for 1 hour, then MOMBr (1.72 g, 13.8 mmol, 1.5 eq.) was added, and the mixture was stirred for another 1 hour. The reaction was monitored by LCMS. After completion of the reaction, it was quenched with H2O (20 mL) and extracted with EtOAc (3 × 20 mL), and the combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, PE: EtOAc = 19: 1) to give 2-bromo-1-(methoxymethoxy)-4-nitrobenzene 2 (2.1 g, 87% yield) as a white solid. M / z: [M+H] + =261.97.

[0172] Step 2: A suspension of 2-bromo-1-(methoxymethoxy)-4-nitrobenzene 2 (1.0 g, 3.81 mmol, 1.0 eq.) in EtOH (10 mL) was treated with Raney nickel (23 mg, 0.38 mmol, 0.1 eq.) at 25 ° C. under a H2 atmosphere for 30 minutes. The reaction was monitored by LCMS. After completion of the reaction, it was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=49:1) to give 3-bromo-4-(methoxymethoxy)aniline 3 (400 mg, 45% yield) as a white solid. [M+H] + =232.1.

[0173] Step 3: A round-bottom flask containing 7-nitro-1H-quinoxaline-2-one 4 (1.0 g, 5.2 mmol, 1.0 eq.) and POCl (5 mL) was placed in an oil bath, heated to 120° C., and refluxed for 3 hours. TLC was used to monitor the reaction. After the reaction was complete, ice water (20 mL) was quenched and extracted with EtOAc (3×20 mL). The combined organic layers were dried and concentrated using anhydrous Na SO. The residue was purified by flash chromatography (silica gel, DCM only) to give 2-chloro-7-nitroquinoxaline 5 (1.0 g, 92% yield) as a white solid. 1 HNMR (400MHz, CDCl3) δ9.22 (s, 1H), 8.88 (d, J = 5.2Hz, 1H), 8.59 (dd, J = 8.8, 2.4Hz, 1H), 8.40 (d, J = 9.2Hz, 1H).

[0174] Step 4: A suspension of 2-chloro-7-nitroquinoxaline 5 (1.0 g, 4.8 mmol, 1.0 eq.) and 1-methyl-4-(4,4,4,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole 6 (1.1 g, 5.2 mmol, 1.1 eq.) in 1,4-dioxane (24 mL) and H2O (8 mL) was treated with tetrakis(triphenylphosphine)palladium (280 mg, 0.2 mmol, 0.05 eq.) and potassium carbonate (1.33 g, 9.6 mmol, 2.0 eq.) at 120°C for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, the mixture was diluted with H2O (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, PE:EtOAc=4:1) to give 2-(1-methylpyrazol-4-yl)-7-nitroquinoxaline 7 (1.2 g, yield 98%) as a yellow solid. [M+H] + =256.0.

[0175] Step 5: A suspension of 2-(1-methylpyrazol-4-yl)-7-nitroquinoxaline 7 (1.2 g, 4.7 mmol, 1.0 eq.) in CH OH (30 mL) and THF (5 mL) was treated with Raney nickel (30 mg, 0.4 mmol, 0.1 eq.) at 25° C. under a H 2 atmosphere for 4 hours. The reaction was monitored by LCMS. After completion of the reaction, it was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, PE: EtOAc = 2: 8) to give 3-(1-methylpyrazol-4-yl)quinoxaline-6-amine 8 (0.95 g, 89% yield) as a yellow-green solid. [M+H] + =226.1.

[0176] Step 6: 3-(1-methylpyrazol-4-yl)quinoxalin-6-amine 8 (440 mg, 1.95 mmol,

[0177] A suspension of 1.0 eq.) in EtOAc:THF:H2O (5+5+5 mL) was treated with Na2CO3 (103 mg,

[0178] 0.97 mmol, 0.5 eq.) was treated at 0°C for 5 minutes, and then phenyl chloroformate 9 (337

[0179] mg, 2.15 mmol, 1.1 eq.). The mixture was stirred at 0°C for another hour.

[0180] After the reaction was completed, it was diluted with H2O (10 mL) and washed with EA (20 mL × 3).

[0181] The residue was purified by flash chromatography (silica gel,

[0182] PE: EtOAc = 1: 1) to obtain a light yellow solid phenyl (3- (1-methyl-1H-pyrazol-4-yl) quinone

[0183] oxalin-6-yl)carbamate 10 (420 mg, 68%). [M+H] + =346.1.

[0184] Step 7: A suspension of phenyl (3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl) carbamate 10 (265 mg, 0.77 mmol, 1.0 eq.) in THF (6 mL) was treated with Et3N (116 mg, 1.15 mmol, 1.5 eq.) at 25°C for 5 minutes, followed by the addition of 3-bromo-4- (methoxymethoxy) aniline 3 (196 mg, 0.85 mmol, 1.1 eq.). The mixture was heated to 70°C for 4 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction was concentrated under reduced pressure and the residue was recrystallized from CHCl (5 mL) to afford 3- (3-bromo-4-hydroxyphenyl) -1- [3- (1-methylpyrazol-4-yl) quinoxalin-6-yl] urea 11 (300 mg, 81% yield) as a yellow solid. [M+H] + =483.1.

[0185] Step 8: A suspension of 3-[3-bromo-4-(methoxymethoxy)phenyl]-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea 11 (400 mg, 0.83 mmol, 1.0 eq.) in THF (6 mL) and CH3OH (6 mL) was treated with concentrated hydrochloric acid at 25°C for 24 hours. After completion of the reaction, the reaction was concentrated under reduced pressure to afford 3-(3-bromo-4-hydroxyphenyl)-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea 12 as a yellow solid (350 mg, 96% yield). [M+H] + =439.2.

[0186] Step 9: A solution of 3-(3-bromo-4-hydroxyphenyl)-1-[3-(1-methylpyrazole-4-yl)quinoxaline-6-yl]urea 12 (240 mg, 0.54 mmol, 1.0 eq.) and 12-bromo-4,7,10-trioxa-1-azadodecane-1-ylcarboxylic acid tert-butyl ester 13 (214 mg, 0.60 mmol, 1.1 eq.) in DMF (4 mL) was treated with potassium carbonate (113 mg, 0.82 mmol, 1.5 eq.) at 80 ° C for 6 hours. LCMS was used to monitor the reaction. After completion of the reaction, it was diluted with EtOAc (20 mL) and washed with brine (20 ml). The organic layer was dried over anhydrous Na SO and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=19:1) to give tert-butyl {1-[2-bromo-4-({[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]carbamoyl}amino)phenyl]-1,4,7,10-tetraoxa-13-azatridecan-13-yl}carboxylate 14 (230 mg, 59% yield) as a brown oil. [M+H] + =714.3.

[0187] Step 10: A solution of tert-butyl {1-[2-bromo-4-({[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]carbamoyl}amino)phenyl]-1,4,7,10-tetraoxa-13-azatridecan-13-yl}carboxylate 14 (230 mg, 0.32 mmol, 1.0 eq.) in CHCl (3 mL) was treated with TFA (0.5 mL) at 25°C for 1 hour. The reaction was monitored by LCMS. After completion of the reaction, the mixture was diluted with EtOAc (10 mL) and washed with saturated NaHCO (10 mL). The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give 3-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)-3-bromophenyl]-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea 15 (190 mg, 96% yield) as a brown oil. [M+H]+=614.2.

[0188] Step 11: A solution of 3-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)-3-bromophenyl]-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea 15 (90 mg, 0.15 mmol, 1.0 eq.) and oxo(2-phenyl-1H-indol-3-yl)acetic acid 16 (59 mg, 0.15 mmol, 1.0 eq.) in CHCl (2 mL) was treated with HATU (84 mg, 0.22 mmol, 1.5 eq.) and DIEA (57 mg, 0.44 mmol, 3.0 eq.) at 25° C. for 2 h. The reaction was monitored by LCMS. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=19:1) and preparative high performance liquid chromatography prep-HPLC (0.05% NH3) to give (R)-N-(2-(2-(2-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)ethoxy)ethoxy)ethoxy)ethyl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine as a pale yellow solid. -6-yl)acetamide JQ1-PEG4-MKK (25.2 mg, yield 17%). [M+H] + =996.2. HPLC:t R 3.327min, 100% purity. 1H NMR (400MHz, DMSO-d6) δ9.22(s,1H),9.11(s,1H),8.85(s,1H),8.61(s,1H),8.29(t,J=5.2Hz,1H),8.26(s,1H),8.23(d,J= 2.0Hz,1H),7.92(d,J=9.2Hz,1H),7.85(d,J=2.4Hz,1H),7.67(dd,J=9.2,2.0Hz,1H),7.48(d,J=8.8Hz,2H),7.42(d,J=8.4H z,2H),7.34(dd,J=9.2,2.0Hz,1H),7.10(d,J=8.8Hz,1H),4.54-4.47(m,1H),4.17-4.10(m,2H),3.95(s,3H),3.81-3.74(m, 2H),3.69-3.61(m,2H),3.59-3.52(m,6H),3.46(t,J=5.6Hz,2H),3.31-3.19(m,4H),2.59(s,3H),2.40(s,3H),1.61(s,3H).

[0189] (2) Synthesis of intermediate AP-PEG-JQ1

[0190] Reaction process

[0191] The specific steps include:

[0192] Step 1: To a mixture of (9H-fluorene-9-yl)methyl (3-hydroxypropyl)carbamate 1 (4.5 g, 15.1 mmol, 1.0 eq.) in CH2Cl2 (50 mL) was added TEA (4.6 g, 45.3 mmol, 3.0 eq.) and 4-nitrobenzylchloroformate 2 (3.6 g, 18.1 mmol, 1.2 eq.). The reaction mixture was stirred at 25 ° C for 2.5 hours. The reaction was monitored by LCMS. After the reaction was completed, the reaction mixture was added to water (50 mL) and extracted with CH2Cl2 (20 mL × 3). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (PE:EtOAc=3:1) to give (9H-fluoren-9-yl)methyl(3-(((4-nitrophenoxy)carbonyl)oxy)propyl)carbamate 3 as a white solid (3.6 g, 51% yield). M / z: [M+Na] + =485.1.

[0193] Step 2: To a mixture of (9H-fluoren-9-yl)methyl (3-(((4-nitrophenoxy)carbonyl)oxy)propyl)carbamate 3 (3.6 g, 7.8 mmol, 1.0 eq.) in CH3CN (15 mL) was added NH3·H2O (3.5 mL). The reaction mixture was stirred at 25°C for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was added to water (30 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (CH2Cl2:CH3OH=5:1) to give (9H-fluoren-9-yl)methyl (3-(carbamoyloxy)propyl)carbamate 4 (2.0 g, 76% yield) as a white solid. M / z:[M+H] + =341.1.

[0194] Step 3: To a mixture of (9H-fluoren-9-yl)methyl (3-(carbamoyloxy)propyl)carbamate 4 (2.0 g, 5.9 mmol, 1.0 eq.) in DMA (5 mL) was added 2,2,2-trichloroethane-1,1-diol 5 (9.8 g, 59.0 mmol, 10.0 eq.). The reaction mixture was stirred at 120 ° C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE: EtOAc = 1: 1) to give (9H-fluoren-9-yl)methyl (3-(((2,2,2-trichloro-1-hydroxyethyl)carbamoyl)oxy)propyl)carbamate 6 (3.4 g, 93% yield) as a yellow oil. M / z: [M+Na] + =509.0.

[0195] Step 4: To a mixture of (9H-fluoren-9-yl)methyl(3-(((2,2,2-trichloro-1-hydroxyethyl)carbamoyl)oxy)propyl)carbamate 6 (3.4 g, 6.9 mmol, 1.0 eq.) in CHCl (25 mL) were added 2 drops of pyridine and thionyl chloride (4.7 g, 40.0 mmol, 5.8 eq.). The reaction mixture was stirred at 25°C for 2.5 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give (9H-fluoren-9-yl)methyl(3-(((1,2,2,2-tetrachloroethyl)carbamoyl)oxy)propyl)carbamate 7 (3.4 g, 97% yield) as a yellow oil.

[0196] Step 5: To a mixture of (9H-fluoren-9-yl)methyl (3-(((1,2,2,2-tetrachloroethyl)carbamoyl)oxy)propyl)carbamate 7 (3.4 g, 6.7 mmol, 1.0 eq.) in CHCl (30 mL) was added 2-aminopyrimidine 8 (6.0 g, 63.6 mmol, 9.5 eq.), and the mixture was stirred at 0°C under nitrogen. The reaction mixture was stirred at 25°C for 3 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE:EtOAc=1:1) to give (9H-fluoren-9-yl)methyl (3-(((2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl)carbamoyl)oxy)propyl)carbamate 9 (2.8 g, 74% yield) as a yellow oil. M / z:[M+H] + =564.1.

[0197] Step 6: To a mixture of (9H-fluoren-9-yl)methyl(3-(((2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl)carbamoyl)oxy)propyl)carbamate 9 (2.8 g, 5.0 mmol, 1.0 eq.) in DMF (6 mL) was added morpholine (2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by HPLC (0.1% FA) to give 3-aminopropyl(2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl)carbamate 10 (2.1 g, 94% yield) as a yellow solid. M / z: [M+H] + =342.0.

[0198] Step 7: (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine A mixture of 2-[2-(2-aminoethoxy)ethoxy]ethanol 11 (167 mg, 1.12 mmol, 1.5 eq.) was stirred at 25° C. for 50 minutes. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by FFC (CH2Cl2:CH3OH=10:1) to give (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine as a yellow oil. -6-yl)-N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)acetamide 13 (360 mg, yield 90%). M / z: [M+H] + =532.2.

[0199] Step 8: To (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine To a solution of 4-methylbenzenesulfonyl chloride (258 mg, 1.35 mmol, 2.0 eq.) in CH2Cl2 (5 mL) was added 4-methylbenzenesulfonyl chloride (258 mg, 1.35 mmol, 2.0 eq.). The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by FFC (CH2Cl2:CH3OH=10:1) to give (S)-2-(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine) as a white solid. -6-yl)acetamido)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate 14 (360 mg, yield 77%). M / z: [M+H] + =686.3.

[0200] Step 9: (S)-2-(2-(2-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine A mixture of 3-aminopropyl N-[2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl]carbamate 10 (150 mg, 0.35 mmol, 2.0 eq.) and KCO (72.5 mg, 0.52 mmol, 3.0 eq.) in ACN (5 mL) was stirred at 65 ° C for 24 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by FFC (CH Cl : CH OH = 10: 1) to give a crude product. The crude product was purified by preparative HPLC (0.1% FA) to give 1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2,4]triazolo[4,3-a][1,4]diazepine as a white solid. -6-yl)-2-oxo-6,9-dioxa-3,12-diazapentadecan-15-yl(2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl)carbamate AP-PEG-JQ1 (26.5 mg, 18% yield). M / z: [M+H] + =855.1. 1 H NMR (400MHz, DMSO-d6) δ8.42(d,J=4.8Hz,2H),8.27(t,J=5.6Hz,1H),7.86(d,J=8.8Hz,1H),7.49(d,J =8.8Hz,2H),7.42(d,J=8.4Hz,2H),7.21(d,J=9.6Hz,1H),6.83(t,J=4.8Hz,1H),6.69(t,J=9.2Hz,1H ),4.51(t,J=7.2Hz,1H),4.06(s,2H),3.57-3.52(m,6H),3.46(t,J=6.0Hz,2H),3.27-3.20(m,5H),2. 83(t,J=4.8Hz,2H),2.73(t,J=7.2Hz,2H),2.59(s,3H),2.41(s,3H),1.82-1.73(m,2H),1.62(s,3H).

[0201] (3) Synthesis of intermediate VDAC binding molecules

[0202] Reaction process

[0203] The specific steps include:

[0204] Step 1: To a solution of 1-bromo-4-(trifluoromethoxy)benzene 1 (1.2 g, 5 mmol, 1.0 eq.) in toluene (20 mL) were added tert-butyl piperazine-1-carboxylate 2 (1.87 g, 10 mmol, 2.0 eq.), Xantphos (0.58 g, 1 mol, 0.2 eq.), Cs CO (3.26 g, 10 mmol, 2.0 eq.) and Pd(OAc) (0.11 g, 0.5 mmol, 0.1 eq.). The reaction mixture was stirred at 110° C. under an N atmosphere for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE / EtOAc=10 / 1) to give tert-butyl 4-(4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate 3 (1.4 g, 80% yield) as a yellow solid. M / z:[M+H-56] + =291.1.

[0205] Step 2: A solution of tert-butyl {4-[4-(trifluoromethoxy)phenyl]piperazine-1-carboxylate 3 (1.4 g, 4.01 mol, 1.0 eq.) in dioxane (20 mL) of HCl was stirred at 25 ° C for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in a solution of NH3 in MeOH (50 mL). The mixture was stirred at 25 ° C for 1 hour. The mixture was concentrated under reduced pressure to give 4-(piperazin-1-yl)phenyldifluoromethylhypofluorite 4 (1.0 g, 100% yield) as a yellow solid. M / z: [M+H] + =247.3.

[0206] Step 3: At 0 ° C, to a solution of 5H-furan-2-one 5 (0.67 g, 8 mmol, 1.0 eq.) in DCM (10 mL) was added a solution of 4-(piperazin-1-yl)phenyldifluoromethyl hypofluorite 4 (1.0 g, 4.0 mmol, 0.5 eq.) in DCM (2 mL). The mixture was stirred at 25 ° C for 16 hours. LCMS showed a good reaction. The mixture was concentrated and purified by silica gel column (PE: EA = 1: 1) to give a yellow solid 4-{4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl}oxolane-2-one 6 (0.8 g, 60% yield). M / z: [M+H] + =331.1.

[0207] Step 4: To a solution of 4-{4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl}oxolan-2-one 6 (0.8 g, 2.4 mmol, 1.0 eq.) in toluene (30 mL) was added AlMe3 (4.8 mL, 2 M, 4.0 eq.) and the mixture was stirred at 25 ° C for 10 minutes. 4-Chloroaniline 7 (0.61 g, 4.8 mmol, 2.0 eq.) was added to the solution. The mixture was stirred at 80 ° C for 16 hours. LCMS showed a good reaction. The mixture was concentrated and purified by silica gel column (EA) to give N-(4-chlorophenyl)-4-hydroxy-3-{4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl}butanamide Vdac (0.64 g, 60% yield) as a white solid. M / z: [M+H] + =458.1. 1 H NMR (400MHz, DMSO-d6) δ10.17(s,1H),7.61(d,J=8.8Hz,2H),7.36-7.30(m,2H),7.16(d,J=8.8Hz,2H),6.97(d,J=9.2Hz,2H),4.54(t,J=5.2H z,1H),3.60-3.54(m,1H),3.48-3.42(m,1H),3.15-3.09(m,5H),2.85- 2.77(m,2H),2.75-2.68(m,2H),2.58-2.53(m,1H),2.40-2.34(m,1H).

[0208] (4) Synthesis of MEC 1 (TSPO-PEG4-MKK)

[0209] Reaction process

[0210] The specific steps include:

[0211] Step 1: To a solution of 2-phenyl-1H-indole 1 (1 g, 5.2 mmol, 1.0 eq.) in THF (20 mL) was added oxalyl chloride (2.31 g, 18.2 mol, 3.5 eq.) at 0 ° C. The mixture was stirred at 25 ° C. under N2 for 16 hours. The reaction was monitored by LCMS. After the reaction was completed, 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 flash chromatography (silica gel, PE: EtOAc = 3: 1) to give methyl 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetate 2 (1.2 g, 83% yield) as a brown solid. [M+H] +=280.0

[0212] Step 2: 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 CH OH (30 mL) and H 2 O (6 mL) was added LiOH · H 2 O (300 mg, 7.16 mmol, 4.0 eq.). The mixture was stirred at 25 ° C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was added to 1N HCl (20 mL) and extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure to give 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetic acid 3 (470 mg, 99% yield) as a yellow solid. M / z: [M+H] + = 266.0.

[0213] Compound 4: See the synthesis of JQ1-PEG4-MKK.

[0214] Step 3: A solution of 3-[4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)-3-bromophenyl]-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea 4 (100 mg, 0.16 mmol, 1.0 eq.) and 2-oxo(2-phenyl-1H-indol-3-yl)acetic acid 3 (47 mg, 0.18 mmol, 1.1 eq.) in CHCl (2 mL) was treated with HATU (93 mg, 0.24 mmol, 1.5 eq.) and DIEA (63 mg, 0.49 mmol, 3.0 eq.) at 25° C. for 1 hour. The reaction was monitored by LCMS. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=19:1) and preparative high performance liquid chromatography prep-HPLC (0.05% NH3) to give N-{1-[2-bromo-4-({[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]carbamoyl}amino)phenyl]-1,4,7,10-tetraoxadodec-12-yl}-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (25.4 mg, 18% yield) as a light yellow solid. [M+H] + =861.1. HPLC:t R 1.014min, 97.66% purity. 1H NMR(400MHz,DMSO-d6)δ12.37(s,1H),9.71(s,1H),9.34(s,1H),9.10(s,1H),8.60(s,1H),8.47(s,1H),8.29–8.18(m,2 H),8.07(d,J=7.2Hz,1H),7.91(d,J=9.2Hz,1H),7.86(d,J=2.4Hz,1H),7.71(d,J=8.4Hz,1H),7.60–7.53(m,2H),7.50- 7.41(m,4H),7.38(d,J=8.4Hz,1H),7.29-7.15(m,2H),7.08(d,J=8.8Hz,1H),4.15-4.09(m,2H),3.95(s,3H),3.79-3.7 2(m,2H),3.66-3.60(m,2H),3.57-3.50(m,4H),3.49-3.44(m,2H),3.23(t,J=6.4Hz,2H),2.92(dd,J=10.4,5.2Hz,2H).

[0215] (5) Synthesis of MEC 2 (MKK-EG2-TSPO)

[0216] Reaction process

[0217] The specific steps include:

[0218] Compound 1: See the synthesis of JQ1-PEG4-MKK.

[0219] Step 1: A solution of 3-(3-bromo-4-hydroxyphenyl)-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea 1 (320 mg, 0.72 mmol, 1.0 eq.) and tert-butyl (2-(2-bromoethoxy)ethyl)carbamate 2 (215 mg, 0.79 mmol, 1.1 eq.) in DMF (6 mL) was treated with potassium carbonate (302 mg, 2.16 mmol, 3 eq.) at 70° C. for 6 hours. The reaction was monitored by LCMS. After completion of the reaction, the mixture was diluted with EA (20 mL) and washed with brine (20 ml). The organic layer was dried over anhydrous Na SO and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=20:1) to give tert-butyl (2-(2-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)ethoxy)ethyl)carbamate 3 (290 mg, 63% yield) as a yellow solid. [M+H] + =627.2.

[0220] Step 2: A solution of tert-butyl 2-(2-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)ethoxy)ethyl)carbamate 3 (290 mg, 0.46 mmol, 1.0 eq.) in CHCl (10 mL) was treated with HCl / ether (3 mL) at 25°C for 10 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction was concentrated under reduced pressure to afford 1-(4-(2-(2-aminoethoxy)ethoxy)-3-bromophenyl)-3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)urea 4 (230 mg, 94% yield) as a brick-red solid. [M+H] + =527.2.

[0221] Compound 5: See the synthesis of MEC 1.

[0222] Step 3: A solution of 1-(4-(2-(2-aminoethoxy)ethoxy)-3-bromophenyl)-3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)urea 4 (230 mg, 0.44 mmol, 1.0 eq.) and 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetic acid 5 (127 mg, 0.48 mmol, 1.1 eq.) in DMF (6 mL) was treated with HATU (249 mg, 0.66 mmol, 1.5 eq.) and DIEA (338 mg, 2.62 mmol, 6.0 eq.) at 25° C. for 8 h. The reaction was monitored by LCMS. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=19:1) and preparative high performance liquid chromatography (prep-HPLC) (0.05% NH3) to give N-(2-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)ethoxy)ethyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide MKK-EG2-TSPO (28 mg, 8.3% yield) as a yellow solid. [M+H] + =773.2. 1H NMR (400MHz, DMSO-d6) δ12.36(s,1H),9.21(s,1H),9.11(s,1H),8.85(s,1H),8.61(s,1H),8.54(t,J=5.6Hz,1H) ,8.26(s,1H),8.23(d,J=2.2Hz,1H),8.10(d,J=7.2Hz,1H),7.93(d,J=8.8Hz,1H),7.85(d,J=2.4Hz,1H),7.67(d d,J=9.2,2.2Hz,1H),7.57(d,J=2.8Hz,2H),7.51-7.46(m,4H),7.36(dd,J=8.8,2.4Hz,1H),7.30-7.20(m,2H),7 .11(d,J=8.8Hz,1H),4.13(d,J=4.8Hz,2H),3.95(s,3H),3.76-3.69(m,2H),3.35(s,2H),2.96(q,J=5.8Hz,2H).

[0223] (6) Synthesis of MEC 3 (sMKK-TSPO)

[0224] Reaction process

[0225] The specific steps include:

[0226] Compound 1: See synthesis of MEC1.

[0227] Compound 2: See the synthesis of JQ1-PEG4-MKK.

[0228] A solution of 3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-amine 2 (100 mg, 0.44 mmol, 1.0 eq.) and oxo(2-phenyl-1H-indol-3-yl)acetic acid 1 (128 mg, 0.48 mmol, 1.1 eq.) in CH Cl (3 mL) was treated with HATU (250 mg, 0.66 mmol, 1.5 eq.) and DIPEA (171 mg, 1.32 mmol, 3.0 eq.) at 25° C. for 1 hour. The reaction was monitored by LCMS. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, CH2Cl2:CH3OH=20:1) and preparative high performance liquid chromatography (prep-HPLC) (0.05% NH3) to give N-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (28.2 mg, 14% yield) as a yellow solid. [M+H] + =473.1.1 H NMR (400MHz, DMSO-d6) δ12.56(s,1H),11.06(s,1H),9.17(s,1H),8.63(s,1H),8.28(s,1H),8.21(dd,J=6.4,2.4Hz,1H) ,8.10(d,J=2.2Hz,1H),7.91(d,J=9.0Hz,1H),7.67-7.59(m,3H),7.53(d,J=6.8Hz,1H),7.34-7.18(m,5H),3.94(s,3H).

[0229] (7) Synthesis of MEC 5 (AP-EG4-TSPO)

[0230] Reaction process

[0231] The specific steps include:

[0232] Compounds 1-3: See the synthesis of MEC1.

[0233] Step 1: A mixture of oxo(2-phenyl-1H-indol-3-yl)acetic acid 3 (1.3 g, 4.9 mmol, 1.0 eq.) in CH2Cl2 (10 mL) was added with DIEA (1.27 g, 9.8 mmol, 2 eq.) and HATU (2.24 g, 5.8 mmol, 1.2 eq.) and stirred at room temperature for 10 minutes. 2-[2-(2-aminoethoxy)ethoxy]ethanol 4 (1.1 g, 7.3 mmol, 1.5 eq.) was then added. The mixture was stirred at 25°C for 1 hour. The reaction was monitored by LCMS. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography on silica gel (CH2Cl2:CH3OH=20:1) to give N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 5 (840 mg, 43% yield) as a yellow solid. [M+H] + =397.1.

[0234] Step 2: To a solution of N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 5 (840 mg, 2.1 mmol, 1.0 eq.), TEA (1.1 g, 10.5 mmol, 5.0 eq.) and DMAP (129.4 mg, 1.1 mmol, 0.5 eq.) in CHCl (5 mL) was added TsCl (1.6 g, 8.4 mmol, 4 eq.). The mixture was stirred at 25° C. for 3 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by FFC (PE:EA=1:1) to give N-{2-[2-(2-{[(4-methylbenzene)sulfonyl]oxy}ethoxy)ethoxy]ethyl}-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 6 (700 mg, 60% yield) as a white solid. M / z: [M+H] + =551.2.

[0235] Compound 7: See the synthesis of AP-PEG-JQ1.

[0236] Step 3: A mixture of N-{2-[2-(2-{[(4-methylbenzene)sulfonyl]oxy}ethoxy)ethoxy]ethyl}-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 6 (200 mg, 0.36 mmol, 1.0 eq.), 3-aminopropyl N-[2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl]carbamate 7 (373.31 mg, 1.1 mmol, 3.0 eq.) and K2CO3 (150.59 mg, 1.1 mol, 3.0 eq.) in ACN (5 mL) was stirred at 70 ° C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by FFC (CH2Cl2:CH3OH=10:1) to give a crude product. The crude product was purified by preparative high performance liquid chromatography (prep-HPLC) (0.1% NH3) to give 3-{[2-(2-{2-[2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamido]ethoxy}ethoxy)ethoxy]amino}propyl N-[2,2,2-trichloro-1-(pyrimidin-2-ylamino)ethyl)carbamate (30.19 mg, 11% yield) as a white solid. M / z: [M+H] + =720.2. HPLC:t R 1.71min, 98.45% purity. 1H NMR (400MHz, DMSO-d6) δ12.37(s,1H),8.49(s,1H),8.43(d,J=4.8Hz,2H),8.07(d, J=7.4Hz,1H),7.82(d,J=8.2Hz,1H),7.59-7.54(m,2H),7.50-7.45(m,4H),7.27-7 .19(m,2H),7.15(d,J=9.2Hz,1H),6.83(m,1H),6.68(m,1H),4.04(s,2H),3.46(s, 4H),3.41(m,2H),3.23(m,2H),2.91(m,2H),2.59(m,2H),2.54(s,2H),1.66(s,2H).

[0237] (8) Synthesis of MEC 7 (MKK-EG4-VDAC)

[0238] Reaction process

[0239] The specific steps include:

[0240] Step 1: To a solution of 1-bromo-4-(trifluoromethoxy)benzene 12 (12.0 g, 0.05 mol, 1.0 eq.) in toluene (200 mL) was added tert-butyl piperazine-1-carboxylate 13 (28.0 g, 0.15 mol, 3.0 eq.), Xantphos (8.6 g, 0.015 mol, 0.3 eq.), Cs CO (64.9 g, 0.19 mol, 3.8 eq.) and Pd(OAc) (1.2 g, 0.74 mol, 14.8 eq.). The reaction mixture was stirred at 110 ° C under an N atmosphere for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE / EtOAc=10 / 1) to give tert-butyl {4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl}carboxylate 14 (14.0 g, 81% yield) as a yellow solid. M / z: [M+H-56] + =291.1.

[0241] Step 2: A solution of tert-butyl {4-[4-(trifluoromethoxy)phenyl]piperazin-1-yl}carboxylate 14 (14.0 g, 0.04 mol, 1.0 eq.) in dioxane (80 mL) of HCl was stirred at 25 ° C for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in NH3 in MeOH (50 mL). The mixture was stirred at 25 ° C for 1 hour. The mixture was concentrated under reduced pressure to give 1-[4-(trifluoromethoxy)phenyl]piperazine 4 (10.0 g, 95% yield) as a yellow solid. M / z: [M+H] + =247.3.

[0242] Step 3: (2E)-4-bromobut-2-enoic acid ethyl ester 1 (600 mg, 3.10 mmol, 1.0 eq.), 1-(1-methylphenyl)-1,4,7,10-tetraoxadodecane-12-ol 2 (1774 mg, 6.20 mmol, 2.0 eq.) and Ag2O (1080 mg, 4.66 mmol, 1.5 eq.) were stirred at 25 ° C in the dark for 48 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with EtOAc (20 mL) and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (PE / EtOAc=2 / 1) to obtain (E)-1-phenyl-2,5,8,11,14-pentaoxaoctadec-16-ene-18-oic acid ethyl ester 3 (600 mg, 48% yield) as a colorless oil. M / z:[M+H] + =397.2.

[0243] Step 4: A solution of (E)-1-phenyl-2,5,8,11,14-pentaoxaoctadec-16-ene-18-oic acid ethyl ester 3 (600 mg, 1.50 mmol, 1.0 eq.), 4-(piperazine-1-yl)phenyldifluoromethyl hypofluorite 4 (561 mg, 2.25 mmol, 1.5 eq.) and TEA (306 mg, 3.00 mmol, 2.0 eq.) in EtOH (0.4 mL) was stirred at 90 ° C for 24 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE / EtOAc=1 / 1) to give ethyl 1-phenyl-16-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-2,5,8,11,14-pentaoxaoctadecane-18-ate 5 (760 mg, 74% yield) as a yellow oil. M / z: [M+H] + =643.3.

[0244] Step 5: To a solution of 1-phenyl-16-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-2,5,8,11,14-pentaoxahexadecan-18-oic acid ethyl ester 5 (600 mg, 0.93 mmol, 1.0 eq.) in EtOH (10 mL) was added Pd / C (99 mg, 0.93 mmol, 1.0 eq.). The reaction mixture was stirred at 25 ° C. under H2 (15 psi) for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give 1-hydroxy-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-oic acid ethyl ester 6 (494 mg, 91% yield) as an orange oil. M / z: [M+H] + =553.2.

[0245] Step 6: To a solution of 1-hydroxy-14-(4-(4-(trifluoromethoxy)phenyl)piperazine-1-yl)-3,6,9,12-tetraoxahexadecan-16-oic acid ethyl ester 6 (494 mg, 0.89 mmol, 1.0 eq.) in DCM (10 mL) was added TEA (271 mg, 2.67 mmol, 3.0 eq.) and TsCl (255 mg, 1.34 mmol, 1.5 eq.). The reaction mixture was stirred at 25 ° C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, water (10 mL) was added to the reaction mixture and extracted with DCM (10 mL×3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE:EtOAc=1:1) to give ethyl 1-(tosyloxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-ate 7 (279 mg, 42% yield) as a yellow oil. M / z: [M+H] + =707.2.

[0246] Compound 8: See synthesis of JQ1-PEG4-MKK

[0247] Step 7: To a solution of ethyl 1-(tosyloxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-ate 7 (279 mg, 0.39 mmol, 0.1 eq.) in DMF (5 mL) was added potassium carbonate (136 mg, 0.98 mmol, 2.5 eq.) and 1-(3-bromo-4-hydroxyphenyl)-3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)urea 8 (347 mg, 0.79 mmol, 2.0 eq.). The reaction mixture was stirred at 70 ° C for 5 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was added to water (10 mL) and filtered. The filter cake was concentrated under reduced pressure to give ethyl 1-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16ate 9 (381 mg, 49% yield) as a yellow oil. M / z: [M+H] + =973.2.

[0248] Step 8: To a solution of 1-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxaline-6-yl)ureido)phenoxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-oic acid ethyl ester 9 (381 mg, 0.39 mmol, 1.0 eq.) in EtOH:HO=4:1 (5 mL) was added LiOH (37 mg, 1.56 mmol, 4.0 eq.). The reaction mixture was stirred at 25 ° C for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, water (5 mL) was added to the reaction mixture and extracted with EtOAc (5 mL×3). The combined organic layers were washed with brine (10 mL), dried over anhydrous NaSO and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (CH2Cl2:CH3OH=10:1) to give 1-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-oic acid 10 (96 mg, 24% yield) as a yellow solid. M / z: [M+H] + =945.1.

[0249] Step 9: To a solution of 1-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)-quinoxalin-6-yl)ureido)phenoxy)-14-(4-(4-(trifluoromethyloxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16oic acid 10 (96 mg, 0.10 mmol, 1.0 eq.) in DMF (3 mL) were added HATU (46 mg, 0.12 mmol, 1.2 eq.), DIEA (19 mg, 19 mg, DIEA (19 mg, 0.15 mmol, 1.5 eq.) and 4-chloroaniline 11 (15 mg, 0.12 mmol, 1.2 eq.). The reaction mixture was stirred at 25° C. for 2 h. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was added Water (10 mL) and extracted with EtOAc (5 mL × 3). The combined organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (CH2Cl2: CH3OH = 10: 1) and preparative high performance liquid chromatography prep-HPLC (0.1% FA) to give a light yellow solid 1- (2-bromo-4- (3- (3- (1-methyl-1H-pyrazol-4-yl) quinoxaline-6-yl) ureido) phenoxy) -N- (4-chlorophenyl) -14- (4- (4- (trifluoromethoxy) phenyl) piperazin-1-yl) -3,6,9,12-tetraoxahexadecane-16-amide MKK-EG4-VDAC (25 mg, 23% yield). M / z: [M + H] + =1054.1. 1 H NMR(400MHz,CH3OD)δ9.00(s,1H),8.43(s,1H),8.27-8.24(m,2H),7.91(d,J=9.2Hz,1H),7.78(d,J=2.8Hz,1H),7 .72(dd,J=9.2,2.4Hz,1H),7.54-7.51(m,2H),7.36(dd,J=8.8,2.8Hz,1H),7.27-7.23(m,2H),7.07-7.01(m,3H), 6.94-6.90(m,2H),4.16-4.13(m,2H),4.00(s,3H),3.86-3.83(m,2H),3.74-3.71(m,2H),3.65-3.56(m,14H),3.1 5-3.13(m,3H),2.97-2.91(m,2H),2.86-2.80(m,2H),2.65(dd,J=14.8,7.2Hz,1H),2.55(dd,J=15.2,6.8Hz,1H).

[0250] (9) Synthesis of MEC13 (Tha-EG4-TSPO)

[0251] Reaction process

[0252] The specific steps include:

[0253] Step 1: To a mixture of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione 1 (200 mg, 0.73 mmol, 1.0 eq.) in DMF (5 mL) stirred under nitrogen was added potassium carbonate (252 mg, 1.82 mmol, 2.5 eq.) and tert-butyl (2-(2-(2-(bromoethoxy)ethoxy)ethoxy)ethyl)carbamate 2 (287 mg, 0.80 mmol, 1.1 eq.). The reaction mixture was stirred at 60° C. for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was stirred at 40° C. for 16 hours. Water (10 mL) was added to the mixture and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE: EtOAc = 0: 1) to give tert-butyl 2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 3 (108 mg, 27% yield) as a yellow solid. M / z: [M+Na] + =572.2.

[0254] Step 2: To a mixture of tert-butyl (2-(2-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 3 (108 mg, 0.19 mmol, 1.0 eq.) in CHCl (2 mL) stirred under nitrogen was added HCl / dioxane (5 mL). The reaction mixture was stirred at 25°C for 3 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give 4-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindolin-1,3-dione 4 (95 mg, 95% yield) as a white solid. M / z: [M+H] + =450.2.

[0255] Step 3: To a mixture of 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetic acid 5 (45 mg, 0.17 mmol, 1.0 eq.) in CHCl (5 mL) were added HATU (77 mg, 0.20 mmol, 1.2 eq.), DIEA (33 mg, 0.25 mmol, 1.5 eq.) and 4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 4 (90 mg, 0.18 mmol, 1.1 eq.). The reaction mixture was stirred at 25° C. for 1 hour. The reaction was monitored by LCMS. After completion of the reaction, water (5 mL) was added to the reaction mixture and extracted with CHCl (5 mL×3). The combined organic layers were washed with brine (10 ml), dried over anhydrous NaSO and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative high performance liquid chromatography (0.1% FA) to obtain N-(2-(2-(-2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide Tha-EG4-TSPO (28.96 mg, 31% yield) as a yellow solid. M / z: [M+H] + =697.2. 1 H NMR(400MHz,DMSO-d6)δ12.45(s,1H),11.11(s,1H),8.53-8.49(m,1H),8.09-8.05(m,1H) ,7.82-7.77(m,1H),7.59-7.54(m,2H),7.53-7.44(m,6H),7.29-7.20(m,2H),5.12-5.04(m ,1H),4.35-4.31(m,2H),3.81-3.77(m,2H),3.66-3.62(m,2H),3.54-3.49(m,4H),3.47-3. 43(m,2H),3.22(t,J=6.4Hz,2H),2.93-2.83(m,3H),2.62-2.52(m,2H),2.06-1.98(m,1H).

[0256] (10) Synthesis of MEC18 (MKK-EG2-VDAC)

[0257] Reaction process

[0258] The specific steps include:

[0259] Step 1: To a solution of ethyl (2E)-4-bromobut-2-enoate 1 (1.0 g, 5.2 mmol, 1.0 eq.) in 2-[2-(benzyloxy)ethoxy]ethanol 2 (2.0 g, 10.4 mmol, 2.0 eq.) was added Ag2O (1.81 g, 7.8 mmol, 1.5 eq.). The mixture was stirred at 25 ° C in the dark for 48 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with EtOAc (20 ml) and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE / EtOAc=2 / 1) to give ethyl (2E)-4-{2-[2-(benzyloxy)ethoxy]ethoxy}but-2-enoate (1.2 g, 75% yield) as a colorless oil. M / z: [M+H] + =309.2.

[0260] Compound 4: See synthesis of Vdac binding molecules

[0261] Step 2: To a solution of ethyl (2E)-4-{2-[2-(benzyloxy)ethoxy]ethoxy}but-2-enoate (500 mg, 1.62 mmol, 1.0 eq.) 3 and TEA (328 mg, 3.24 mmol, 2.0 eq.) in EtOH (1 mL) was added 4-(piperazin-1-yl)phenyldifluoromethylhypofluorite 4 (481 mg, 1.94 mmol, 1.2 eq.). The mixture was stirred at 90° C. for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE / EtOAc=1 / 1) to give ethyl 4-(2-(2-(benzyloxy)ethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoate 5 (600 mg, 67% yield) as a yellow oil. M / z:[M+H] + =555.3.

[0262] Step 3: To a solution of ethyl 4-(2-(2-(benzyloxy)ethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoate 5 (600 mg, 1.08 mmol, 1.0 eq.) in MeOH (10 mL) was added Pd / C (95 mg, 1.08 mmol, 1.0 eq.). The reaction mixture was stirred under H2 (15 psi) at 25°C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give ethyl 4-(2-(2-hydroxyethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoate 6 (500 mg, 99% yield) as a yellow oil. M / z: [M+H] + =465.2.

[0263] Step 4: To a solution of ethyl 4-(2-(2-hydroxyethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoate 6 (500 mg, 1.07 mmol, 1.0 eq.) in DCM (10 mL) was added TEA (326 mg, 3.22 mmol, 3.0 eq.) and TsCl (307 mg, 1.61 mmol, 1.5 eq.). The reaction mixture was stirred at 25 ° C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, water (10 ml) was added to the reaction mixture and extracted with DCM (10 ml×3). The combined organic layers were washed with brine (20 ml), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE:EtOAc=1:1) to give ethyl 4-(2-(2-(tosyloxy)ethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoate 7 (450 mg, 67% yield) as a colorless oil. M / z: [M+H] + =619.2.

[0264] Compound 8: See synthesis of JQ1-PEG4-MKK

[0265] Step 5: To a solution of ethyl 4-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoate (140 mg, 0.23 mmol, 1 eq.) in DMF (5 mL) was added potassium carbonate (125 mg, 0.90 mmol, 4 eq.) and 3-(3-bromo-4-hydroxyphenyl)-1-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]urea (100 mg, 0.23 mmol, 1.0 eq.). The reaction mixture was stirred at 70° C. for 5 hours. The reaction was monitored by LCMS. After completion of the reaction, water (10 ml) was added to the reaction mixture and filtered. The filter cake was concentrated under reduced pressure to give ethyl 1-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-ate 9 (180 mg, 90% yield) as a yellow oil. M / z: [M+H] + =885.2.

[0266] Step 6: To a solution of 1-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxaline-6-yl)ureido)phenoxy)-14-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-3,6,9,12-tetraoxahexadecan-16-oic acid ethyl ester 9 (150 mg, 0.17 mmol, 1.0 eq.) in THF:H2O=4:1 (5 mL) was added LiOH·H2O (28 mg, 0.67 mmol, 4.0 eq.). The reaction mixture was stirred at 25°C for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, water (5 mL) was added to the reaction mixture and extracted with EtOAc (5 mL×3). The combined organic layers were washed with brine (10 ml), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give 4-(2-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)ethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoic acid 10 (130 mg, 89% yield) as a yellow solid. M / z: [M+H] + =857.2.

[0267] Step 7: To a solution of 4-(2-(2-(2-bromo-4-(3-(3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl)ureido)phenoxy)ethoxy)ethoxy)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanoic acid 10 (100 mg, 0.11 mmol, 1.0 eq.) in DMF (3 mL) were added HATU (53 mg, 0.14 mmol, 1.2 eq.), DIEA (30 mg, 0.23 mmol, 2 eq.) and 4-chloroaniline 11 (14 mg, 0.11 mmol, 1 eq.). The reaction mixture was stirred at 25° C. for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, water (10 ml) was added to the reaction mixture and extracted with EtOAc (5 ml×3). The combined organic layers were washed with brine (15 ml), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative TLC (CHCl:CHOH = 10:1) and preparative high-performance liquid chromatography (prep-HPLC) (0.1% NH·HO) to obtain 4-{4-[1-(2-[2-[2-bromo-4-({[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]carbamoyl}amino)phenoxy]ethoxy)ethoxy)-3-[(4-chlorophenyl)carbamoyl]propan-2-yl]piperazin-1-yl}phenyl difluoromethyl hypofluorite MKK-EG2-VDAC (40 mg, 35% yield) as a white solid. M / z: [M+H]+ 966.2. 1 HNMR(400MHz,DMSO-d6)δ10.13(s,1H),9.21(s,1H),9.11(s,1H),8.85(s,1H),8.60(s,1H),8.27-8.20(m,2H),7 .93(d,J=9.0Hz,1H),7.85(d,J=2.8Hz,1H),7.69-7.31(m,1H),7.60(d,J=8.8Hz,2H),7.38-7.29(m,3H),7.13(d ,J=8.8Hz,2H),7.07(d,J=9.2Hz,1H),6.92(d,J=9.2Hz,2H),4.17-4.07(m,2H),3.95(s,3H),3.79-3.74(m,2H), 3.67-3.51(m,4H),3.29(s,2H),3.05(s,4H),2.80(s,2H),2.67(d,J=2.0Hz,2H),2.54(s,2H),2.44-2.31(m,1H).

[0268] (11) Synthesis of MEC19 (MKK-Vdac)

[0269] Reaction process

[0270] The specific steps include:

[0271] Step 1: To a solution of 2-bromo-4-nitrophenol 1 (2.0 g, 9.2 mmol, 1.0 eq.) and (2E)-4-bromobut-2-enoic acid ethyl ester 2 (3.55 g, 18.4 mmol, 2.0 eq.) in ACN (30 mL) was added K2CO3 (3.81 g, 27.6 mmol, 3.0 eq.), and the mixture was stirred at 25 ° C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, the reaction was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, PE: EtOAc = 4: 1) to give (2E)-4-(2-bromo-4-nitrophenoxy)but-2-enoic acid ethyl ester 3 (2.45 g, 80% yield) as a white solid. M / z: [M+H] + =330.0.

[0272] Step 2: To a suspension of (2E)-4-(2-bromo-4-nitrophenoxy)but-2-enoic acid ethyl ester 3 (2.35 g, 7.1 mmol, 1.0 eq.) and 4-(piperazine-1-yl)phenyldifluoromethyl hypofluorite 4 (2.22 g, 7.8 mmol, 1.1 eq.) in DMF (4 mL) was added EtN (3.59 g, 35.5 mmol, 5.0 eq.) and the mixture was stirred at 90 ° C for 24 hours. The reaction was monitored by LCMS. After completion of the reaction, it was diluted with EA (50 mL) and washed with H O (100 mL) and brine (100 ml) in sequence, and the organic layer was dried over anhydrous Na SO and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, PE:EtOAc=4:1) to give ethyl 4-(2-bromo-4-nitrophenoxy)-3-[4-(4-{[(difluoromethyl)-fluoroinoxy]oxy}phenyl)piperazin-1-yl]butanoate 5 (1.0 g, 24% yield). [M+H] + =576.1.

[0273] Step 3: To a solution of ethyl 4-(2-bromo-4-nitrophenoxy)-3-[4-(4-{[(difluoromethyl)-fluoroquinoline]oxy}phenyl)piperazin-1-yl]butanoate 5 (1.0 g, 1.7 mmol, 1.0 eq.) in EtOH (5 mL) and H O (1 mL) was added LiOH H O (0.36 g, 8.5 mmol, 5.0 eq.) and the mixture was stirred at 25 ° C for 3 hours. The reaction was monitored by TLC. After completion of the reaction, it was diluted with ice water (30 mL), the pH was adjusted to 5, and extracted with EtOAc (320 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated to give 4-(2-bromo-4-nitrophenoxy)-3-[4-(4-{[(difluoromethyl)oxy}phenyl)piperazin-1-yl]butanoic acid 6 (0.96 g, 100% yield) as a pale yellow solid. [M+H] + =548.0.

[0274] Step 4: To a solution of 4-(2-bromo-4-nitrophenoxy)-3-[4-(4-{[(difluoromethyl)-fluoroinoxy]oxy}phenyl)piperazin-1-yl]butanoic acid 6 (0.96 g, 1.7 mmol, 1.0 eq.) and 4-chloroaniline 7 (0.24 g, 1.8 mmol, 1.1 eq.) in DCM (10 mL) was added HATU (0.71 g, 1.8 mmol, 1.1 eq.) and DIEA (0.66 g, 5.0 mmol, 3.0 eq.) at 25° C., and the desired solution was stirred for 1 hour. The reaction was monitored using LCMS. After completion of the reaction, it was concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, DCM only) to give 4-{4-[1-(2-bromo-4-nitrophenoxy)-3-[(4-chlorophenyl)carbamoyl]propan-2-yl]piperazin-1-yl}phenyl difluoromethyl hypofluorite 8 (0.79 g, 71% yield) as a yellow solid. [M+H] + =657.0.

[0275] Step 5: A solution of 4-{4-[1-(2-bromo-4-nitrophenoxy)-3-[(4-chlorophenyl)carbamoyl]propan-2-yl]piperazin-1-yl}phenyl difluoromethyl hypofluorite 8 (200 mg, 0.30 mmol, 1.0 eq.) in EtOH (2 mL) was treated with Raney Nickel at 25° C. under H 2 atmosphere for 2 hours. The reaction was monitored by LCMS. After completion of the reaction, the mixture was filtered and concentrated under reduced pressure to afford 4-{4-[1-(4-amino-2-bromophenoxy)-3-[(4-chlorophenyl)carbamoyl]propan-2-yl]piperazin-1-yl}phenyl difluoromethyl hypofluorite 9 (180 mg, 75% yield) as a yellow oil. [M+H]+ =627.1.

[0276] Compound 10: See the synthesis of JQ1-PEG4-MKK

[0277] Step 6: A solution of 4-{4-[1-(4-amino-2-bromophenoxy)-3-[(4-chlorophenyl)carbamoyl]propan-2-yl]piperazin-1-yl}phenyldifluoromethylhypofluorite 9 (123 mg, 0.36 mmol, 1.5 eq.) in THF (2 mL) was treated with EtN (72 mg, 0.72 mmol, 3.0 eq.) at 25°C for 5 minutes, followed by the addition of 4-{4-[1-(4-amino-2-bromophenoxy)-3-[(4-chlorophenyl)carbamoyl]propan-2-yl]piperazin-1-yl}phenyldifluoromethylhypofluorite 10 (150 mg, 0.24 mmol, 1.0 eq.) and the mixture was heated to 70°C for 16 hours. The reaction was monitored by LCMS. After completion of the reaction, it was evaporated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (prep-HPLC) (0.05% NH3) to give 4-(4-{1-[2-bromo-4-({[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]carbamoyl}amino)phenoxy]-3-[(4-chlorophenyl)carbamoyl]propan-2-yl}piperazin-1-yl)phenyl difluoromethyl hypofluorite MKK-Vdac (38.7 mg, 18% yield) as a white solid. [M+H] + =878.1. HPLC:t R 2.149min, 98.14% purity. 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),9.22(s,1H),9.11(s,1H),8.85(s,1H),8.61(s,1H),8.26(s,1H),8. 22(d,J=2.0Hz,1H),7.93(d,J=8.8Hz,1H),7.83(d,J=2.4Hz,1H),7.67(dd,J=9.2,2.4Hz,1H),7.63(d,J=9 .2Hz,2H),7.39-7.32(m,3H),7.16(d,J=6.8Hz,3H),6.99(d,J=9.2Hz,2H),4.26-4.12(m,2H),3.95(s,3H) ,3.62-3.52(m,1H),3.14-3.06(m,4H),3.01-2.91(m,2H),2.81-2.70(m,3H),2.60(dd,J=14.8,6.0Hz,1H). 19F NMR (400 MHz, DMSO) δ -57.21.

[0278] Example 2 Comparison of the ability of different MEC molecules to repair mitochondrial damage (restore mitochondrial membrane potential) in HeLa cells

[0279] In this example, the MEC molecules synthesized by the present invention and their ability to repair CCCP-induced mitochondrial damage (restoring mitochondrial membrane potential) in HeLa cells at a concentration of 100 nanomolar are scored in Table 1. Here, +++, strong; ++, moderate; +, weak; -, no.

[0280] The method involves measuring mitochondrial membrane potential using the Mitochondrial Membrane Potential Assay Kit (JC-1). HeLa cells (cervical cancer cell line) were seeded into 24-well plates at a density of 50,000 per well. Different MECs and AUTAC4 were co-treated with 20 micromolar carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 3 hours and then stained according to the JC-1 kit instructions. After staining for 30 minutes, the cells were washed with PBS and photographed using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). Images were analyzed using the Nikon camera's built-in software.

[0281] Table 1 Mitochondrial damage repair ability of MEC molecules

[0282] As shown in Table 1, when MAP3K1 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 has the best effect, followed by EG2. In the absence of the linker molecule, MEC molecules basically have no effect on repairing damaged mitochondria. The choice of mitochondrial outer membrane protein is not important. Both TSPO and VDAC can fully support the role of MEC molecules in repairing damaged mitochondria. On the other hand, the choice of E3 ligase is crucial. CRBN or APC CDC20 Neither MEC13 nor MEC5, designed as E3 ligases, could repair damaged mitochondria. Consistent with literature reports, AUTAC 4 had the ability to repair damaged mitochondria at 10 micromolar concentrations, but was completely inactive at 100 nanomolar concentrations.

[0283] Example 3 Comparison of the effects of different MEC molecules on restoring damaged mitochondrial membrane potential in various cells

[0284] Changes in mitochondrial membrane potential are the gold standard for distinguishing healthy and damaged mitochondria.

[0285] (1) HeLa cells

[0286] The JC-1 and TMRE methods were used to evaluate the restorative effects of different MEC small molecules on mitochondrial membrane potential after CCCP-induced mitochondrial damage in HeLa cells, as shown in Figure 4.

[0287] Specifically, the following steps are included: staining with the JC-1 probe is performed according to the method of Example 2. Tetramethylrhodamine ethyl ester (TMRE) dye is a membrane-permeable cationic fluorescent probe that can specifically identify mitochondrial membrane potential, thereby attaching to mitochondria and producing bright fluorescence. At certain concentrations, rhodamine dyes have low toxicity to cells and are therefore commonly used to detect mitochondria in animal cells, plant cells, and microorganisms. Cell treatment is consistent with the description of the JC-1 staining method. TMRE is prepared into a 5 mM stock solution according to the reagent manufacturer's instructions. The cells are incubated with the solution for 20 minutes in the dark, washed once with PBS, and the fluorescence changes between different wells are captured using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). The images are analyzed using the software provided with the Nikon camera.

[0288] The results showed that at 100 nanomolar concentrations, MEC1 and MEC7 had the strongest effect on repairing damaged mitochondrial membrane potential, followed by MEC2 and MEC18, MEC3 and MEC19, and MEC5 and MEC13, which were essentially inactive. A mixture of individual MEC1 end molecules (NC, including 100 nanomolar each of IKAM-1 and 2-phenylindole-3-glyoxylamide) showed no activity, as expected, confirming that MEC requires a chimeric molecular form to function. AUTAC4 had the ability to repair mitochondrial damage at 10 micromolar concentrations, but was ineffective at 100 nanomolar concentrations, consistent with literature reports.

[0289] (2) SN4741 neural cells

[0290] JC-1 was used to detect mitochondrial membrane potential and to evaluate the effects of MEC1 and MEC7 small molecules on the β-amyloid peptide 1-42 (Aβ) in SN4741 cells. 1-42 )-induced mitochondrial damage, as shown in Figure 5.

[0291] The specific steps include: using the mitochondrial membrane potential detection kit (JC-1) to detect mitochondrial membrane potential. SN4741 cells (mouse dopaminergic neurons) were divided into 24-well plates at a density of 30,000 per well, and Aβ was added. 1-42After co-treatment with 10 μM (10 μM) and different MECs (100 nM) for 72 h, the cells were stained according to the instructions of the JC-1 kit for 30 min and then washed with PBS. Finally, the cells were photographed under a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan), and the images were analyzed using the software provided with the Nikon camera.

[0292] The results showed that Aβ 1-42 It can significantly damage SN4741 cells and reduce mitochondrial membrane potential. MEC1 and MEC7 can effectively restore mitochondrial membrane potential at 100 nanomolar concentrations, proving that these two MEC molecules have a repairing effect on mitochondrial damage caused by β-amyloid peptide 1-42.

[0293] (3) SH-SY5Y neural cells

[0294] (3-1) The TMRE probe was used to evaluate the changes in mitochondrial membrane potential in human neuroblastoma cells (SH-SY5Y) after co-treatment with CCCP (20 μM) and different MEC small molecules (100 nmol) for 6 hours.

[0295] Specifically, the following steps are included: SH-SY5Y cells are seeded at a density of 100,000 per well in a 24-well plate, and after overnight culture, different MEC molecules are co-incubated with CCCP for 6 hours, and then TMRE staining is performed according to the operating method of Hela cells in Example 2.

[0296] As shown in Figure 6 , the results showed that MEC1 had a significant effect in restoring CCCP-damaged mitochondrial membrane potential, followed by MEC2 and MEC3, while MEC5 had basically no repair effect.

[0297] (3-2) The JC-1 method was used to evaluate the repair function of MEC1 and MEC7 small molecules on CCCP-induced mitochondrial damage in SH-SY5Y cells.

[0298] Specifically, the following steps were included: SH-SY5Y (human neuroblastoma cells) cells were seeded at a density of 100,000 per well in a 24-well plate, cultured overnight, MEC1 molecules were co-incubated with CCCP for 6 hours, and then JC-1 staining was performed according to the operating method of Hela cells in Example 2.

[0299] The results are shown in FIG7 . At 100 nanomolar concentrations, both MEC1 and MEC7 small molecules can restore mitochondrial membrane potential.

[0300] (4) Vascular smooth muscle cells

[0301] The JC-1 staining method was used to evaluate the restorative effect of different MEC small molecules on mitochondrial membrane potential after mitochondrial damage caused by angiotensin II in vascular smooth muscle cells.

[0302] Specifically, the following steps were included: MOVAS cells (mouse aortic vascular smooth muscle cells) were seeded at a density of 100,000 per well in a 24-well plate, stimulated with 1 micromolar angiotensin II for 24 hours, and then treated with 100 nanomolar MEC7, MEC13, MEC18, MEC19, AUTAC4, and MEC7-Cont (a simple mixture of IKAM-1 and Vdac binding molecules) for 6 hours. Referring to the JC-1 staining protocol in Example 2, the changes in mitochondrial membrane potential of the cells after treatment with different molecules were evaluated.

[0303] As shown in Figure 8, after treatment with 100 nanomolar MEC molecules, MEC1 and MEC7 had the strongest effect on restoring mitochondrial membrane potential, followed by MEC18, MEC19, and MEC13 and AUTAC4. A single MEC7 doublet (MEC7cont) had no ability to restore mitochondrial membrane potential, as expected.

[0304] The above data fully demonstrate that among the synthetic MEC molecules, MEC1 and MEC7 have the strongest effects on repairing mitochondrial damage. The following examples will focus on MEC1.

[0305] Example 4: Effect of MEC1 on repairing damaged mitochondria in various cells

[0306] 1) MEC1 itself does not cause mitochondrial damage and has no cytotoxicity

[0307] Mitochondrial membrane potential

[0308] A. HeLa cells

[0309] The JC-1 staining method was used to evaluate the effect of MEC1 treatment on mitochondrial membrane potential in undamaged Hela cells, specifically comprising the following steps: Hela cells were seeded at a density of 50,000 per well in a 24-well plate, and the cells were incubated with MEC1 at different concentrations for 3 hours. The cells were stained and analyzed according to the Hela cell staining method of Example 2.

[0310] As shown in FIG9 , the results showed that in undamaged Hela cells, MEC1 (1, 10, 100 nanomoles) molecules had no significant effect on mitochondrial membrane potential after 3 hours of treatment.

[0311] B.SH-SY5Y neural cells

[0312] The TMRE probe was used to evaluate the effect of MEC1 small molecule treatment on mitochondrial membrane potential in SH-SY5Y cells, specifically comprising the following steps: referring to the treatment method of Hela cells in Example 3, TMRE staining was performed on SH-SY5Y cells.

[0313] As shown in FIG10 , the results showed that in undamaged SH-SY5Y cells, treatment with MEC1 molecules at concentrations ranging from 1 to 100 nM for 6 h did not affect the mitochondrial membrane potential.

[0314] MitoSox

[0315] MitoSOX Red superoxide indicator is a novel fluorescent dye that specifically targets mitochondria in living cells. Mitochondrial superoxide oxidizes the MitoSOX reagent, producing bright red fluorescence. The MitoSOX probe was used to evaluate the effect of MEC1 small molecule incubation on mitochondrial ROS in intact HeLa cells. The following steps were performed: HeLa cells were seeded at a density of 50,000 per well in a 24-well plate. After overnight incubation, the cells were treated with various concentrations of MEC1 for 3 hours. The cells were then stained in the dark for 20 minutes according to the MitoSOX Red (superoxide indicator) instructions, washed once with PBS, and imaged using a fluorescence microscope.

[0316] As shown in FIG11 , the results showed that administration of 1 nM to 10 μM MEC1 small molecules to undamaged Hela cells for 3 h had essentially no effect on mitochondrial ROS.

[0317] ATP production capacity

[0318] Firefly luciferase requires ATP to generate luminescent light. When both firefly luciferase and luciferin are in excess, luminescence is proportional to ATP concentration within a certain concentration range. This principle was used to evaluate the effects of 24-hour treatment with various concentrations of the MEC1 small molecule on mitochondrial ATP production in intact HeLa cells. The following steps were performed: HeLa cells were seeded at a density of 50,000 per well in a 24-well plate. After an overnight culture, the cells were incubated with various concentrations of MEC1 or AUTAC4 for 24 hours. After discarding the supernatant, 500 μL of ATP assay buffer was added to each well. Protein quantification and luminescence detection were performed using a multi-function microplate reader (BioTek Synergy H1) using the ATP assay kit (Shanghai Biotech Co., Ltd., S0026). ATP quantification was performed based on the assay results.

[0319] As shown in Figure 12, the results showed that the MEC1 small molecule had no effect on mitochondrial ATP production at concentrations between 1 nM and 1 μM (the concentration range that effectively repairs damaged mitochondria), but had a certain inhibitory effect on mitochondrial ATP production at 10 μM. A simple mixture of the two end molecules of MEC1, A (IKAM-1) and T (2-phenylindole-3-glyoxylamide), had no effect on mitochondrial ATP production. The control molecule AUTAC4 had no effect on mitochondrial ATP production at concentrations of 100 nM and 10 μM.

[0320] Cell proliferation

[0321] The CCK8 method was used to detect the cell viability of MEC1 small molecules in the concentration range of 1nM to 10μM in undamaged Hela cells after incubation for 24 hours. The method specifically includes the following steps: Hela cells were seeded at a density of 10,000 per well in a 96-well plate, and after overnight culture, the cells were incubated with different concentrations of MEC1 for 24 hours. Subsequently, 10ul of CCK8 reagent was added to each well. After incubation for 2 hours at 37°C, 5% CO2, and 90% humidity, the absorbance was measured at a wavelength of 450nm using a multi-function microplate reader (BioTek Synergy H1).

[0322] As shown in FIG13 , the results showed that MEC1 did not affect cell viability in the concentration range of 1 nM to 1 μM, and only at 10 μM did MEC1 have a certain (about 20%) cytotoxic effect.

[0323] 2) MEC1 repairs mitochondrial damage, enhances mitochondrial function, and inhibits cell death in various damaged cells

[0324] Mitochondrial membrane potential

[0325] A. HeLa cells

[0326] The method of staining mitochondrial membrane potential using TMRE probe was used to evaluate the repair effect of different concentrations of MEC small molecules on mitochondrial damage caused by CCCP (20 μM) treatment for 3 hours in HeLa cells, which specifically included the following steps: staining and photographing according to the TMRE staining method of HeLa cells in Example 3.

[0327] As shown in Figure 14, the results showed that the MEC1 small molecule repaired the loss of mitochondrial membrane potential caused by CCCP in a concentration-dependent manner at 1-100 nM, with the best effect at 100 nanomolar. However, at 1 μM and 10 μM, this recovery effect decreased with increasing MEC1 concentration, suggesting that the MEC small molecule we designed has the same "Hook" effect as traditional protac small molecules.

[0328] B.SN4741 neural cells

[0329] The method of using TMRE probe to stain mitochondrial membrane potential in SN4741 (mouse dopaminergic neurons) cells was used. CCCP (20 μM) or oligomycin (Oligomycin) + antimycin (Antimycin) were treated for 6 hours to cause mitochondrial damage. The effect of co-treatment with different concentrations of MEC1 small molecules on the recovery of mitochondrial membrane potential was evaluated. Specifically, the following steps were included: SN4741 cells were seeded at a density of 100,000 per well in a 24-well plate, MEC1 molecules were co-incubated with CCCP or Oligomycin + Antimycin for 6 hours, and then stained and photographed according to the TMRE staining method of Hela cells in Example 3.

[0330] As shown in FIG15 , the results showed that the MEC1 small molecule had a concentration-dependent effect on repairing mitochondrial membrane potential at 1-100 nM on mitochondrial damage induced by CCCP or Oligomycin+Antimycin, with the best effect at 100 nanomolar.

[0331] C.SH-SY5Y cells

[0332] The method of staining mitochondrial membrane potential using TMRE probe and JC-1 probe was used to evaluate the repair effect of MEC1 small molecule co-treatment on mitochondrial damage caused by CCCP (20 μM) treatment for 6 hours in SH-SY5Y cells, which specifically included the following steps: staining and photographing were performed according to the treatment of SH-SY5Y cells in Example 4.

[0333] As shown in FIG16 , the results of the two probe detections both showed that the effect of MEC1 in restoring mitochondrial membrane potential was concentration-dependent, with the best effect at 100 nanomolar.

[0334] D.AML-12 hepatocytes

[0335] JC-1 was used to detect mitochondrial membrane potential and evaluate the effect of MEC1 on mitochondrial membrane potential in CCCP-treated and untreated AML-12 cells, specifically comprising the following steps: AML-12 cells were cultured with DMDM-F12 supplemented with 15% fetal bovine serum and 1% insulin, and after the cells adhered to the wall in a large dish, they were digested and then divided into 24-well plates at a rate of 100,000 cells per well. After overnight, the cells were divided into four treatment groups, each of which was an untreated control group (Cont), a group treated with 100 nanomolar MEC1 small molecule for 6 hours (MEC1), a group treated with 20 micromolar CCCP for 6 hours (CCCP), and a combined group treated with CCCP for 6 hours and then with 100 nanomolar MEC1 for 4 hours (CCCP+MEC1). After the cells were treated according to the above method, they were stained with a JC-1 kit according to the instructions, washed with PBS after 30 minutes of staining, and finally, the mitochondrial membrane potential of each group of cells was photographed using a fluorescence microscope.

[0336] As shown in FIG17 , the results showed that the MEC1 small molecule could restore the mitochondrial membrane potential after CCCP damage in hepatocytes, but had no effect on the mitochondrial membrane potential in intact cells.

[0337] E. HEK293T cells

[0338] The method of staining mitochondrial membrane potential using JC-1 probe was used to evaluate the restorative effect of co-treatment with different concentrations of MEC1 small molecules on mitochondrial membrane potential in HEK293T cells after mitochondrial damage caused by CCCP (20 μM) treatment for 6 h. The method specifically included the following steps: staining and photographing were performed according to the treatment of SH-SY5Y cells in Example 4.

[0339] As shown in Figure 18, the results showed that MEC1 had a significant effect of restoring mitochondrial membrane potential at 10nM to 100nM, while the effect decreased at 1μM, suggesting that the MEC1 small molecule has a "Hook" effect in HEK293T cells.

[0340] ●Intracellular ROS and mitochondrial ROS (MitoSox)

[0341] A. HeLa cells

[0342] DCFH-DA and Mitosox probes were used to stain the overall intracellular ROS and mitochondrial ROS, respectively. After 3 h of co-incubation with 20 μM CCCP and 1 nM, 10 nM, and 100 nM MEC1 small molecules, the ROS level was evaluated. The specific steps included the following steps: Hela cells were inoculated and treated with small molecules according to the treatment in Example 2, and then stained according to the instructions of DCFH-DA and MitoSOX. The probes were incubated with the cells for 20 minutes in the dark, washed once with PBS, and photographed under a fluorescence microscope.

[0343] As shown in FIG19 , the results showed that the MEC1 small molecule inhibited the production of global ROS and mitochondrial ROS in HeLa cells in a concentration-dependent manner, with the best effect at 100 nanomolar.

[0344] B.SH-SY5Y cells

[0345] The DCFH-DA probe was used to stain the overall ROS in SH-SY5Y cells. After treatment with 20 μM CCCP and co-incubation with 10 nM, 50 nM, and 100 nM MEC1 small molecules for 6 h, ROS levels were assessed. The specific steps included the following: the cultured SH-SY5Y cells were treated with small molecules according to the method for SH-SY5Y in Example 3, and then DCFH-DA staining and imaging were performed according to the method described in Figure 19.

[0346] As shown in FIG20 , the results showed that the MEC1 small molecule had the ability to inhibit overall ROS in a concentration-dependent manner, with the best effect at 100 nanomolar.

[0347] C.SN4741 cells

[0348] Mitochondrial reactive oxygen species in SN4741 cells were detected using Mitosox, which specifically included the following steps: cultured SN4741 cells were treated according to the method for SN-4741 cells in Example 3, and then MitoSox staining and imaging were performed according to the method described in Figure 19.

[0349] As shown in FIG21 , the results showed that 100 nM of MEC1 and MEC7 small molecules could effectively inhibit the increase of SN4741 mitochondrial reactive oxygen species induced by Aβ.

[0350] ATP production capacity

[0351] a. Evaluate the effects of varying concentrations of MEC1, small molecules at either end of MEC1, or AUTAC4 on mitochondrial ATP production in CCCP-damaged HeLa cells. Specifically, the following steps were performed: HeLa cells were seeded at a density of 50,000 cells per well in a 24-well plate. After overnight culture, the cells were incubated with 50 μM CCCP and varying concentrations of MEC1, small molecules at either end of MEC1, or AUTAC4 for 24 hours. The supernatant was discarded, and 500 μL of ATP assay buffer was added to each well. Protein quantification and luminescence detection were performed using a multi-function microplate reader (BioTek Synergy H1) according to the ATP assay kit (Shanghai Beyotime Biotechnology Co., Ltd., S0026). ATP quantification was performed based on the assay results.

[0352] As shown in Figure 22, CCCP significantly reduced ATP production. The MEC1 small molecule significantly restored ATP production at 10 and 100 nM, but had no significant effect at 1 nM. Consistent with literature reports, the positive control molecule AUTAC4 had some effect in restoring ATP production at 10 μM but was ineffective at 100 nM. As expected, a simple mixture of the two end molecules of MEC1, A (IKAM-1) and T (2-phenylindole-3-glyoxylamide), failed to restore ATP production.

[0353] b. The effect of different concentrations of MEC1 small molecules on the ATP production capacity of SH-SY5Y cells treated with CCCP (50 μM) for 24 hours was evaluated. The specific steps included the following: SH-SY5Y cells were seeded at a density of 50,000 per well in a 24-well plate. After overnight culture, the cells were incubated with 50 μM CCCP and different concentrations of MEC1 for 24 hours. After discarding the supernatant, 500 μL of ATP detection lysate was added to each well. Protein quantification and luminescence detection were performed using a multi-function microplate reader (BioTek Synergy H1) according to the method of the ATP detection kit (Shanghai Beyotime Biotechnology Co., Ltd. S0026).

[0354] Cell proliferation

[0355] The CCK8 method was used to detect the cell viability of MEC1 small molecules in the concentration range of 1nM to 10μM in Hela cells damaged by CCCP after incubation for 24 hours. The method specifically includes the following steps: Hela cells were seeded at a density of 10,000 per well in a 96-well plate, and after overnight culture, they were incubated with 50 micromolar CCCP and different concentrations of MEC1 for 24 hours. Subsequently, 10ul of CCK8 reagent was added to each well. After incubation for 2 hours at 37°C, 5% CO2, and 90% humidity, the absorbance was measured at a wavelength of 450nm using a multi-function microplate reader (BioTek Synergy H1).

[0356] As shown in Figure 23, the results showed that CCCP significantly induced cell death. The MEC1 small molecule gradually inhibited CCCP-induced cell death in the 1-100 nM concentration range, but had little effect at 1 μM and 10 μM. At 10 μM, it actually promoted cell death, demonstrating the "hook" effect of MEC1 and potential toxic effects at high concentrations. A simple mixture of the two end molecules of MEC1, A (IKAM-1) and T (2-phenylindole-3-glyoxylamide), did not inhibit cell death.

[0357] Example 5: MEC1 small molecule repairs mitochondrial damage in HeLa cells by enhancing mitochondrial autophagy

[0358] 1) MEC1 does not degrade TSPO or induce autophagy in HeLa cells without mitochondrial damage

[0359] Western Blot was used to evaluate the changes in mitochondrial inner and outer membrane proteins and autophagy levels in undamaged Hela cells after treatment with MEC1 small molecules for 6 hours. The method specifically includes the following steps: Hela cells were plated at 50,000 per well in a 24-well plate. After overnight attachment, the cells were treated according to the following methods: an untreated control group, treatment with 20 micromolar CCCP for 30 minutes, washed and replaced with fresh culture medium, and treatment with CCCP for 30 minutes followed by treatment with different concentration gradients (1, 5, 10, 50, 100 nanomolar) of MEC1 for 6 hours. After removing the cell supernatant, 80 μL Sample Buffer was added to each well and the cell lysate was collected. The samples were boiled at 100°C and the expression of related proteins was detected by Western blotting.

[0360] As shown in FIG24 , the results showed that different concentrations of MEC1 small molecules neither degraded mitochondrial membrane proteins (intrinsic protein TIM23 and outer membrane protein TSPO) nor enhanced the autophagy level (LC3-II level) in cells without mitochondrial damage.

[0361] 2) MEC1 degrades the mitochondrial outer membrane protein TSPO and the inner membrane protein TIM23 in cells with mitochondrial damage in an autophagy-dependent but proteasome-independent manner

[0362] A. HeLa cells

[0363] First, Western Blot and immunofluorescence methods were used to evaluate the changes in mitochondrial membrane proteins in Hela cells that were pretreated with 20 μM CCCP for 30 minutes and washed away, and then treated with MEC1 small molecules for 6 hours. The specific steps included the following: Hela cells were plated in 24-well plates at 50,000 per well. After overnight attachment, the cells were treated according to the following methods: an untreated control group, a control group that was treated with 20 micromolar CCCP for 30 minutes, washed away and replaced with fresh culture medium, and a control group that was treated with CCCP for 30 minutes and then treated with different concentration gradients (1, 5, 10, 50, 100 nanomolar) of MEC1 for 6 hours. For western blot, after removing the cell supernatant, 80 μL of Sample 1 was added to each well. The cell lysate was collected after buffer, and the samples were boiled at 100°C and then the expression of the mitochondrial inner and outer membranes was detected by WB; for immunofluorescence, Hela cells treated with MEC1 small molecules were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and then permeabilized with 0.2% permeabilizer for 10 minutes, washed with PBS, blocked with 5% BSA, incubated with TSPO antibody overnight for four times, washed three times with PBS, incubated with fluorescent secondary antibody, and finally incubated with nuclear fuel DAPI for 30 minutes. Finally, images were taken using a fluorescence microscope.

[0364] As shown in Figure 26, the results showed that the MEC1 small molecule degraded the mitochondrial inner membrane protein TIM23 and the outer membrane protein TSPO in a concentration-dependent manner (Figure 25a). Immunofluorescence results showed that the MEC1 small molecule caused a decrease in TSPO levels after CCCP-induced damage, indicating degradation (Figure 25b). To further explore the molecular pathways by which MEC1 degrades mitochondrial membrane proteins, we used autophagy inhibitors CQ and wortmannin and proteasome inhibitor MG132 in combination with MEC1 to treat CCCP-pretreated Hela cells. As shown in Figures 25c and 25d, Western Blot results showed that the effect of MEC1 on degrading mitochondrial membrane proteins was blocked by CQ and wortmannin but not by MG132, demonstrating that the effect of MEC1 depends on the autophagy pathway rather than the proteasome pathway.

[0365] B.SH-SY5Y cells

[0366] According to the method described in FIG25 , the degradation of TIM23 and TSPO in SH-SY5Y cells was evaluated by pre-treatment with CCCP (20 μM) for 30 minutes and then adding different concentrations of MEC1 for different treatment times.

[0367] As shown in Figure 26, the results showed that MEC1 small molecule degraded TIM23 and TSPO in a concentration (Figure 26a-b) and time-dependent manner. MEC1 small molecule alone had no significant effect on degrading TSPO or TIM23 after treatment with different concentrations for 12 hours (Figure 26c).

[0368] 3) MEC1 further enhances CCCP-induced autophagy

[0369] HeLa cells were treated as described in FIG26 , except that different antibodies were used for incubation. In this figure, LC3 antibody was used for incubation at 4° C. overnight.

[0370] As shown in Figure 27, western blot results showed that 100 nM MEC1 further enhanced the conversion of LC3 type I to type II caused by CCCP treatment for 30 minutes in HeLa cells, indicating that MEC1 further enhanced CCCP-induced cellular autophagy.

[0371] 4) MEC1 restores CCCP-damaged mitochondrial membrane potential independently of the proteasome but dependent on autophagy

[0372] 4-1) Mitochondrial membrane potential and mitochondrial ROS were stained using JC-1 and Mitosox probes, respectively, to evaluate the effect of MEC1 on restoring CCCP-damaged mitochondrial membrane potential, specifically comprising the following steps: Hela cells were treated as described in Example 2, incubated with MEC1, CCCP, and the autophagy inhibitor BafA1 or the proteasome inhibitor MG132 for 3 hours, and then stained according to the JC-1 and Mitosox instructions.

[0373] As shown in Figure 28, the results showed that the autophagy inhibitor BafA1 significantly inhibited the effect of MEC1 small molecules in repairing mitochondrial membrane potential (Figure 28a) and reducing mitochondrial ROS generation (Figure 28b) in CCCP-damaged Hela cells, while the proteasome inhibitor MG132 could not, demonstrating that the effect of MEC1 small molecules in restoring mitochondrial membrane potential and reducing mitochondrial ROS generation depends on autophagy rather than the proteasome pathway.

[0374] 4-2) To further evaluate whether the role of MEC1 in repairing damaged mitochondria is dependent on autophagy, we constructed HeLa cells in which ATG5 (a gene essential for autophagy) was knocked out. Western Blot analysis confirmed that complete knockout of the ATG5 gene resulted in no ATG5 protein production (Figure 29a).

[0375] (1) The effect of different concentrations of MEC1 on the mitochondrial ATP production capacity after CCCP injury in ATG5-knockout Hela cells was evaluated. Specifically, the following steps were performed: ATG5-knockout Hela cells were seeded at a density of 50,000 per well in a 24-well plate. After overnight culture, the cells were incubated with 50 μM CCCP and different concentrations of MEC1 for 24 hours. After discarding the culture supernatant, 500 μL of ATP detection lysate was added to each well. Protein quantification and luminescence detection were performed using a multifunctional microplate reader (BioTek Synergy H1) according to the method of the ATP detection kit (Shanghai Biyuntian Biotechnology Co., Ltd. S0026). ATP quantification was performed based on the test results.

[0376] As shown in Figure 29(b), CCCP significantly reduced the ATP level in ATG5-knockout Hela cells, while MEC1 small molecules could not restore the ability to produce ATP at 1-100 nM, further demonstrating that the role of MEC1 small molecules in restoring the ability to produce ATP depends on autophagy.

[0377] (2) JC-1 and Mitosox probes were used to stain mitochondrial membrane potential and mitochondrial ROS, respectively, to evaluate the role of MEC1 in repairing CCCP-damaged mitochondria after ATG5 knockout, specifically comprising the following steps: Hela cells were treated as described in Example 2, MEC1 and CCCP were co-incubated for 3 hours, and then stained according to the instructions of JC-1 and Mitosox.

[0378] As shown in Figure 29(c) and (d), CCCP significantly damaged mitochondria in ATG5 knockout cells, resulting in decreased mitochondrial membrane potential and increased mitochondrial reactive oxygen species levels, while MEC1 had no restorative effect, demonstrating that the role of MEC1 small molecules in repairing mitochondrial damage depends on autophagy.

[0379] 5) MEC1 enhances mitophagy in damaged cells

[0380] 5-1) In Hela cells overexpressing GFP-LC3, mitochondria were located using a Mitotracker probe, and the effects of MEC1 small molecules with and without CCCP treatment on mitophagy were evaluated, specifically comprising the following steps: 50,000 Hela cells overexpressing GFP-LC3 were plated per well in a 24-well plate. After overnight attachment, the cells were treated as follows: an untreated control group (Cont), a group treated with 100 nanomolar MEC1 small molecules alone for 6 hours (MEC1), a group treated with 20 micromolar CCCP alone for 3 hours (CCCP), and a combination group treated with CCCP and 100 nanomolar MEC1 for 3 hours (CCCP+MEC1). After treatment as described above, the cells were stained with a mitochondrial staining kit according to the instructions, washed with PBS after 30 minutes of staining, and finally, the green fluorescent autophagosomes GFP and red fluorescent mitochondria of each group of cells were photographed using a fluorescence microscope.

[0381] As shown in Figure 30, after treatment with MEC1 alone, Mitotracker and GFP-LC3 did not colocalize, indicating that the MEC1 small molecule itself does not induce mitophagy. CCCP treatment can cause colocalization of Mitotracker and GFP-LC3 to a certain extent, and MEC1 combined with CCCP treatment further enhances this colocalization, indicating that MEC1 enhances mitophagy in damaged HeLa cells.

[0382] 5-2) Keima proteins exhibit different fluorescence signals at acidic and neutral pH. Therefore, mitochondrial-localized Keima (also known as mitoKeima) can indicate mitochondria entering lysosomes via the autophagic pathway, providing a direct reflection of the extent of mitophagy. MitoKeima was stably expressed in HeLa cells overexpressing parkin. Mitophagy was induced by CCCP treatment, and changes in fluorescence signals at 440 nm (neutral pH) and 586 nm (acidic pH) were observed. The following steps were performed: HeLa cells overexpressing Parkin and mitoKeima were seeded at a density of 50,000 per well in a 24-well plate. Cells were treated with CCCP for 3 hours or without treatment and then treated with various concentrations of MEC1 for 6 hours. Fluorescence changes at 440 nm and 586 nm were observed under a fluorescence microscope without staining and analyzed using software.

[0383] As shown in FIG31 , the results showed that when MEC1 was added after CCCP treatment, the red fluorescence of the 586 nm channel was enhanced in a concentration-dependent manner, indicating that MEC1 enhanced CCCP-induced mitophagy.

[0384] 5-3) In addition, Mtphagy Dye is chemically bound to the mitochondria in the cell and emits weak fluorescence. When mitochondria undergo autophagy, the damaged mitochondria fuse with lysosomes, the pH drops, and becomes acidic. At this time, Mtphagy Dye produces strong fluorescence. In Hela cells overexpressing GFP-parkin, the cells were incubated with the Mtphagy Dye probe for 1 hour, washed off, and then pretreated with CCCP for 30 minutes. Finally, different concentrations of MEC1 small molecules were added. The specific steps include: Hela cells overexpressing GFP-parkin protein were seeded at a density of 50,000 per well in a 24-well plate. After overnight culture, the Mtphagy Dye probe was first loaded into the cells and incubated for 1 hour, washed off, and then treated with different concentrations of MEC1 molecules for 6 hours without further staining. The changes in red fluorescence were observed under a fluorescence microscope and analyzed uniformly using software.

[0385] As shown in Figure 32, CCCP pretreatment for 30 minutes induced a certain degree of mitophagy. Adding MEC1 significantly enhanced mitophagy, which increased with increasing MEC1 concentration. However, the enhancement weakened at a MEC1 concentration of 10 μM, again demonstrating a "hook effect."

[0386] Example 6 Verification of the mechanism by which MEC1 small molecules repair damaged mitochondria by enhancing mitochondrial autophagy

[0387] 1) MEC1 binds to TSPO

[0388] The Cellular Thermal Shift Assay (CETSA) is an assay used to measure the binding efficiency of drugs to target proteins within cells. Its principle is that target proteins often have a protective effect when bound to drug molecules. Specifically, as temperature increases, proteins denature and aggregate, which can be removed by centrifugation. However, once the protein is bound to the drug, the amount of denatured protein decreases at the same temperature.

[0389] Specifically, the following steps were included: Based on this principle, HeLa cells were treated according to the method described in Figure 19. The HeLa cell extract was divided into five equal parts, and the cell extracts were incubated with different concentrations of MEC1 small molecules on ice for 1 hour, then incubated at 55°C for 5 minutes, and then centrifuged at 20,000g for 30 minutes. The supernatant was aspirated for immunoblotting.

[0390] As shown in Figure 33, the results showed that MEC1 had a concentration-dependent protective effect on TSPO protein in vitro, but had no effect on UQURC1, proving that MEC1 can bind to TSPO.

[0391] 2) Ability of MEC1 to bind MAP3K1

[0392] Using CETSA, purified His-MAP3K1-CTD protein was added to extracts from MAP3K1- / - HeLa cells overexpressing Flag-MAP3K1. The mixture was divided equally into five aliquots and incubated with various concentrations of MEC1 molecules, denatured at 55°C for 5 minutes, and then centrifuged. Sample preparation involved the following steps: Based on this principle, HeLa cells were treated as described in Figure 19. The HeLa cell extract was divided equally into five aliquots and incubated with various concentrations of MEC1 molecules on ice for 1 hour, followed by incubation at 55°C for 5 minutes, and centrifuged at 20,000 g for 30 minutes. The supernatant was aspirated for immunoblotting.

[0393] As shown in Figure 34, western blot results showed that MEC1 had a concentration-dependent binding effect on Flag-MAP3K1 and His-MAP3K1-CTD proteins.

[0394] 3) MEC1 forms a terminary complex with MAP3K1 and TSPO

[0395] Adoption of GST The pull-down assay technique was used to detect the interaction between GST-TSPO and His-MAP3K1-CTD under conditions where MEC1 was present or absent. Specifically, the following steps were performed: GST-TSPO protein (1 μg), GST-TSPO protein (1 μg) + His-MAP3K1-CTD protein (1 μg), GST-TSPO protein (1 μg) + MEC1 (20 nmol / L), or GST-TSPO protein (1 μg) + His-MAP3K1-CTD protein (1 μg) + MEC1 (20 nmol / L) were added to 100 μl of IP buffer, and the mixture was incubated at room temperature for half an hour. 10 μl of GST beads were added and incubated at 4°C for 6 hours. The supernatant was collected after centrifugation at 300 g for 5 minutes, and the precipitate was washed three times with IP buffer before sample preparation. The His-MAP3K1-CTD protein in the supernatant and precipitate was detected by immunoblotting, and the GST-TSPO protein signal in the precipitate was used as a control.

[0396] As shown in Figure 35, western blot results showed that GST-TSPO and His-MAP3K1-CTD had obvious interaction only in the presence of MEC1, proving that MEC1, MAP3K1 and TSPO formed a ternary complex.

[0397] 4) Temporal effect of MEC1 recruitment of MAP3K1 to TSPO

[0398] In Hela cells treated with 10uM CCCP for 30min to induce mitochondrial damage, 100nM MEC1 small molecule was used for 10min, 30min and 60min respectively. As shown in Figure 36, the proximity ligation technique (PLA) showed that the interaction between MAP3K1 and TSPO existed 10min after MEC1 treatment of the cells, and the interaction was further enhanced after 30min and 60min.

[0399] 5) MEC1 enhances mitochondrial K63 ubiquitination

[0400] By extracting mitochondria, Hela cells were treated with 100nM MEC1 small molecule for 3h, 10uM CCCP for 30min, or 10uM CCCP for 30min, washed out and then treated with 100nM MEC1 small molecule for 3h, and then the cell pellets were collected to extract cell mitochondria.

[0401] As shown in Figure 37, the results showed that mitochondrial K63 ubiquitination was significantly enhanced after treatment with MEC1 alone or CCCP alone, and further enhanced after combined treatment with MEC1 and CCCP. On the other hand, K48 ubiquitination did not change significantly.

[0402] 6) MEC1 enhances K63 ubiquitination of TSPO and VDAC

[0403] By co-immunoprecipitation (co-IP) method, empty plasmid, HA-tagged ubiquitination plasmid, HA-tagged K63 ubiquitination plasmid (the expressed tagged ubiquitin molecule can only be ubiquitinated at the K63 site, and the K at other sites has been mutated) and HA-tagged K48 ubiquitination plasmid (the expressed tagged ubiquitin molecule can only be ubiquitinated at the K48 site, and the K at other sites has been mutated) were transfected into 293T cells respectively. After 24 hours of transfection, the cells were treated with 10uM CCCP for 30 minutes, washed off and treated with 100nM MEC1 small molecule for 3 hours. TSPO and VDAC1 were detected after immunoprecipitation with HA tag antibody.

[0404] As shown in FIG38 , the results showed that both CCCP and MEC1 led to increased K63 ubiquitination of TSPO and VDAC, and combined treatment of the two further enhanced TSPO and VDAC K63 ubiquitination.

[0405] 7) MEC1 repairs CCCP-damaged mitochondria in a manner dependent on MAP3K1

[0406] MAP3K1 knockout Hela cells were constructed, and Western blotting (WB) was used to verify that MAP3K1 protein was not expressed in the MAP3K1 knockout Hela cells, as shown in FIG39 .

[0407] WT HeLa, MAP3K1- / -Hela and MAP3K1- / -Hela cells transfected with Flag-tagged MAP3K1 were treated with 20uM CCCP for 3h to induce mitochondrial damage and the cells were co-treated with 20uM CCCP and 100nM MEC1 small molecules for 3h. The specific steps included the following: WT Hela and MAP3K1- / -Hela were divided into 24-well plates at a rate of 50,000 cells per well, and Flag-tagged MAP3K1 was transfected into MAP3K1- / -Hela using lipo3000. After 48 hours, each cell line was treated with drugs according to the above method. After treatment, the mitochondrial membrane potential of the cells was stained with JC-1 and photographed under a fluorescence microscope.

[0408] As shown in Figure 40, the results show that MEC1 cannot restore CCCP-induced mitochondrial membrane potential in MAP3K1- / - Hela cells. Furthermore, MEC1 cannot repair the loss of mitochondrial membrane potential caused by CCCP damage in MAP3K1- / - Hela cells. Evaluation of total cellular ROS, mitochondrial ROS, and cell proliferation yielded the same conclusion, indicating that MEC1 small molecules are unable to repair mitochondrial damage in MAP3K1- / - Hela cells.

[0409] As shown in Figure 41, the results showed that MEC1 completely restored the role of MEC1 in repairing CCCP-damaged mitochondrial membrane potential in MAP3K1- / - Hela cells transfected with Flag-tagged MAP3K1.

[0410] MAP3K1- / -Hela cells were treated with 10uM CCCP for 30min and 10uM CCCP for 30min, respectively, then washed off and treated with 100nM MEC1 small molecules for 3h. The method for extracting mitochondria included the following steps: WT Hela and MAP3K1- / -Hela were cultured in large dishes, and after the cells adhered, each cell line was treated with drugs according to the above method. After the treatment, the cell pellets were collected, the cell mitochondria were separated, and the cell ubiquitination and the mitochondrial inner and outer membranes were detected by WB.

[0411] As shown in FIG42 , the results showed that MEC1 had no effect on the ubiquitination of mitochondrial K63 and K48 in MAP3K1- / - Hela cells.

[0412] The cells were treated in the same manner as above, and the cell lysates were collected and analyzed by WB. The results, as shown in FIG43 , showed that MEC1 had no degradation effect on the mitochondrial outer membrane protein TSPO and the inner membrane protein TIM23 in MAP3K1- / - Hela cells.

[0413] 8) MEC1 repairs CCCP-damaged mitochondria in a TSPO-dependent manner

[0414] TSPO knockout Hela cells were constructed, and WB showed that TSPO protein was not expressed in TSPO knockout Hela cells, as shown in Figure 44.

[0415] WT HeLa and TSPO- / -Hela cells were treated with 20uM CCCP for 3h to induce mitochondrial damage and 20uM CCCP and 100nM MEC1 small molecule were co-treated for 3h. The mitochondrial membrane potential was detected using JC-I and TMRE. The method specifically included the following steps: 50,000 WT Hela and TSPO- / -Hela cells were plated in each well of a 24-well plate. After the cells adhered, each cell line was treated with drugs according to the above method. After treatment, the mitochondrial membrane potential of the cells was stained with JC-1 and TMRE and then photographed using a fluorescence microscope.

[0416] As shown in Figure 45, the results show that MEC1 can effectively restore CCCP-damaged mitochondrial membrane potential in WT HeLa cells but not in TSPO- / - HeLa cells, demonstrating that MEC1's role in repairing damaged mitochondria is dependent on TSPO. As expected, the effect of MEC7 is independent of TSPO.

[0417] WT HeLa and TSPO- / -Hela cells were treated with 20uM CCCP for 3h to induce mitochondrial damage and the cells were co-treated with 20uM CCCP and 100nM MEC1 small molecule for 3h, and DCFH-DA was used to detect intracellular ROS, which specifically included the following steps: The specific implementation steps are as shown in the corresponding content of Figure 29 above.

[0418] As shown in Figure 46, the results show that MEC1 effectively inhibits ROS generated by CCCP treatment in WT HeLa cells but not in TSPO- / - HeLa cells. MEC1 also fails to restore ATP production in TSPO- / - HeLa cells damaged by CCCP. These results strongly support the requirement of TSPO for MEC1 to repair damaged mitochondria.

[0419] A plasmid encoding full-length TSPO with a myc tag was constructed and transfected into TSPO- / -Hela cells. Western blotting revealed that TSPO was successfully expressed in TSPO- / -Hela cells, as shown in FIG47 .

[0420] WT HeLa, TSPO- / -Hela, and TSPO- / -Hela cells expressing myc-tagged TSPO were treated with 20uM CCCP for 3h to induce mitochondrial damage and co-treated with 20uM CCCP and 100nM MEC1 small molecule for 3h, respectively. The specific steps include:

[0421] WT Hela and TSPO Hela were seeded into 24-well plates at a rate of 50,000 cells per well. Myc-tagged TSPO was transfected into TSPO- / -Hela cells using lipo3000. After 48 hours, each cell line was treated with drugs as described above. After treatment, the cells were stained with JC-1 and mitochondrial reactive oxygen species and photographed using a fluorescence microscope.

[0422] As shown in Figure 48, the results showed that MEC1 could not restore CCCP-damaged mitochondrial membrane potential in TSPO- / - Hela cells, nor reduce mitochondrial ROS generated by CCCP treatment, but could do so in WT Hela cells and TSPO- / - Hela cells transfected with TSPO protein. This further validated the key role of TSPO in MEC1's repair of damaged mitochondria.

[0423] 9) MEC1 repairs CCCP-damaged mitochondria in a manner dependent on NBR1

[0424] Specifically, the following steps were included: WT Hela were divided into 24-well plates at a rate of 50,000 cells per well, NBR1 knockdown siRNA was exogenously transferred into WT Hela using lipo3000, and after 48 hours, the cells in each well were treated as follows: untreated control group (Cont), cells treated with 20 μM CCCP for 3 hours, and cells treated with 20 μM CCCP and 100 nmol MEC1 for 3 hours, then the cell pellets were collected for WB and mitochondrial membrane potential staining.

[0425] The three figures shown in Figure 49 respectively demonstrate that NBR1-specific siRNA effectively knocks down NBR1 expression in HeLa cells, resulting in the loss of MEC1's ability to inhibit CCCP-induced mitochondrial ROS production and repair CCCP-damaged mitochondrial membrane potential. This demonstrates that MEC1's action requires the selective autophagy receptor protein NBR1.

[0426] 10) MEC1 repairs CCCP-damaged mitochondria in a Nur77-dependent manner

[0427] As shown in Figure 50, WT HeLa cells were treated with 100 nM of the MEC1 small molecule alone or 20 uM of CCCP for 3 hours to induce mitochondrial damage, and the cells were treated with 20 uM CCCP and 100 nM of the MEC1 small molecule together for 3 hours. Immunofluorescence detection of Nur77 protein was performed, which specifically included the following steps: WT HeLa cells were plated at 50,000 per well in a 24-well plate, and after the cells attached, each cell line was treated with the drug according to the above method. After treatment, Nur77 and cell nuclei in the cells were stained with Nur77 antibody and DAPI, and then photographed under a fluorescence microscope. WT HeLa cells were plated at 10,000 per well in a 96-well plate, and after the cells attached, each cell line was treated with the drug according to the above method. After treatment, the cells were stained with the proximity ligation technique and then photographed under a fluorescence microscope. 50,000 WT Hela cells were plated per well in a 24-well plate, and Nur77 knockdown siRNA was transfected into WT Hela cells using lipo3000. After the cells adhered to the wall, each cell line was treated with drugs according to the above method. After treatment, the cell pellets were collected for WB and mitochondrial reactive oxygen species staining.

[0428] The three figures shown in Figure 50 respectively show that CCCP treatment in HeLa cells causes Nur77 to be exported from the nucleus, MEC1 recruits a large amount of Nur77 to the mitochondrial outer membrane when CCCP causes mitochondrial damage, and knockdown of Nur77 causes the disappearance of the role of MEC1 in repairing damaged mitochondria.

[0429] Example 7: MEC1 small molecule inhibits NLRP3 inflammasome by enhancing mitochondrial autophagy in macrophages (BMDM)

[0430] 1) MEC1 does not affect cell proliferation or induce cell death in macrophages

[0431] The CCK-8 cell proliferation assay was used to evaluate the cell proliferation of MEC1 in mouse bone marrow-derived macrophages (BMDM) treated with and without lipopolysaccharide (LPS), as shown in FIG51 .

[0432] The method specifically comprises the following steps: culturing and differentiating bone marrow cells with a DMEM medium containing 10% fetal bovine serum (FBS) and 20 ng / ml of macrophage colony stimulating factor (M-CSF); the cells that adhere to the wall after 7 days are BMDM cells. 60,000 cells were plated per well in 96-well plates. After overnight, the medium was replaced with reduced-serum Opti-MEM medium and the cells were treated as follows: 100 ng / mL lipopolysaccharide (LPS) alone (Mock), LPS treatment for 3 hours followed by 1 nmol MEC1 for 24 hours (LPS+1nM MEC1), LPS treatment for 3 hours followed by 10 nmol MEC1 for 24 hours (LPS+10nM MEC1), LPS treatment for 3 hours followed by 100 nmol MEC1 for 24 hours (LPS+100nM MEC1), and LPS treatment for 3 hours followed by 1 μmol MEC1 for 24 hours (LPS+1μM MEC1). Each well was assayed in triplicate. 10 μL of CCK-8 assay reagent was then added to each well. The cells were incubated in the dark for 2 hours, and the absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader.

[0433] The results showed that MEC1 small molecules did not affect the proliferation of macrophages.

[0434] Lactate dehydrogenase (LDH) cytotoxicity assay was used to evaluate the cytotoxicity of MEC1 in mouse bone marrow-derived macrophages (BMDM) treated with or without lipopolysaccharide (LPS), as shown in FIG52 .

[0435] The specific steps involved: BMDM cells were plated into 96-well plates at a rate of 100,000 cells per well and treated in triplicate according to the following groups: 100 ng / mL lipopolysaccharide (LPS) alone (Mock), LPS treatment for 3 hours followed by 10 μM nigericin for 1 hour (LPS+Nig), LPS treatment for 3 hours followed by 10 nmol MEC1 for 6 hours (LPS+10 nM MEC1), LPS treatment for 3 hours followed by 50 nmol MEC1 for 6 hours (LPS+50 nM MEC1), and LPS treatment for 3 hours followed by 100 nmol MEC1 for 6 hours (LPS+100 nM MEC1), with triplicate wells per group. Cells were then treated according to the instructions of the Lactate Dehydrogenase Cytotoxicity Assay Kit, and absorbance was measured at 490 nm using a BioTek Synergy H1 microplate reader.

[0436] The results showed that Nigericin induced BMDM cell death after LPS pretreatment, while MEC1 did not induce macrophage cell death.

[0437] 2) MEC1 does not affect the basal autophagy level of macrophages

[0438] Western Blot experiments were performed to evaluate the autophagy of MEC1 in mouse bone marrow-derived macrophages BMDM treated with and without LPS, as shown in FIG53 .

[0439] The specific steps involved: BMDM cells were plated into 24-well plates at a rate of 300,000 cells per well. After overnight, the culture medium was replaced with reduced-serum Opti-MEM medium. The cells were then treated as follows: untreated (Cont); 100 ng / mL lipopolysaccharide (LPS) alone (Mock); a combination of LPS treatment for 3 hours followed by 10 nmol MEC1 for 6 hours (LPS+10nM MEC1); a combination of LPS treatment for 3 hours followed by 50 nmol MEC1 for 6 hours (LPS+50nM MEC1); and a combination of LPS treatment for 3 hours followed by 100 nmol MEC1 for 6 hours (LPS+100nM MEC1). Cell lysates were then collected, and the levels of LC3I and LC3II in the cells were assayed by Western Blot.

[0440] The results showed that MEC1 treatment did not affect the conversion of LC3 type I and type II, indicating that MEC1 did not affect the basal autophagy level of BMDM.

[0441] 3) MEC1 enhances mitochondrial autophagy induced by NLRP3 inflammasome activation in macrophages

[0442] Mitochondrial autophagy dye Mtphagy Dye staining experiment was used to evaluate the mitophagy of MEC1 in mouse bone marrow-derived macrophages BMDM treated with and without lipopolysaccharide (LPS) and nigericin, as shown in FIG54 .

[0443] Specifically, the following steps were included: BMDM cells were plated into 24-wells at a rate of 300,000 cells per well, and cells were stained overnight using the staining method described in Example 5-3. The culture medium was then replaced with a serum-reduced Opti-MEM medium, and cells were treated according to the following groupings: a 100 ng / ml lipopolysaccharide (LPS)-only LPS treatment group (Mock), a 3-hour LPS treatment group followed by a 30-minute treatment with 5 μM nigericin (LPS+Nig), a 3-hour LPS treatment group followed by a 3-hour treatment with 100 nmol MEC1 followed by a 30-minute treatment with 5 μM nigericin (LPS+Nig+MEC1), and images were captured using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). Images were analyzed using the software provided with the Nikon camera. The scale of the fluorescence images is 20 μm.

[0444] The results showed that LPS+Nig caused mitochondrial damage by activating NLRP3 inflammasome, and MEC1 could significantly enhance the red fluorescence of Mtphagy Dye after LPS+Nig mitochondrial damage, indicating that MEC1 can increase the level of mitochondrial autophagy caused by NLRP3 inflammasome activation.

[0445] 4) MEC1 inhibits LPS+Nig-induced NLRP3 inflammasome activation and cell pyroptosis (wild-type BMDM)

[0446] ELISA was used to evaluate the release of IL-1β and TNF-α by MEC1 in mouse bone marrow-derived macrophages BMDM treated with or without lipopolysaccharide (LPS) and nigericin, as shown in FIG55 .

[0447] The specific steps include: BMDM cells are divided into 24 wells at a ratio of 300,000 cells per well, and the culture medium is replaced with reduced serum medium Opti-MEM after overnight. The cells are treated according to the following grouping methods: 100 ng / ml lipopolysaccharide (LPS) alone (Mock), LPS treatment for 3 hours followed by 5 micromolar nigericin treatment for 30 minutes (LPS+Nig), LPS treatment for 3 hours, 1 nanomolar MEC1 treatment for 6 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+1nM MEC1), LPS treatment for 3 hours, 10 nanomolar MEC1 treatment for 6 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+10nM MEC1), LPS treatment for 3 hours, 50 nanomolar MEC1 treatment for 6 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+50nM MEC1). The cells were treated with LPS for 3 hours, followed by 100 nanomolar MEC1 for 6 hours, and finally 5 micromolar Nigericin for 30 minutes (LPS+Nig+100 nM MEC1). The cell culture supernatants were then collected and assayed for IL-1β and TNF-α levels according to the instructions of the mouse IL-1β and TNF-α ELISA kits. The absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader, and data were analyzed using Elisa Calc software.

[0448] The results showed that MEC1 could inhibit the release of NLRP3-dependent inflammatory factor IL-1β in a concentration-dependent manner, but did not affect the release of NLRP3-independent inflammatory factor TNF-α, indicating that MEC1 can inhibit the activation of NLRP3 inflammasome caused by LPS+Nig.

[0449] Lactate dehydrogenase (LDH) cytotoxicity assay was used to evaluate the pyroptosis of MEC1 in mouse bone marrow-derived macrophages (BMDM) treated or untreated with lipopolysaccharide (LPS) and nigericin, as shown in FIG56( a ).

[0450] Specifically, the following steps were included: BMDM cells were divided into 96-well plates at a volume of 100,000 cells per well in 200 μl, and the culture medium was replaced with reduced serum medium Opti-MEM overnight. The cells were then treated according to the following groupings: untreated cell group (Cont), maximum enzyme activity group (Max), 100 ng / ml lipopolysaccharide (LPS)-treated group (Mock), LPS-treated group for 3 hours followed by 10 μM nigericin (Nigericin)-treated group (LPS+Nig), LPS-treated group for 3 hours followed by 10 nmol MEC1-treated group for 6 hours, and finally 10 μM Nigericin-treated group for 1 hour (LPS+Nig+10 nM MEC1), LPS-treated group for 3 hours followed by 50 nmol MEC1-treated group for 6 hours, and finally 10 μM Nigericin-treated group for 1 hour (LPS+Nig+50 nM MEC1). Cells were treated with LPS for 3 hours, followed by 100 nanomolar MEC1 for 6 hours, and finally 10 micromolar Nigericin for 1 hour (LPS + Nigericin + 100 nM MEC1). Each group consisted of triplicate wells. Cells were then treated according to the instructions of the Lactate Dehydrogenase Cytotoxicity Assay Kit, and absorbance was measured at 490 nm using a BioTek Synergy H1 microplate reader.

[0451] The results showed that LPS+Nig induced cell pyroptosis and released LDH, while MEC1 inhibited the release of LDH in a concentration-dependent manner, indicating that MEC1 inhibited cell pyroptosis caused by LPS+Nig.

[0452] Western Blot experiments were performed to evaluate the pyroptosis of MEC1 in mouse bone marrow-derived macrophages BMDM treated with or without lipopolysaccharide (LPS) and nigericin, as shown in FIG56( b ).

[0453] The specific steps include: BMDM cells are divided into 24-wells at a ratio of 300,000 cells per well, and the culture medium is replaced with reduced serum culture medium Opti-MEM after overnight, and the cells are treated according to the following grouping methods: 100 ng / ml lipopolysaccharide (LPS) treatment group (Mock), LPS treatment for 3 hours followed by 5 micromolar nigericin treatment for 30 minutes (LPS+Nig), LPS treatment for 3 hours, followed by 10 nanomolar MEC1 treatment for 6 hours, and finally 5 micromolar nigericin treatment for 30 minutes (LPS+Nig+10nM MEC1), LPS treatment for 3 hours, followed by 50 nanomolar MEC1 treatment for 6 hours, and finally 5 micromolar nigericin treatment for 30 minutes (LPS+Nig+50nM MEC1), LPS treatment for 3 hours, followed by 100 nanomolar MEC1 treatment for 6 hours, and finally 5 micromolar nigericin treatment for 30 minutes (LPS+Nig+100nM MEC1). The cell lysate was then collected and the content of Gasdermin D in the cells was detected by Western Blot.

[0454] The results showed that LPS+Nig induced BMDM cell pyroptosis mediated by Gasdermin D cleavage, while 100 nanomoles of MEC1 inhibited the cleavage of Gasdermin D, indicating that MEC1 inhibited cell pyroptosis caused by LPS+Nig.

[0455] 5) MEC1 inhibits LPS-induced CAPS NLRP3 inflammasome activation and pyroptosis (CAPS mutant BMDM)

[0456] CAPS is an autoinflammatory disease caused by NLRP3 mutations. The NLRP3 inflammasome with CAPS mutations only requires a single signal, LPS, to be activated.

[0457] An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the release of IL-1β by MEC1 in CAPS BMDM macrophages derived from CAPS mice treated with or without LPS, as shown in FIG57( a ).

[0458] The specific steps include culturing differentiated bone marrow cells in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 20 ng / mL macrophage colony-stimulating factor (M-CSF). Adherent cells after 7 days are designated as CAPS BMDM cells. 300,000 cells per well are plated in 24-well plates. Overnight, the medium is replaced with reduced-serum Opti-MEM medium. Cells are then divided into the following groups: untreated cells (Cont); 100 ng / mL lipopolysaccharide (LPS)-treated group (LPS); a combination of LPS treatment for 3 hours followed by 10 nmol MEC1 for 6 hours (LPS+10 nM MEC1); a combination of LPS treatment for 3 hours followed by 50 nmol MEC1 for 6 hours (LPS+50 nM MEC1); and a combination of LPS treatment for 3 hours followed by 100 nmol MEC1 for 6 hours (LPS+100 nM MEC1). The cell culture supernatant was then collected and the IL-1β content in the supernatant was detected according to the instructions of the mouse IL-1β ELISA kit. Finally, the absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader, and the data were analyzed using Elisa Calc software.

[0459] The results showed that MEC1 could inhibit LPS-induced IL-1β release in CAPS BMDM in a concentration-dependent manner, indicating that MEC1 could inhibit NLRP3 inflammasome activation.

[0460] Mitochondrial superoxide was detected using Mitosox to evaluate the mitochondrial ROS levels of MEC1 in CAPS mouse bone marrow-derived macrophages (CAPS BMDM) treated with and without LPS, as shown in FIG57( b ).

[0461] The cells were treated as described above and then stained according to the instructions of the Mitosox kit. After 30 minutes of staining, the cells were washed with PBS and photographed using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). The images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence image is 20 μm.

[0462] The results showed that the red fluorescence of CAPS BMDM was enhanced after LPS treatment, while MEC1 concentration-dependently weakened the red fluorescence, indicating that MEC1 can reduce the level of mitochondrial ROS in a concentration-dependent manner.

[0463] Western Blot experiments were performed to evaluate the pyroptosis of MEC1 in CAPS BMDM macrophages derived from CAPS mice treated with or without lipopolysaccharide (LPS), as shown in FIG57( c ).

[0464] The cells were treated in the same manner as above, and then the cell lysate was collected and the gasdermin D content in the cells was detected by Western Blot.

[0465] The results showed that LPS induced Gasdermin D cleavage-mediated pyroptosis of CAPS BMDM cells, while MEC1 concentration-dependently inhibited Gasdermin D cleavage, indicating that MEC1 inhibited LPS-induced cell pyroptosis.

[0466] 6) MEC1 attenuates mitochondrial damage induced by NLRP3 inflammasome activation in macrophages

[0467] Mitochondrial membrane potential was detected using JC-1, and the effect of MEC1 on mitochondrial membrane potential in mouse bone marrow-derived macrophages BMDM treated with or without lipopolysaccharide (LPS) and nigericin was evaluated, as shown in FIG58 .

[0468] Specifically, the following steps were included: BMDM cells were plated into 24-well plates at a ratio of 300,000 cells per well. After overnight, the culture medium was replaced with reduced-serum Opti-MEM medium, and the cells were treated according to the following groupings: 100 ng / ml lipopolysaccharide (LPS) alone (Mock), LPS treatment for 3 hours followed by 5 μM nigericin treatment for 30 minutes (LPS+Nig), LPS treatment for 3 hours followed by 100 nmol MEC1 treatment for 3 hours and finally 5 μM nigericin treatment for 30 minutes (LPS+Nig+MEC1). The cells were then stained according to the instructions of the JC-1 kit, washed with PBS after 30 minutes of staining, and photographed using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). Images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence image is 20 μm.

[0469] The results showed that LPS+Nig treatment led to a decrease in red fluorescence and an increase in green fluorescence, indicating a loss of mitochondrial membrane potential, while 100 nanomoles of MEC1 enhanced the red fluorescence after LPS+Nig treatment and weakened the green fluorescence, indicating that MEC1 can restore the mitochondrial membrane potential.

[0470] Reactive oxygen species (ROS) detection experiments were used to evaluate the effect of MEC1 on overall ROS in mouse bone marrow-derived macrophages (BMDM) treated or not with lipopolysaccharide (LPS) and nigericin, as shown in Figure 59(a).

[0471] The cells were treated as described above and then stained according to the ROS kit instructions. After 30 minutes of staining, the cells were washed with PBS and photographed using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). The images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence image is 20 μm.

[0472] The results showed that LPS+Nig treatment led to enhanced green fluorescence and increased cellular ROS levels, while 100 nanomolar MEC1 effectively reduced green fluorescence, indicating that MEC1 inhibited the overall ROS level.

[0473] Mitochondrial superoxide was detected using Mitosox to evaluate the mitochondrial ROS level of MEC1 in mouse bone marrow-derived macrophages (BMDM) cells treated with or without lipopolysaccharide (LPS) and nigericin, as shown in FIG59( b ).

[0474] The specific steps include: BMDM cells are divided into 24 wells at a ratio of 300,000 cells per well, and the culture medium is replaced with reduced serum medium Opti-MEM after overnight. The cells are treated according to the following grouping methods: 100 ng / ml lipopolysaccharide (LPS) alone (Mock), LPS treatment for 3 hours followed by 5 micromolar nigericin treatment for 30 minutes (LPS+Nig), LPS treatment for 3 hours, followed by 10 nanomolar MEC1 treatment for 3 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+10nM MEC1), LPS treatment for 3 hours, followed by 50 nanomolar MEC1 treatment for 3 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+50nM MEC1), LPS treatment for 3 hours, followed by 100 nanomolar MEC1 treatment for 3 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+100nM MEC1), and LPS treatment for 3 hours, followed by 100 nanomolar MEC1 treatment for 3 hours, and finally 5 micromolar Nigericin treatment for 30 minutes (LPS+Nig+100nM MEC1). The cells were then stained according to the instructions of the Mitosox kit, washed with PBS after 30 minutes of staining, and photographed under a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). The images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence image is 20 μm.

[0475] The results showed that LPS+Nig treatment led to enhanced red fluorescence and increased mitochondrial ROS levels, while MEC1 could reduce red fluorescence in a concentration-dependent manner, indicating that MEC1 inhibited mitochondrial ROS levels.

[0476] Lysotracker was used to detect lysosomes and evaluate the effect of MEC1 on lysosomes in mouse bone marrow-derived macrophages BMDM treated or not with lipopolysaccharide (LPS) and nigericin, as shown in FIG60 .

[0477] The cells were treated as described above and subsequently stained according to the Lysotracker kit instructions. After 30 minutes of staining, the cells were washed with PBS and photographed using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). Images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence images is 20 μm.

[0478] The results showed that LPS+Nig treatment led to weakened green fluorescence and lysosomal damage, while MEC1 could enhance green fluorescence in a concentration-dependent manner, indicating that MEC1 could repair lysosomal damage.

[0479] 7) The effect of MEC1 on repairing damaged mitochondria in BMDM is dependent on autophagy

[0480] An ELISA experiment was used to evaluate the effect of the autophagy inhibitor Bafilomycin A1 on the repair of damaged mitochondria by MEC1 in mouse bone marrow-derived macrophages BMDM treated with or without LPS+Nigericin, as shown in Figure 61(a).

[0481] The specific steps include: BMDM cells are divided into 24 wells at a ratio of 300,000 cells per well, and the culture medium is replaced with reduced serum medium Opti-MEM after overnight. The cells are treated according to the following grouping methods: 100 ng / ml lipopolysaccharide (LPS) treatment group (Mock), LPS treatment for 3 hours followed by 5 μM nigericin treatment for 30 minutes (LPS+Nig), LPS treatment for 3 hours, 100 nmol MEC1 treatment for 6 hours, and finally 5 μM nigericin treatment for 30 minutes (LPS+Nig+MEC1), LPS treatment for 3 hours, combined treatment with 100 nmol MEC1 and 100 nmol BafilomycinA1 for 6 hours, and finally 5 μM nigericin treatment for 30 minutes (LPS+Nig+MEC1+BafA1), and then the cell culture supernatant is collected, and the IL-1β content in the supernatant is detected according to the instructions of the mouse IL-1β ELISA kit, and finally the BioTek Synergy The absorbance was measured at 450 nm using an H1 microplate reader, and the data were analyzed using Elisa Calc software.

[0482] The results showed that MEC1 inhibited the release of IL-1β, while the autophagy inhibitor BafA1 blocked the inhibitory effect of MEC1 on IL-1β, indicating that the role of MEC1 in repairing damaged mitochondria depends on the autophagy pathway.

[0483] Western Blot experiments were performed to evaluate the effect of the autophagy inhibitor BafilomycinA1 on the repair of damaged mitochondria by MEC1 in mouse bone marrow-derived macrophages BMDM treated with or without LPS+Nigericin, as shown in Figure 61(b).

[0484] The cells were treated as above, and the cell lysates were collected for Western Blot detection of TSPO and TIM23.

[0485] The results showed that MEC1 could cause the degradation of TSPO and TIM23, while the autophagy inhibitor BafA1 blocked the effect of MEC1 on TSPO and TIM23 degradation, indicating that the role of MEC1 in repairing damaged mitochondria depends on the autophagy pathway.

[0486] An ELISA experiment was used to evaluate the effect of proteasome inhibitor MG132 on the repair of damaged mitochondria by MEC1 in mouse bone marrow-derived macrophages BMDM treated with or without LPS+Nigericin, as shown in Figure 62(a).

[0487] Specifically, the following steps were included: BMDM cells were divided into 24-well plates at a ratio of 300,000 cells per well, and the culture medium was replaced with reduced serum medium Opti-MEM after overnight. The cells were treated according to the following groupings: 100 ng / ml lipopolysaccharide (LPS) alone (Mock), LPS was first treated for 3 hours and then 5 micromolar nigericin was treated for 30 minutes (LPS+Nig), LPS was first treated for 3 hours, 100 nanomolar MEC1 was added for 6 hours, and finally 5 micromolar nigericin was added for 30 minutes (LPS+Nig+MEC1), LPS was first treated for 3 hours, 100 nanomolar MEC1 and 20 micromolar MG132 were added for 6 hours, and finally 5 micromolar nigericin was added for 30 minutes (LPS+Nig+MEC1+MG132), and the cell culture supernatant was then collected, and the IL-1β content in the supernatant was detected according to the instructions of the mouse IL-1β ELISA kit, and finally the BioTek Synergy The absorbance was measured at 450 nm using an H1 microplate reader, and the data were analyzed using Elisa Calc software.

[0488] The results showed that MEC1 could inhibit the release of IL-1β, and the proteasome inhibitor MG132 could not block the inhibitory effect of MEC1 on IL-1β, indicating that the role of MEC1 in repairing damaged mitochondria is not dependent on the proteasome pathway.

[0489] Western Blot experiments were performed to evaluate the effect of proteasome inhibitor MG132 on the repair of damaged mitochondria by MEC1 in mouse bone marrow-derived macrophages BMDM treated with or without LPS+Nigericin, as shown in Figure 62(b).

[0490] The cells were treated as above, and the cell lysates were collected for Western Blot detection of TSPO and TIM23.

[0491] The results showed that MEC1 could cause the degradation of TSPO and TIM23, while the proteasome inhibitor MG132 could not block the effect of MEC1 on TSPO and TIM23 degradation, indicating that the role of MEC1 in repairing damaged mitochondria is not dependent on the proteasome pathway.

[0492] Mitochondrial superoxide was detected using Mitosox to evaluate the effects of Bafilomycin A1 and MG132 on mitochondrial ROS in mouse bone marrow-derived macrophages BMDM treated with or without LPS+Nigericin, as shown in Figure 63(a).

[0493] Specifically, the following steps were included: BMDM cells were divided into 24-well plates at a rate of 300,000 cells per well, and the culture medium was replaced with reduced serum medium Opti-MEM overnight. The cells were then treated according to the following groupings: 100 ng / ml lipopolysaccharide (LPS) alone (Mock), LPS was first treated for 3 hours and then treated with 5 μM nigericin for 30 minutes (LPS+Nig), LPS was first treated for 3 hours and then treated with 100 nmol MEC1 for 3 hours, and finally treated with 5 μM Nigericin for 30 minutes (LPS+Nig+MEC1), LPS was first treated for 3 hours and then treated with 100 nmol MEC1 for 3 hours, and finally treated with 5 μM Nigericin for 30 minutes (LPS+Nig+MEC1), and LPS was first treated for 3 hours and then treated with 100 nmol MEC1 for 3 hours. The cells were treated with 100 nmol MEC1 and 100 nmol BafilomycinA1 for 3 hours, and then 5 μmol Nigericin was added for 30 minutes (LPS+Nig+MEC1+BafA1). The cells were first treated with LPS for 3 hours, and then 100 nmol MEC1 and 20 μmol MG132 were added for 3 hours, and then 5 μmol Nigericin was added for 30 minutes (LPS+Nig+MEC1+MG132). The cells were then stained according to the instructions of the Mitosox kit, washed with PBS after 30 minutes of staining, and finally photographed with a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). The images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence image is 20 μm.

[0494] The results showed that MEC1 inhibited mitochondrial ROS levels, BafA1+MEC1 red fluorescence was restored, while MG132+MEC1 red fluorescence could not be restored, indicating that the effect of MEC1 in repairing damaged mitochondria depends on the autophagy pathway rather than the proteasome pathway.

[0495] Reactive oxygen species (ROS) detection experiments were used to evaluate the effects of Bafilomycin A1 and MG132 on overall ROS in MEC1 in mouse bone marrow-derived macrophages (BMDM) treated with or without LPS+Nigericin, as shown in Figure 63(b).

[0496] The cells were treated as described above and then stained according to the ROS kit instructions. After 30 minutes of staining, the cells were washed with PBS and photographed using a fluorescence microscope (Nikon TI fluorescence microscopy; Nikon, TI-DH, Japan). The images were analyzed using the software provided with the Nikon camera. The scale bar of the fluorescence image is 20 μm.

[0497] The results showed that MEC1 reduced the overall ROS level, the green fluorescence of BafA1+MEC1 was restored, and the green fluorescence of MG132+MEC1 could not be restored, indicating that the effect of MEC1 in repairing damaged mitochondria depends on the autophagy pathway rather than the proteasome pathway.

[0498] Example 8: MEC1 small molecule inhibits NLRP3 inflammasome activation in mice

[0499] 1) MSU-induced peritonitis model

[0500] Eight-week-old C57Bl6 mice were randomly divided into a model group and a drug administration group, with the drug administration groups being MEC1 1 μg group and MEC1 5 μg group. The drug administration group was intraperitoneally injected with MEC1 small molecules on the previous day, and the model group was intraperitoneally injected with normal saline in parallel. On the second day, the drug administration group was intraperitoneally injected with MEC1 small molecules, and the model group was injected with normal saline in parallel. One hour later, MSU suspension was intraperitoneally injected at a dose of 50 mg / kg. Six hours later, peritoneal lavage fluid was collected, and the monocyte ratio was detected by flow cytometry, and the IL-1β level was detected by ELISA. The specific steps included:

[0501] Flow cytometric detection of monocytes: rinse the mouse peritoneal cavity with 1 ml PBS buffer and collect the collected cells into a 1.5 ml EP tube, centrifuge at 2000 rpm for 10 min; discard the supernatant, add 1 ml red blood cell lysis buffer, and centrifuge for 2-3 min; neutralize with 9 ml DMEM, centrifuge at 2000 rpm for 10 min; discard the supernatant, resuspend in 1 ml DMEM, filter into a 1.5 ml EP tube, centrifuge at 3000 rpm for 1 min; discard the supernatant, block with 80 μl serum on ice for 30 min; mix well, take 5 μl / tube to 100 μl PBS + Ab (ly6G pp5.5), and keep on ice for 30 min; add 1 ml PBS, centrifuge at 3000 rpm for 5 min, then resuspend with 300 μl PBS, filter into a flow cytometer tube and prepare for detection.

[0502] Elisa detects IL-1β levels:

[0503] 1. Remove the unused microporous strips from the plate frame, put the remaining strips back into the aluminum foil bag containing desiccant, and reseal for storage.

[0504] 2. Add 350 μL of 1x wash buffer to each well, let it stand for 40 seconds, and then discard the liquid. This step is a total of 3 washes.

[0505] 3. Add 100 μL of standard / sample diluent (R1) to the blank wells.

[0506] 4. Add 100 μL of standards or samples of different concentrations to the other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 hours.

[0507] 5. Prepare the biotinylated antibody (100x) working solution 15 minutes before use.

[0508] 6. Discard the liquid in the wells and repeat the washing steps in step 2.

[0509] 7. Add biotinylated antibody working solution (100 μL / well) to each well, cover with new sealing film, and incubate at 37°C for 1 hour.

[0510] 8. Prepare the streptavidin-HRP (100x) working solution 15 minutes before use.

[0511] 9. Discard the liquid in the wells and repeat the washing steps in step 2.

[0512] 10. Add streptavidin-HRP working solution (100 μL / well) to each well, cover with new sealing film, and incubate at 37°C for 30 minutes.

[0513] 11. Preheat the microplate reader.

[0514] 12. Discard the liquid in the wells and repeat the washing steps in step 2.

[0515] 13. Add TMB substrate (100 μL / well) to the wells. Incubate at 37°C in the dark for 15-20 minutes. 14. Add stop solution (50 μL / well) and immediately place in a microplate reader. Measure the OD value of each well at 450 nm within 5 minutes.

[0516] As shown in Figure 64, the results showed that the monocyte level and IL-β level in the MEC15μg group were significantly lower than those in the model group.

[0517] 2) ALUM-induced peritonitis model

[0518] Eight-week-old C57Bl6 mice were randomly divided into a model group and a drug-treated group, with the drug-treated groups being MEC15μg group and MEC112.5μg group. The drug-treated group was intraperitoneally injected with MEC1 small molecules on the previous day, and the model group was intraperitoneally injected with normal saline in parallel. On the second day, the drug-treated group was intraperitoneally injected with MEC1 small molecules, and the model group was injected with normal saline in parallel. One hour later, ALUM suspension was intraperitoneally injected at a dose of 50 mg / kg. Six hours later, peritoneal lavage fluid was collected, and the monocyte ratio was detected by flow cytometry, and the IL-1β level was detected by ELISA. The specific steps included: Elisa detection of IL-1β level and flow cytometric detection were the same as those of the MSU-induced peritonitis model.

[0519] As shown in FIG64 , the results showed that the monocyte level and IL-β level in the MEC15 μg group were lower than those in the model group.

[0520] 3) LPS+D-Gal-induced acute liver injury model

[0521] Nine-week-old C57Bl6 mice were randomly divided into a model group and a drug-treated group. The drug-treated groups were treated with MEC1 0.125 mg / kg, 0.5 mg / kg, and 2 mg / kg. The drug-treated group was intraperitoneally injected with MEC1 small molecules on the previous day, and the model group was intraperitoneally injected with normal saline in parallel. On the second day, the drug-treated group was intraperitoneally injected with MEC1 small molecules, and the model group was injected with normal saline in parallel. One hour later, 80 μg / kg LPS + 200 mg / kg D-Gal were intraperitoneally injected. Five hours later, blood was collected and the supernatant was centrifuged for transaminase detection.

[0522] As shown in Figure 65, LPS+D-Gal caused severe liver damage, with both AST and ALT transaminases significantly elevated. MEC1 inhibited liver damage in a dose-dependent manner, with the optimal dose being 0.5 mg / kg. However, the effect decreased at 2 mg / kg, suggesting that the "hook effect" evident in cell experiments also existed in living animals.

[0523] Example 9 Preventive and therapeutic effects of MEC1 small molecule in a high-fat-induced obesity model in mice

[0524] 1) Body Weight Change: Four-week-old male C57Bl6 mice were acclimated for one week and divided into a normal chow diet group and a 60% high-fat diet group. Body weight changes were measured weekly. Starting from the ninth week, the high-fat diet group was further divided into a high-fat control group (HFD) and a high-fat treatment group (HFD + MEC1 5μg). The high-fat treatment group received intraperitoneal injections of MEC1 (5μg) every other day, while the normal chow diet group and the high-fat control group received intraperitoneal injections of saline in parallel until week 19. The MEC1 dosing solution was prepared as follows: 5 mg of MEC1 powder was dissolved in 500 μl of DMSO to make a 10 mg / ml stock solution. This solution was diluted with saline containing 1% Tween-80 and 100 μl was injected intraperitoneally into each mouse.

[0525] As shown in Figure 66(a), the results showed that from the day of administration, the weight gain of the high-fat group was significantly slower than that of the high-fat control group, and its weight gain rate returned to the level of the normal diet group. Representative images of mice at week 19 are shown in Figure 66(b).

[0526] 2) Metabolic cages: Oxygen consumption (VO2) and carbon dioxide production (VCO2) were monitored continuously for 48 hours in normal mice (ND), mice fed a high-fat diet (HFD), and mice treated with MEC1 (HFD+MEC1). Values ​​were normalized to body weight. Mice of the same age and sex but fed a conventional diet (ND) served as controls. The following steps were performed:

[0527] Each mouse to be tested was placed in an independent cage of the integrated laboratory monitoring system (Columbus Instruments). The mice had free access to water and corresponding feed and maintained a 12-h light-dark cycle at a constant temperature of 22±0.5°C. During the feeding process, the system was used to measure the oxygen consumption and carbon dioxide production of individual mice in the cage for 48 consecutive hours to reflect the energy expenditure of individual mice.

[0528] As shown in Figure 67, the results showed that compared with the control mice fed with conventional feed, the VO2 and VCO2 of high-fat fed mice (HFD) were significantly decreased, but the carbon dioxide production and oxygen consumption of the mice in the MEC1 administration group (HDF+MEC1) were restored to a considerable extent, suggesting that MEC1 can promote fat energy consumption in obese mice.

[0529] 3) Glucose tolerance test (GTT)

[0530] The specific steps include:

[0531] The mice were fasted for 16 hours, but were not deprived of water. They were intraperitoneally injected with 50% high glucose solution at a dose of 1.5 mg / g. The tail vein blood glucose of the mice was measured using a Sinocare blood glucose meter with blood glucose test strips before injection (0 minute), 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes after injection.

[0532] As shown in Figure 68(a), the blood glucose levels of the high-fat control group were significantly higher than those of the normal diet group after 30 minutes, reflecting decreased glucose clearance. However, glucose tolerance was restored in mice given MEC1. Calculation of the area under the curve over 120 minutes, as shown in Figure 68(b), revealed statistically significant differences between the high-fat diet group and the high-fat model group.

[0533] 4) The mice were dissected and their epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) were removed and their weights were measured. As shown in Figure 69, the results showed that the fat weights of the HDF+MEC1 group were significantly lower than those of the HFD group.

[0534] 5) Blood was collected from mice by removing their eyeballs. Approximately 600 μl of whole blood was collected into a 1.5 ml EP tube. The blood was allowed to stand for 5 minutes, then centrifuged at 3000 rpm for 10 minutes. The supernatant was transferred to another fresh 1.5 ml EP tube and assayed using an automated biochemical analyzer for ALT, AST, total cholesterol, triglycerides, and low-density lipoprotein (LDL). ALT and AST are indicators of liver function, while total cholesterol, triglycerides, and LDL are important indicators of fat metabolism.

[0535] As shown in Figure 70, the results showed that compared with the HFD group, the above indicators in the HFD+MEC1 group were lower than those in the HFD group, suggesting that MEC1 can reduce liver fat synthesis and inhibit liver damage caused by high fat.

[0536] 6) Liver and adipose tissue were fixed with 4% paraformaldehyde and then stained with H&E. The following steps were performed: ① The liver tissue was fixed in formaldehyde solution, dehydrated with graded ethanol, and then embedded in paraffin. Before staining, the tissue was cut into 4 μm slices and dewaxed before H&E staining as follows:

[0537] ②Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0538] ③ Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol for 5 minutes each, and then stain them in eosin solution for 5 minutes.

[0539] ④ Dehydration and sealing: The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min until transparent, and then sealed with neutral gum.

[0540] As shown in Figure 71, the results showed that the HFD group had significant lipid droplet vacuoles in liver tissue, while the HFD+MEC1 group had smaller lipid droplets compared to the HFD group. Results in adipose tissue showed that adipocytes in the HFD group were significantly enlarged, while the HFD+MEC1 group had an increased number of adipocytes and a smaller size compared to the HFD group. Scale bar: 40 μm.

[0541] 7) Mitochondria in liver and adipose tissue were observed using an electron microscope (Tecnai G2Spirit).

[0542] As shown in Figure 72, the results show that in liver and adipose tissue, the normal diet group (ND) exhibited normal mitochondrial morphology, with no shrinkage and well-distributed cristae. However, the high-fat diet group (HFD) exhibited significantly shrunken mitochondria, smaller mitochondria, and indistinguishable cristae. However, treatment with MEC1 restored mitochondrial morphology, with visible cristae. Scale bar: 5 μm.

[0543] 8) Transcriptome sequencing: To further explore the mechanism of MEC1 in treating obesity, we performed RNA sequencing on liver tissue. The specific steps included:

[0544] ①Liver RNA extraction

[0545] Add chloroform (1 / 5 the volume of RNAiso Plus) to the liver tissue homogenate lysate, tightly cap the centrifuge tube, and mix until the solution emulsifies and turns milky white. Let it stand at room temperature for 5 minutes. Centrifuge at 12,000g for 15 minutes at 4°C. Carefully remove the tube from the centrifuge. The homogenate will now separate into three layers. Aspirate the supernatant and transfer it to a fresh tube. Add 0.5-1 times the volume of RNAiso Plus to the supernatant. Invert the tube to mix thoroughly and let it stand on ice for 10 minutes. Transfer the sample that has been treated with isopropanol to the RNA extraction column in batches. Centrifuge at 12,000g for 1 minute, discarding the liquid in the collection tube until all the sample has passed through the column. Add ethanol to the RNA extraction column and centrifuge at 12,000g for 1 minute, discarding the liquid in the collection tube. Repeat this step once. After discarding the liquid in the collection tube for the final time, spin the column once at 12,000g for 1 minute to completely remove the ethanol from the extraction column.

[0546] ②RNA library construction and quality control:

[0547] Total RNA extracted from liver tissue was enriched for poly(A)-tailed mRNA using Oligo(dT) magnetic beads, followed by random shearing of the resulting mRNA with divalent cations in Fragmentation Buffer. First-strand cDNA was synthesized using the fragmented mRNA as a template and random oligonucleotides as primers using M-MuLV reverse transcriptase. The RNA strand was then degraded with RNase H, and second-strand cDNA was synthesized using dNTPs using DNA polymerase I. The purified double-stranded cDNA was end-repaired, A-tailed, and ligated with sequencing adapters. cDNAs of approximately 370 to 420 bp were screened using AMPure XP beads, amplified by PCR, and purified again using AMPure XP beads to generate a library. After library construction, a Qubit2.0 Fluorometer was used for preliminary quantification, and the library was diluted to 1.5 ng / ul. The insert size of the library was then detected using an Agilent 2100 bioanalyzer. Once the insert size was in line with expectations, qRT-PCR was used to accurately quantify the effective concentration of the library (the effective concentration of the library was greater than 2 nM) to ensure library quality.

[0548] ③Data analysis

[0549] Image data from sequencing fragments generated by a high-throughput sequencer is converted into sequence data (reads) using CASAVA base calling. The raw data obtained from sequencing contain a small number of reads with sequencing adapters or low-quality reads. To ensure the quality and reliability of data analysis, the raw data must be filtered. This includes removing reads with adapters, reads containing N (N indicates that the base information cannot be determined), and low-quality reads (reads with Qphred <= 20 bases accounting for more than 50% of the total read length). Furthermore, Q20, Q30, and GC content are calculated for the clean data. All subsequent analyses are based on high-quality clean data.

[0550] Sequencing reads were aligned to the mouse reference genome GRCm38 using STAR-2.5.2b software. Briefly, expression values ​​were normalized and differential expression analysis was performed using the DeSeq2 package. Heatmaps were generated using z-scores after normalizing counts using the Pheatmap library in R. Gene set enrichment analysis (GSEA) was performed using software (version 4.2.3) downloaded from MSigDB to assess whether specific genes were significantly enriched in MEC1-treated liver tissue from HFD mice compared to controls.

[0551] Analysis of the expression levels of lipid synthesis-related genes in Figure 73 (a) shows that the expression levels of a considerable number of lipid synthesis-related genes in the high-fat model group (HFD) mice were significantly higher than those in the conventional diet group (ND), but the expression levels of these genes were reduced in the high-fat administration group (HFD+MEC1). Gene set enrichment score analysis (ES, see Figure 73 (b)) further supports the conclusion that lipid synthesis-related genes tend to be highly expressed in the high-fat model group mice, but MEC1 administration curbs this tendency. These results suggest that MEC1's inhibitory effect on obesity in mice fed a high-fat diet may be achieved by reducing lipid synthesis in liver tissue.

[0552] 9) The transcriptome sequencing results showed that the expression of mitochondrial autophagy-related genes was analyzed. As shown in Figure 74, the MEC1 group significantly upregulated autophagy-related genes such as LC3 (Map1lc3b), ATG13, NBR1, ATG9A, and ULK1 compared with the HFD group, further demonstrating that the MEC1 small molecule has an ameliorative effect on obesity by enhancing mitochondrial autophagy.

[0553] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solutions of the present invention may be subjected to various simple modifications, including combining the various technical features in any other appropriate manner. These simple modifications and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A chimeric molecule for enhancing mitophagy, characterized in that, the structure of the chimeric molecule is shown in Formula 1: M-L-O Formula 1 wherein, M represents a ligand of MAP3K1, L represents a linker chain, and O represents a ligand of an outer mitochondrial membrane protein; The structural formula of M includes: L is an alkoxy chain, including: -(CH 2 CH 2 O) a -, -(CH 2 CH 2 CH 2 O) b -, where a and b are natural numbers greater than or equal to 1 respectively.

2. The chimeric molecule for enhancing mitophagy according to claim 1, characterized in that, the outer mitochondrial membrane proteins include TSPO, VDAC, MIRO1, MFN, etc.

3. The chimeric molecule for enhancing mitophagy according to claim 2, characterized in that, The structural formula of O includes: wherein, X is a halogen.

4. The chimeric molecule for enhancing mitophagy according to claim 1, characterized in that, The structural formula of the chimeric molecule is shown in Formula 2 or Formula 3 as follows: Among them, n is a natural number from 1 to 10; X 1 is a halogen; Among them, m is a natural number from 1 to 10; X 2 , X 3 , X 4 are all halogens.

5. The chimeric molecule for enhancing mitophagy according to claim 1, characterized in that, the X is selected from at least one of fluorine, chlorine, bromine, and iodine.

6. The chimeric molecule for enhancing mitophagy according to claim 4, characterized in that, n is a natural number from 1 to 5.

7. The chimeric molecule for enhancing mitophagy according to claim 4, characterized in that, m is a natural number from 1 to 5.

8. The chimeric molecule for enhancing mitophagy according to claim 4, characterized in that, The chimeric molecule is selected from:

9. A pharmaceutical composition, characterized in that, it comprises the chimeric molecule for enhancing mitophagy according to any one of claims 1-6, and optionally a pharmaceutical excipient.

10. Use of the chimeric molecule for enhancing mitophagy according to any one of claims 1-8, and the pharmaceutical composition according to claim 9 in the preparation of a drug for preventing or treating a disease of mitochondrial dysfunction.

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