Application of Ivesinol derivative in preparation of autophagy activated cell model

Abstract

The invention relates to the technical field, in particular to application of an Ivesinol derivative in preparation of an autophagy activated cell model. By screening a compound library, a compound with the effect of improving the autophagy level of cancer cells and normal cells is obtained, and the compound is a small molecule compound specifically targeting autophagy process protein. Compared with other compounds with similar structures or some intermediate compounds in the synthesis process, the compound provided by the invention has a more ideal autophagy enhancement effect, which is specifically reflected on the effect of promoting the conversion of LC3-I to LC3-II in cells and the degradation of p62 protein. When the compound is used for treating cells, a corresponding autophagy activated cell model can be formed. According to the scheme, the technical problem that an autophagy model capable of avoiding non-target activation and signal channel crosstalk is difficult to effectively establish due to the lack of an efficient and specific autophagy regulating agent can be solved, and the method has ideal popularization and application prospects.

Description

Technical Field

[0001] The invention relates to the technical field, and in particular to application of ivesinol derivatives in preparing autophagy activated cell models. Background Art

[0002] Autophagy is a self-degradation process in cells, through which cells can remove damaged proteins and organelles and recycle these components. Autophagy plays an important role in maintaining the stability of the intracellular environment and coping with stress conditions such as nutrient deficiency. When cells sense hunger or other stress signals, the autophagy mechanism in the cell will be activated. This usually involves some specific protein complexes, such as the ULK1 complex, which start the autophagy process after receiving the signal. After the autophagy process begins, a double-layer membrane structure gradually forms in an area of ​​the cytoplasm, which is called the isolation membrane or phagophore. As the process develops, the isolation membrane will gradually expand and wrap around the target substance or organelle that needs to be degraded. When the isolation membrane completely wraps around the target substance, it closes into a closed vesicle, the autophagosome. The autophagosome then fuses with the lysosome to form an autophagolysosome. In the autophagolysosome, the target substance is broken down into smaller molecules, such as amino acids and fatty acids, by hydrolases in the lysosome. These molecules are then released back into the cytoplasm for reuse by the cell. Autophagy is not only essential for the health of individual cells, but is also associated with the development of a variety of diseases, including neurodegenerative diseases, cardiovascular diseases, cancer, etc. Therefore, studying the mechanism of autophagy is of great significance for understanding the pathophysiology of these diseases and developing new treatments.

[0003] Due to the importance of studying autophagy, establishing a cell model of autophagy is of great significance for scientific research, which is mainly reflected in the following aspects:

[0004] Understanding basic biological processes: Cellular autophagy is a complex biological process involving multiple signaling pathways and regulatory factors. By establishing a cell model, researchers can explore in detail the initiation, progression, and termination mechanisms of autophagy, as well as the modes of action of various molecules in the autophagy process. This is of great significance for a comprehensive understanding of how cells maintain their homeostasis through autophagy.

[0005] Disease mechanism research: Many diseases, such as neurodegenerative diseases (Alzheimer's disease, Parkinson's disease), cardiovascular diseases, diabetes, cancer, etc., are related to abnormal cellular autophagy. By constructing specific cell models, scientists can simulate the changes in autophagy under these disease states and deeply analyze the specific role of autophagy in the occurrence and development of diseases, thus providing a theoretical basis for the prevention and treatment of diseases.

[0006] Drug screening and development: Establishing a cell autophagy model helps to screen and evaluate potential therapeutic drugs. Through high-throughput screening technology, candidate drugs that can regulate autophagy activity can be quickly identified from a large number of compounds. This is crucial for the development of new therapies for diseases caused by abnormal autophagy.

[0007] Validating hypotheses and testing new approaches: Under laboratory conditions, cellular models allow researchers to precisely control experimental conditions and test various hypotheses about autophagy.

[0008] In short, establishing a cell model of autophagy is not only helpful for basic scientific research, but also provides important technical support for clinical applications and drug development. It is an indispensable part of modern life science research. Especially in some cancer treatment practices, researchers have found that the increase in autophagy levels is related to the drug resistance of cancer cells. Cancers with increased autophagy levels need new treatment methods to overcome the above problems. Therefore, it is particularly necessary to establish relevant experimental models for increased autophagy levels for the above research.

[0009] In the prior art, cell autophagy activity regulators are usually used to prepare relevant autophagy cell models. Cell autophagy activity regulators can be used as carrier models for autophagy research and observation, and have the potential to be applied to disease treatment. In recent years, they have been widely used in scientific research on disease diagnosis and treatment. However, the various existing autophagy activity regulators are not developed for autophagy. They mainly target upstream signals or affect lysosomal function. There are problems such as non-target target activation, signal pathway crosstalk, and large side effects. There is still a lack of small molecule compounds that specifically target proteins in the autophagy process. In fact, there are currently no intervention measures specifically for regulating autophagy that can be used in humans. Therefore, it is of great significance to discover and explore new, efficient and specific autophagy regulators. Summary of the invention

[0010] The purpose of the present invention is to provide an application of ivesinol derivatives in preparing a cell model of autophagy activation, so as to solve the technical problem that it is difficult to effectively establish an autophagy cell model that can avoid non-target target activation and signal pathway crosstalk due to the lack of efficient and specific autophagy regulators in the prior art.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] Application of ivesinol derivatives in preparing cell models of autophagy activation.

[0013] Furthermore, the structural formula of the ivesinol derivative is shown in Formula I:

[0014]

[0015] Furthermore, the cells were cultured in a culture medium containing 0.5-10 μM Ivesinol derivatives for 1-8 hours to obtain an autophagy activated cell model.

[0016] Furthermore, the cells were cultured in a medium containing 10 μM Ivesinol derivatives for 4 hours to obtain an autophagy-activated cell model.

[0017] Furthermore, the cells include cancer cells and / or normal cells.

[0018] Furthermore, the cells include MCF-7 cells and / or HEK293 cells.

[0019] Furthermore, the culture medium includes DMEM culture medium containing 10% fetal bovine serum.

[0020] Furthermore, the cells are cultured in a DMEM medium containing 10% fetal bovine serum for 12-16 hours until the cells grow to a confluence of 60-70%, and then the Ivesinol derivative is added.

[0021] Furthermore, in a cell model of autophagy activation, the conversion of LC3-I to LC3-II was increased, and the level of p62 protein was decreased.

[0022] Furthermore, the cell model is a breast cancer cell model with enhanced autophagy level.

[0023] The present technical solution also provides an autophagy activator, the structural formula of which is shown in Formula I:

[0024]

[0025] The technical principle of this technical solution is:

[0026] At present, the research on new autophagy activity regulators is mainly focused on three directions: small chemical molecules, active ingredients of natural products and synthesis of new chimeras. Among them, small chemical molecules and active ingredients of natural products can be collectively referred to as small molecule compounds. Small molecule compounds are an important resource for discovering new autophagy activity regulators because they are easy to administer, have a short onset time and most of them are reversible. This study screened out new autophagy regulators from a small molecule compound library, and studied their active phenotypes, structural basis and mechanism of action to better understand autophagy and explore potential clinical application scenarios. The various existing autophagy activity regulators are not developed for autophagy, but mainly target upstream signals or affect lysosomal function. There are problems such as non-target target activation, signal pathway crosstalk, and large side effects. There is still a lack of small molecule compounds that specifically target proteins in the autophagy process.

[0027] The compounds screened in this study can specifically target proteins in the autophagy process. By using the late autophagy inhibitor bafilomycin A1 and the compounds screened in this scheme to co-treat wild-type MCF-7 cells, the experimental results showed that the compounds screened in this scheme can increase both cell LC3 synthesis and autophagy flux. This means that the compound is an early activator of autophagy, and its target may be in the early steps or upstream pathways of autophagy. The compounds screened in this scheme are used to treat cancer cells or normal cells at a certain concentration for a period of time, which will promote the conversion of LC3-I to LC3-II and the degradation of p62 protein in cells, forming a cell model of autophagy activation. The compounds screened in this technical scheme produce more significant reaction phenotypes than the conventional autophagy activator Torin1 in scientific research experiments, have higher LC3-II levels and similar p62 levels than the mature autophagy activator Torin1, and the compounds in this scheme also produce more clustered fluorescent particles in the screening experiment. Therefore, the compounds proposed in this scheme have potential application value in future scientific research experiments.

[0028] As an important way to maintain cell homeostasis, autophagy can be activated by conditions such as hypoxia and oxidative stress, and then damaged organelles, cell residues, misfolded proteins, etc. are decomposed to provide energy to cells. Malignant tumors can regulate the development of tumors by activating biomolecules and pathways related to cell autophagy. In this technical scheme, the compounds screened by this scheme can effectively activate the autophagy level of breast cancer cells MCF-7, thereby affecting the development of cancer cells, treatment effects, drug resistance, recurrence and metastasis, etc. The use of the compounds of this scheme to treat breast cancer cells MCF-7 can form a breast cancer cell model with increased autophagy levels. Increased autophagy levels may have multiple effects such as increased cancer cell resistance, increased tumor growth or metastasis activity, and promotion of cell apoptosis. Therefore, the breast cancer cell model with increased autophagy levels is different from the ordinary breast cancer cell model, reflecting the different states of breast cancer disease. Different treatment methods are needed for breast cancer with increased autophagy levels, so it is necessary to use such models to conduct relevant mechanism studies and drug screening. For example, in many types of cancer, increased autophagy levels can lead to increased drug resistance in cancer cells. In this case, conventional chemotherapy drugs cannot be used to treat cancer, and new treatments that are suitable for cancers with increased autophagy levels need to be explored. Therefore, the model established in this protocol is of great significance for studying and screening drugs for treating different types of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the experimental results of screening small molecule compounds that regulate cell autophagy activity in Example 1 (A: counting the number of LC3 particles in the fluorescence microscopy results; B: fluorescence microscopy results after pEGFP-LC3 MCF-7 cells were treated with compound B2, Torin1 positive control, CQ positive control, and DMSO negative control for 4 hours, CQ: chloroquine; DMSO: dimethyl sulfoxide; C: structural formula of compound B2; D: Western blotting results after MCF-7 cells were treated with compound B2, Torin1, CQ, and DMSO for 4 hours, GAPDH: internal reference protein; E: WB results after MCF-7 cells were treated with compound B2 and A1 for 4 hours).

[0030] Figure 2 The results of the analysis of the autophagy regulating activity of Ivesinol and its derivatives in Example 1 (A: Western blot results of MCF-7 cells treated with symmetrical compounds for 4 hours; B: p62:GAPDH statistical graph in three independent experiments with symmetrical compounds; C: Western blot results of MCF-7 cells treated with asymmetrical compounds for 4 hours; D: p62:GAPDH statistical graph in three independent experiments with asymmetrical compounds; E: Western Blot results of MCF-7 cells after treatment with autophagy activators).

[0031] Figure 3 This is the LC3 protein turnover detection result under the treatment of compound B2 and / or Baf A1 in Example 3.

[0032] Figure 4 These are the concentration- and time-dependent study results on the activation of cellular autophagy by compound B2 of Example 4 (A: Western blot results of MCF-7 cells treated with different concentrations of the compound for 4 hours; B: Western blot results of HEK293 cells treated with different concentrations of the compound for 4 hours; C: Western blot results of MCF-7 cells treated with compound B2 for different time periods; D: laser confocal microscopy results of pEGFP-LC3 MCF-7 cells treated with compound B2 for different time periods; E: counting the number of LC3 particles in the fluorescence microscopy results). DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0034] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below.

[0035] The term "autophagy-activated cell model" refers to a cell model formed by increasing the level of autophagy in cells through drug treatment and other means. The increase in autophagy level is mainly reflected in the increase in the conversion of LC3-I to LC3-II and the enhanced degradation of p62 protein. Autophagy-activated cell models are important tools for studying the mechanism and function of cellular autophagy. Through these models, researchers can explore the activation mechanism of autophagy under different conditions, its biological function, and its role in disease.

[0036] The term "Chloroquine (CQ)" refers to a widely used antimalarial drug that is also used to treat autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Chloroquine is a commonly used autophagy inhibitor used to study the role of autophagy in cellular physiological and pathological processes. By comparing the autophagy levels of treated and untreated cells, changes in autophagy under specific conditions can be evaluated. Chloroquine can enhance the effects of chemotherapy and radiotherapy by inhibiting autophagy, and therefore shows potential in the treatment of certain cancers. Chloroquine is a weakly alkaline compound that can penetrate the cell membrane and enter the lysosome, raising the pH value inside the lysosome, thereby inhibiting the activity of lysosomal enzymes. This leads to the obstruction of the fusion of autophagosomes with lysosomes, and the cargo in the autophagosome cannot be effectively degraded. Due to the obstruction of the fusion of autophagosomes with lysosomes, autophagosomes accumulate in the cell, which manifests as an interruption of the autophagic flow. The inhibitory effect of chloroquine on autophagy can be evaluated by detecting the accumulation of LC3-II and the level of p62.

[0037] The term "Torin1" refers to a highly selective mTOR (Mechanistic Target of Rapamycin) inhibitor, which is widely used to study cell autophagy and other biological processes related to the mTOR signaling pathway. mTOR is a key cell signaling molecule that participates in the regulation of multiple biological processes such as cell growth, proliferation, metabolism and autophagy. Torin1 is an autophagy activator.

[0038] The term "Bafilomycin A1 (Baf A1)" refers to a potent autophagy inhibitor that mainly blocks the autophagy process by inhibiting the acidification of lysosomes to prevent the fusion of autophagosomes with lysosomes. Bafilomycin A1 is widely used in cellular autophagy research, especially in exploring the integrity of the autophagic flux and lysosomal function. The integrity of the autophagic flux can be assessed by comparing the levels of LC3-II and p62 before and after Bafilomycin A1 treatment. The accumulation of LC3-II and the increase of p62 after treatment indicate that the autophagic flux is blocked.

[0039] The term "MCF-7 cells" refers to a widely used breast cancer cell line.

[0040] The term "HEK293 cells (Human Embryonic Kidney 293 cells)" refers to a widely used immortalized human embryonic kidney cell line.

[0041] The term "pEGFP-LC3 MCF-7 cells" refers to a cell line formed by transfecting MCF-7 cells with the plasmid pEGFP-LC3. pEGFP-LC3 is a commonly used green fluorescent protein (GFP)-tagged LC3 plasmid that is often used to study cellular autophagy. By transfecting pEGFP-LC3 into MCF-7 cells, the formation and distribution of autophagosomes can be observed in real time under a fluorescence microscope.

[0042] The term "LC3 protein" refers to microtubule-associated protein 1 light chain 3. LC3 protein is a very important class of proteins in the study of cellular autophagy, and it plays a key role in the autophagy process. LC3-I: This is the non-lipidated form of LC3 and exists in the cytoplasm. It is the initial form of LC3 protein and its main function is to serve as a precursor protein in the autophagy process. LC3-II: When the cell initiates the autophagy process, LC3-I will be cut off a small C-terminal sequence under the action of Atg4 enzyme, and then combined with phosphatidylethanolamine (PE) under the catalysis of Atg7 and Atg3 enzymes to form lipidated LC3-II. LC3-II can be integrated into the autophagosome membrane and participate in the formation and maturation of autophagosomes. LC3-PE: The form of LC3-II combined with phosphatidylethanolamine is stably embedded in the autophagosome membrane until the autophagosome fuses with the lysosome and is degraded. LC3 as an autophagy marker Due to the specific localization of LC3-II on the autophagosome membrane, LC3 protein has become an important marker for detecting and studying cellular autophagy.

[0043] The term "p62", also known as SQSTM1 (Sequestosome 1), is a multifunctional protein that plays an important role in the process of cellular autophagy. Existing technologies often use Western Blot to detect the expression levels of p62 and LC3-II to evaluate the activity of cellular autophagy.

[0044] The following is further described in detail through specific implementation methods:

[0045] Example 1: Screening of autophagy modulating compounds

[0046] In the previous study, a new synthetic small molecule compound library was constructed, which contains 66 small molecule compound libraries (CN117024262A Synthesis and application of natural product Ivesinol and its derivatives). Ivesinol and its derivatives have much better inhibitory activity against drug-resistant Enterococcus faecium and Staphylococcus aureus than the current first-line clinical drugs. However, the existing technology has not yet studied and reported its other functions besides antibacterial function.

[0047] The free prototypic LC3 (pro-LC3) in the cytoplasm is cut into LC3-I by ATG4 after autophagy activation, and then couples with phosphatidylethanolamine on the autophagosome membrane to form a lipidated form of LC3, called LC3-II. Based on this mechanism, researchers have developed a fusion molecular probe of LC3 protein and GFP green fluorescent protein, which makes autophagy biogenesis easy to visualize and monitor, and the detection method is a conventional form of existing technology. Once the autophagy flux changes, many clustered green fluorescent particles (puncta) will form in the cell. At present, GFP-LC3 fluorescent probe labeling technology occupies a major position in phenotypic screening experiments to identify autophagy regulators.

[0048] The fusion molecular probe of LC3 protein and GFP green fluorescent protein was used to visualize the occurrence of autophagy. The human breast cancer MCF-7 cells with fluorescent probe pGFP-LC3 were treated with small molecule compounds at a final concentration of 20 μM for 4 hours. The green aggregated particles were observed by fluorescence microscopy to screen potential regulators of cell autophagy activity. At the same time, Western Blot was used to detect the LC3 protein level of cells after compound treatment to verify the screening results. The experimental results can be found in Figure 1 The selected small molecule compounds are specifically shown in Formula I (Compound B2). The inventors screened the compounds in the library and found that 7 small molecule compounds produced clustered fluorescent particles after treating cells, among which Compound B2 had the largest number of particles. The positive control all produced clustered fluorescent particles, while the remaining 59 compounds and the negative control group only had a diffuse fine sand-like signal background.

[0049]

[0050] The green aggregated particles were observed under fluorescence microscopy to screen potential regulators of cell autophagy activity. Figure 1 The experimental results show that the compound of formula I (i.e., compound B2) has a significant effect, and the effect of compound A5 / A3 / A2 / A1 / A4 / B1 is not as good as B2. In the group treated with chloroquine (CQ), an autophagy inhibitor, LC3-I and LC3-II increased due to the blocking of autophagy lysosome fusion and degradation; the autophagy activator Torin1 group promoted autophagy lysosome degradation, and LC3-I and LC3-II decreased, which are consistent with the change characteristics of typical LC3 under the corresponding regulatory mechanism. Among them, compounds B2 / A5 / A3 / A2 / A1 / A4 / B1 are all end products or intermediates involved in the prior patent CN117024262A.

[0051] The synthesis method of formula I is described in detail in Chinese patent CN117024262A. The specific compound of formula I conforms to the general structural formula of IVE-2 in the prior patent, and R in the general structural formula of IVE-2 is 5 and R 6 isobutyryl, more specifically, compound T-2 in patent CN117024262A. The general synthesis process is shown in Formula II (the general synthesis process of compound B2), which is generally described as follows:

[0052] Compound 1 (2,4,6-trihydroxybenzaldehyde) in Formula II undergoes methylation reaction (a) to obtain compound 2; Compound 2 undergoes aldehyde reduction reaction (b) to obtain compound 3; Compound 3 and compound 4 (isobutyryl chloride) undergo Friedel-Crafts acylation reaction (c) to obtain compound 5. Compound 6 (phloroglucinol) in Formula II undergoes methylation (d) to obtain compound 7; Compound 7 undergoes Friedel-Crafts acylation reaction (e) to obtain compound 8; Compound 8 undergoes aldehyde introduction reaction (f) to obtain compound 9, and then the aldehyde group of compound 9 is reduced (g) to obtain compound A1. Compound 5 in Formula II and compound A1 undergo coupling reaction (h) to obtain compound B2 and / or B1 and / or C1. Among them, compound B2 is a drug with the function of promoting autophagy. The specific synthesis method of compound B2 (Formula I) can be found in Example 3 "Synthesis of Derivative T-2" in patent CN117024262A. Since the specific synthesis method has been disclosed, it will not be repeated here.

[0053] in addition, Figure 1 The experimental data of compound A5 / A3 / A2 / A1 / A4 are also shown. A5 / A3 / A2 / A1 / A4 are intermediate products in the synthesis process. The structural formula of A1 refers to the corresponding compound in formula II. The structural formula of compound A4 / A3 / A2 refers to the corresponding compound in formula III (compound 6 is reacted to obtain compound 10 / A4 / A3 / A2, which is then reacted to obtain compound D1). The structural formula of compound A5 refers to the corresponding compound in formula IV (compound 11 is reacted to form compound 12, which is then reacted to form compound A5). None of these comparative compounds have the effect of regulating cell autophagy.

[0054]

[0055] Example 2: Drug activity comparison experiment

[0056] Because LC3 is also involved in atypical autophagy and has non-autophagic functions, changes in LC3 levels only indicate autophagic carrier flux, not actual autophagic flux. At the same time, the autophagy receptor SQSTM1 / p62 can also be used as a protein marker for detecting autophagy. p62 and its bound ubiquitinated cargo are bound to complete autophagosomes and degraded in autophagic lysosomes, thus serving as an indicator of autophagic cargo degradation. Combining the LC3 and p62 change trends, it can be determined whether the cell autophagic flow is regulated. Further screening studies were conducted in this embodiment.

[0057] In the process of drug screening, the inventors conducted experimental studies on a variety of compounds with similar structures. The LC3 and p62 signals of wild-type MCF-7 cells treated with symmetrical compounds (B1-B8) and asymmetrical compounds (C1-C7) at a concentration of 20 μM for 4 hours were detected by Western Blot experiments. The structural formula of compound B1 specifically refers to the corresponding compound of formula II, compound B2 is formula I of this scheme, the structural formula of compound B3 / B4 specifically refers to the corresponding compound of formula VI, the structural formula of compound B5-B8 specifically refers to the corresponding compound of formula V; the structural formula of compound C1 specifically refers to the corresponding compound of formula II, and the structural formula of C2-C7 specifically refers to the corresponding compound in formula V. The above compounds have certain structural similarities and are all Ivesionol and its derivatives, but they show different effects on autophagy.

[0058]

[0059] The experimental results can be found in Figure 2 Through the experimental results and the statistical graph of the p62:GAPDH ratio in three independent experiments, it can be observed that compared with the negative control (DMSO group), the LC3-II / LC3-I ratio increased and the p62 / GAPDH ratio decreased in cells treated with compounds B2, B6-B8, C1-C5 and C7, indicating that the conversion of LC3-I to LC3-II and the degradation of p62 protein occurred in these 10 experimental groups, that is, cellular autophagy was activated. At the same time, it was also observed that other compounds did not show relevant responses or produced a slight autophagy inhibition effect.

[0060] In addition, we compared the above 10 compounds with autophagy stimulating activity in the same Western Blot experiment (20μM concentration of compounds treated wild-type MCF-7 cells for 4 hours). All compound treatment groups showed an increase in LC3-II and a decrease in p62, and it was found that the change trend in compound B2 group was the most obvious. For specific experimental results, see Figure 2. The above compounds have certain structural similarities, and only compound B2 shows the most significant ability to activate autophagy, which is specifically manifested in the degradation of p62 protein and the conversion of LC3-I to LC3-II. More specifically, the only difference between compound B1 and compound B2 selected in this scheme is the position of the two methoxy groups, which results in B1 basically not having the effect of promoting the conversion of LC3-I to LC3-II and the degradation of p62 protein, that is, B1 has no regulatory effect on cell autophagy. The hydroxyl group (-OH) at the 3 / 3' position instead of the methoxy group (-OMe) can enhance the activity of the compound in promoting autophagy. In addition, compared with B2 of this scheme, compound B6 has the same side chains at the 1 / 1' and 3 / 3' positions, and only the 4 / 4' side chain is slightly different. The experimental results show that the autophagy activation activity of compound B2 is significantly higher than that of compound B6, which indicates that the R / R' side chain group at 4 / 4' is the key to the level of autophagy activation of such compounds, and the R / R' side chain at 4 / 4' may be the most contributing active site.

[0061] In summary, compound B2 exerts the best autophagy activation effect, that is, it has the strongest biological activity in activating cell autophagy among these analogs. In addition, compound B2 is active in the range of 5-40 μM, with a good concentration correlation in the range of 5-20 μM, and the above experimental data show that compound B2 produces a more significant reaction phenotype than Torin1, a conventional autophagy activator in scientific research experiments. Therefore, the working concentration of compound B2 was subsequently set at 10 μM.

[0062] The compound B2 group has higher LC3-II levels and similar p62 levels than the mature autophagy activator Torin1. It was also noted that compound B2 also produced more clustered fluorescent particles in the screening experiment. Combining these two aspects, compound B2 is more convenient for observing autophagy activity phenotype experiments than Torin1, a commonly used autophagy activator in scientific research experiments. In addition, compound B2 has good concentration- and time-dependent biological activity. Therefore, the proposed compound B2 in this scheme has potential application value in future scientific research experiments.

[0063] Example 3: Study on the mechanism of action of compound B2 as an early activator of autophagy

[0064] In the previous experiment, compound B2 had the strongest biological activity for activating cell autophagy among a series of similar compounds. Therefore, the following method was used to detect the effect of compound B2 on autophagy-related proteins under different treatment conditions by Western blotting and confocal microscopy observation, so as to understand the characteristics of its ability to activate autophagy. In recent years, in order to describe in detail the changes in intracellular autophagic flow, researchers have proposed an experiment called LC3 turnover, the principle of which is to detect LC3 and p62 proteins in the presence and absence of sufficient inhibitors and analyze the trend of changes. Among them, the selection of inhibitors is wide, including compounds that inhibit proteases, regulate lysosomal pH, block the fusion of autophagosomes and lysosomes, and knock out membrane proteins (LAMP2) associated with lysosomes. Compared with protease inhibitors, bafilomycin A1 (1-100nM) can be added immediately before autophagy induction. In the turnover experiment, if a change in autophagic flux occurs, the content of LC3-II will be higher in the presence of inhibitors.

[0065] In order to study the changes in intracellular autophagic flux in detail, the LC3 turnover experiment was used to analyze the changes in autophagic flux. The wild-type MCF-7 cells were treated with 100nM concentration of late autophagy inhibitor bafilomycin A1 (Baf A1) and 10μM concentration of compound B2 alone or in combination for 4 hours. The results showed that the degree of increase in the LC3-II / LC3-I ratio and p62 protein level was from high to low in the bafilomycin A1 alone treatment group, the combined treatment group and the B2 alone treatment group, indicating that B2 treatment can increase both the synthesis and conversion of cellular LC3-I and the autophagic flux. This means that compound B2 activates autophagic targets in the early steps or upstream pathways of autophagy. For detailed experimental results, see Figure 3 Compound B2 is an early activator of autophagy, which targets the early steps or upstream pathways of autophagy.

[0066] Example 4: Study on the concentration- and time-dependent activation of autophagy by compound B2

[0067] In order to analyze the activation of cell autophagy by different concentrations of compound B2, we treated MCF-7 and HEK293 cells with compound B2 at gradient concentrations (0.5μM, 1μM, 2μM, 5μM, 10μM) within the working concentration for 4 hours, and then detected the signals of autophagy-related proteins LC3 and p62 by WB experiments. The results showed that with the increase of compound B2 treatment concentration, LC3-II increased and p62 protein decreased in both MCF-7 and HEK293 cells, indicating that compound B2 has the ability to activate cell autophagy in a concentration-dependent manner in both tumor cells and normal cells within the working concentration. For detailed experimental results, see Figure 4 A and B.

[0068] In addition, the results were different when the treatment time (1 hour, 2 hours, 4 hours, 6 hours, and 8 hours) of B2 at the same working concentration (10 μM) was different. Within 4 hours, as the treatment time increased, LC3-II increased and p62 protein decreased; when the treatment time exceeded 6 hours, both LC3-II and p62 protein recovered from the previous trend, but were still different from the negative control group. For detailed experimental results, see Figure 4 CE.

[0069] In combination with the above content, the method for establishing a cell model with enhanced autophagy is as follows:

[0070] MCF-7 cells are added to DMEM medium containing 10% fetal bovine serum (FBS) at a seeding density of 40,000 to 60,000 cells / mL (preferably 40,000 cells / mL). After culturing for 12-16 hours (preferably 16 hours), the 24-well plate grows to 60-70%, and compound B2 is added at a final concentration of 0.5-10 μM (preferably 10 μM) and cultured for 1-8 hours (preferably 4 hours) to obtain the autophagy activation model of MCF-7 cells.

[0071] HEK293 cells were added to DMEM medium containing 10% fetal bovine serum (FBS) at a seeding density of 60,000 to 80,000 cells / mL (preferably 6 cells / mL), and cultured for 12-16 hours (preferably 16 hours). Compound B2 was added at a final concentration of 0.5-10 μM (preferably 10 μM) and cultured for 1-8 hours (preferably 4 hours) to obtain an autophagy activation model of HEK293 cells.

[0072] In summary (Examples 1-4), this scheme screened out a target small molecule compound B2 with good autophagy activation activity from the newly synthesized small molecule compound library. Using the MCF-7 cell line that stably expresses the GFP-LC3 probe, a cell imaging-based screening method was used to find that 7 compounds in the newly synthesized compound library can significantly induce the formation of cellular autophagy marker GFP-LC3 dots; statistical analysis found that compound B2 can significantly enhance the formation of cellular autophagosomes. WB results showed that compound B2 treatment can increase the LC3-II / LC3-Ⅰ ratio in MCF-7 cells and significantly reduce the level of p62. The above results show that compound B2 can significantly activate cellular autophagy.

[0073] Based on western blot experiments, it was found that 10 compounds could increase the LC3-II / LC3-Ⅰ ratio and reduce the p62 level in MCF-7 cells, among which compound B2 had the most obvious trend. The above results show that compound B2 has the best activity in activating cell autophagy in this derivative library. Through structure-activity analysis and discussion, it was found that the activity of compound B2 originated from the chemical skeleton of the pyrogallol dimer and the three side chain groups. Among them, the activity of the pyrogallol analog chemical skeleton dimer structure is better than the monomer structure; the R / R' side chain group at the 4 / 4' position of the three side chain groups affects the main activity of the compound, and a carbonyl structure is required.

[0074] Based on the LC3 turnover experiment, the co-treatment group in the WB results can increase LC3-II and p62 proteins. Based on the concentration gradient experiment, the WB results showed that compound B2 increased the concentration-dependent LC3-Ⅱ / LC3-Ⅰ ratio and reduced the p62 level in both MCF-7 and HEK293 cell lines. Based on the time gradient experiment, the WB results showed that compound B2 increased the time-dependent LC3-II / LC3-Ⅰ ratio and reduced the p62 level in MCF-7 cells; and confocal microscopy observed the time-dependent induction of the formation of GFP-LC3 dots in MCF-7 cells that stably expressed GFP-LC3. The above results show that compound B2 can activate the early steps of cell autophagy in a concentration- and time-dependent manner.

[0075] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Application of Ivesinol derivatives in the preparation of autophagy-activated cell models.

2. The use of the ivesinol derivative according to claim 1 in preparing a cell model for autophagy activation, characterized in that: The structural formula of ivesinol derivatives is shown in Formula I:

3. The use of the ivesinol derivative according to claim 2 in preparing a cell model for autophagy activation, characterized in that: The cells were cultured in a medium containing 0.5-10 μM Ivesinol derivatives for 1-8 hours to obtain an autophagy activated cell model.

4. The use of the ivesinol derivative according to claim 3 in preparing a cell model for autophagy activation, characterized in that: The cells were cultured in a medium containing 10 μM Ivesinol derivatives for 4 hours to obtain an autophagy activated cell model.

5. Use of the ivesinol derivative according to claim 4 in preparing a cell model for autophagy activation, characterized in that: The cells include cancer cells and / or normal cells.

6. Use of the ivesinol derivative according to claim 5 in preparing a cell model for autophagy activation, characterized in that: The cells include MCF-7 cells and / or HEK293 cells.

7. Use of the ivesinol derivative according to claim 6 in preparing a cell model for autophagy activation, characterized in that: The culture medium comprises a DMEM culture medium containing 10% fetal bovine serum; the cells are cultured in the DMEM culture medium containing 10% fetal bovine serum for 12-16 hours until the cells grow to a confluence of 60-70%, and then the Ivesinol derivative is added.

8. Use of the ivesinol derivative according to claim 7 in preparing a cell model for autophagy activation, characterized in that: In cell models of activated autophagy, the conversion of LC3-I to LC3-II was increased, and the level of p62 protein was decreased.

9. Use of the ivesinol derivative according to claim 8 in preparing a cell model for autophagy activation, characterized in that: The cell model is a breast cancer cell model with enhanced autophagy level.

10. An autophagy activator, characterized in that: Its structural formula is shown in Formula I:

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