Application of phellinus linteus in preparation of TRIM24 protein inhibitor
By binding linalool to the TRIM24 protein, the lack of TRIM24 protein-targeting biological agents in existing technologies has been solved, achieving inhibition of tumor cell proliferation and reduction of drug resistance, and providing a safe and effective tumor treatment option.
Patent Information
- Application Number
- CN202511386043.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-26
- Publication Date
- 2025-11-14
AI Technical Summary
The lack of effective TRIM24 protein-targeting biological agents in the current technology makes it difficult to solve the problems of tumor recurrence and drug resistance in many cancer patients after surgery, and common targeted drugs have problems of drug resistance or toxic side effects.
Using linalool as a TRIM24 protein inhibitor, it inhibits the activity of TRIM24 protein by binding to it, thereby reducing the proliferation and drug resistance of tumor cells.
Phellinus linteus can directly bind to the TRIM24 protein, inhibiting its role in promoting tumor proliferation and reducing the tolerance of tumor cells to anti-tumor drugs, thus providing a safe and inexpensive tumor treatment option.
Smart Images
Figure CN120938993A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, specifically to the application of linalool in the preparation of TRIM24 protein inhibitors. Background Technology
[0002] Malignant tumors have become one of the most serious threats to human health. Colorectal cancer, as one of the most common cancers in humans, ranks third in incidence and second in mortality worldwide. Currently, surgery, chemotherapy, radiotherapy, and even immunotherapy are commonly used in clinical practice to eliminate most tumor cells. However, many patients still cannot be cured after surgery. The reasons are: (1) Most patients are diagnosed at an advanced stage, and surgical resection has limited effect. The residual cancer cells after surgery will form new lesions, thereby causing tumor recurrence and proliferation; (2) Common targeted drugs (Regorafenib, Bevacizumab, Cetuximab, Panitumumab, etc.) have various defects such as drug resistance or toxic side effects, resulting in some patients being unable to undergo surgery and having no drugs available. There is an urgent need to develop new targeted anticancer agents.
[0003] TRIM24 is a member of the Trim family of proteins and belongs to the transactivator group. Recent studies have shown that TRIM24 is closely related to the development and progression of various tumors, including liver cancer, gastric cancer, lung cancer, bladder cancer, leukemia, and thyroid cancer. Overexpression of TRIM24 often indicates a worse prognosis and higher malignancy in cancer patients, and can be used as a prognostic indicator. TRIM24 can exert transcriptional inhibition of tumor cell apoptosis, induce cell proliferation, invasion, and proliferation, and even induce chemoresistance in tumor cells through pathways such as AKt phosphorylation and histone deacetylation. For example, TRIM24 mainly upregulates the expression of cyclin A, B, D1, E, p-Rb, CDK4, Ki67, Bcl-2, Snail, Slug, vimentin, and β-catenin, as well as other cell cycle proteins and anti-apoptotic proteins. Simultaneously, it inhibits tumor suppressor genes such as P27, P53, Bax, Caspase-8, and EMT-related proteins, leading to abnormal cell proliferation. TRIM24, as a transcriptional co-regulator, plays a crucial regulatory role in controlling cell growth, proliferation, differentiation, and tumor formation, proliferation, and recurrence. TRIM24 can influence the tumor cell cycle by regulating tumor-related proteins and signaling pathways, promoting cell proliferation, inhibiting apoptosis, promoting tumor proliferation and recurrence, and even inducing chemoresistance. It is significant in various tumor cell types. These findings indicate that TRIM24 has a broad role in tumorigenesis and development, making it a promising target for cancer therapy. However, there are currently very few biologics that directly target TRIM24. Therefore, developing inhibitors of the TRIM24 protein that exert effects both in vitro and in vivo is essential.
[0004] Plant natural products, as endogenous small molecules, participate in important physiological activities, such as regulating plant growth and development and resisting pests and diseases. They are also a major source of innovative drug molecules for treating major human diseases. Mulberrin is a natural compound extracted from plants such as mulberry trees. However, to date, there is no research on mulberrin targeting the TRIM24 protein. Summary of the Invention
[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides the application of linalool in the preparation of TRIM24 protein inhibitors.
[0006] The technical solution of the present invention is as follows:
[0007] This invention first provides the application of linalool in the preparation of TRIM24 protein inhibitors.
[0008] The mulberry ketone described in this invention is a known compound, with the English name Mulberrin and its structural formula as follows:
[0009]
[0010] This invention also provides the application of linalool in the preparation of drugs with anticancer effects.
[0011] Preferably, the cancer is colorectal cancer.
[0012] Preferably, the linalool, as a TRIM24 protein inhibitor, exerts its anti-cancer effect by binding to the TRIM24 protein.
[0013] This invention also provides the application of linalool in reducing the resistance of tumor cells to anti-tumor drugs.
[0014] Preferably, the application of linalool in reducing the resistance of tumor cells to antitumor drugs induced by high TRIM24 expression.
[0015] Preferably, the application of linalool as a TRIM24 protein inhibitor in reducing the resistance of tumor cells to antitumor drugs caused by high TRIM24 expression.
[0016] Preferably, the protein inhibitor or drug comprises a therapeutically effective amount of linalool and a pharmaceutically acceptable carrier.
[0017] Preferably, the protein inhibitor or drug can be formulated as an oral or injectable preparation.
[0018] Preferably, the oral preparations include capsules, tablets, granules, powders, pills, drop pills, sustained-release preparations, oral liquids, mixtures, and syrups.
[0019] Preferably, the oral preparation includes liquid injection, powder for injection, and tablet for injection.
[0020] Beneficial effects:
[0021] (1) TRIM24 protein is associated with various cancers, such as colorectal cancer and lung cancer. TRIM24 protein is highly expressed in colorectal cancer. In this invention, it was found that linalool can directly bind to TRIM24 protein and can be used to treat tumor drug resistance caused by high TRIM24 expression.
[0022] (2) This invention discovered that linalool can directly bind to the TRIM24 protein. Therefore, linalool can be used as a TRIM24 protein inhibitor to address the problem of tumor proliferation caused by TRIM24 protein activation. In addition, this invention also found that as the concentration of linalool increases, the tumor cell proliferation ability decreases, providing an effective approach for subsequent research on TRIM24 protein inhibitors or anti-tumor drugs.
[0023] (3) This invention utilizes mulberry extract as an inhibitor of TRIM24 protein to inhibit tumor cell proliferation. It has the following advantages: ① Mulberry extract directly acts on TRIM24 protein to inhibit its activity; ② Mulberry extract has high safety, low price, and good prospects for development and utilization. Attached Figure Description
[0024] Figure 1 The figure shows the results of an experiment demonstrating that linalool can inhibit the proliferation of colon cancer cells.
[0025] Figure 2 The figure shows the experimental results of SPR experiments demonstrating that linalool can directly bind to the TRIM24 protein.
[0026] Figure 3 The figure shows the experimental results of cell thermal migration experiments demonstrating that linalool can increase the thermal stability of TRIM24 protein.
[0027] Figure 4 Figure showing the experimental results of successfully constructing TRIM24 knockout cells and control cells.
[0028] Figure 5 The cell proliferation experiment demonstrated that knocking out TRIM24 weakened the inhibitory effect of linalool on colon cancer proliferation (the inhibitory effect of linalool was weakened in knockout cells). Experimental results are shown in the figure.
[0029] Figure 6 This diagram illustrates the molecular docking simulation of the binding pattern between linalool and TRIM24 protein. Detailed Implementation
[0030] The present invention will be further described in detail below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.
[0031] 1. Both linalool and linalool can inhibit the proliferation of colon cancer cells. Figure 1 )
[0032] Cell proliferation detection
[0033] a) The cells were seeded into a 96-well plate at a density of 3000 cells per well;
[0034] b) After cell adhesion, cells were treated with different concentrations of drugs for 24h, 48h, or 72h;
[0035] c) Remove the culture medium from the 96-well plate, wash twice with PBS, add culture medium containing 10% WST-1, and incubate at 37°C for 2 hours;
[0036] d) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell proliferation inhibition rate.
[0037] 2. To determine whether linalool binds to TRIM24 protein, we used the SPR experiment for analysis. Figure 2 ) and Cell thermal migration assay (CETSA) Figure 3 This demonstrates that linalool binds to the TRIM24 protein:
[0038] Purified TRIM24 protein (catalog number: Ag5417) was purchased from the ProteinTech website. Analysis was performed using SPR (Spray-Return) assays, and the results are as follows: Figure 1 As shown, this indicates that there is a direct interaction between mulberry rosin and the TRIM24 protein, meaning that mulberry rosin can bind to the TRIM24 protein.
[0039] Figure 2 Experimental steps: Ligand immobilization
[0040] The activator is prepared by mixing 400 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) with 100 mM NHS (N-hydroxysuccinimide) just before injection.
[0041] The mixture was injected into the chip at a flow rate of 20 μL / min, and the chip was activated for 240 seconds. TRIM24 protein was diluted to 30 μg / mL with immobilization buffer and then injected into the sample channel at a flow rate of 20 μL / min. Finally, the chip was blocked with 1M ethanolamine hydrochloride at a flow rate of 20 μL / min for 240 seconds.
[0042] A multi-cycle method was used to analyze the analytes: Mulberrin was diluted to six concentrations (50, 25, 12.5, 6.25, 3.125, and 0 μM) using the same analyte buffer. Mulberrin was injected into the sample channel at a flow rate of 20 μL / min, with a binding phase lasting 240 seconds followed by a dissociation phase lasting 360 seconds. Both binding and dissociation processes were performed within the analyte buffer. Six analyte cycles were repeated in ascending order of concentration. After each cycle of interaction analysis, the sensor chip surface was regenerated for 10 seconds at a flow rate of 150 μL / min using 10 mM glycine-hydrochloric acid buffer as the injection buffer to remove the analytes. The injection and regeneration steps were then repeated for the next concentration of Mulberrin.
[0043] Figure 3 Experimental Procedure: Cells were collected after treatment with lindane (50 μM) or DMSO for 1 hour and resuspended in PBS supplemented with a mixture of protease inhibitors (cocktail, Sigma, St. Louis, MO, USA, 1:100) and PMSF (Beyotime, 1 mM). The resulting cell suspension was aliquoted into 50 μL portions, heated at a specified temperature for 3 minutes, and then cooled to room temperature for 5 minutes. The samples were then lysed using three freeze-thaw cycles in liquid nitrogen and a 25°C water bath. To separate soluble components, the heated lysates were centrifuged at 16,000 × g for 15 minutes at 4°C. Soluble proteins in the supernatant were analyzed by Western blot, with band intensities normalized to the lowest temperature band intensity.
[0044] Western blot experimental procedures
[0045] Add an appropriate amount of 5× loading buffer to each sample, mix well, and boil in water at 95℃ for 10 min; cool the sample on ice and centrifuge for a few seconds to prepare for electrophoresis loading; use 80V at the beginning of the electrophoresis stage, and increase the voltage to 120V after the sample enters the separating gel until the molecular weight of the protein to be detected is separated, then prepare for membrane transfer; cut a PVDF membrane 8.5cm long and 5cm wide, and activate it in methanol for a few seconds; place the electrophoresis gel on the black side of the transfer clamp, then place the activated PVDF membrane, filter paper, etc. in sequence, place the transfer clamp into the transfer tank, add transfer buffer, and start the transfer; transfer at 235mA for 2.5h. The transfer was performed at low temperature. After the transfer, the PVDF membrane was blocked with 5% skim milk on a horizontal shaker for 2 hours. After blocking, the PVDF membrane was washed with TBST, and the region containing the target protein was cut off. The corresponding primary antibody was added, and the membrane was incubated overnight at 4°C. The PVDF membrane was washed three times with TBST buffer on a shaker for 10 minutes each time. The corresponding secondary antibody diluted with 5% skim milk was added, and the membrane was incubated at room temperature for 2 hours. The PVDF membrane was washed three times with TBST buffer on a shaker for 10 minutes each time. The prepared chemiluminescent reagent was evenly dropped onto the PVDF membrane and developed using a chemiluminescence image analyzer.
[0046] 3. Experiments have confirmed that linalool does indeed exert its anti-cancer effect through TRIM24.
[0047] We purchased the TRIM24 knockout plasmid (Beyotime, product number: L06905) and constructed a stable TRIM24 knockout cell line (sgTRIM24) and control cells (sgcon) in colorectal cancer cells. Figure 4 The process of constructing stable cells is as follows:
[0048] (1) Plate 293T cells and prepare for plasmid transfection when the density reaches about 50%.
[0049] (2) 293T cells were simultaneously transfected with the TRIM24 knockout plasmid and pMDLg / pRRE (Addgene)
[0050] A mixed plasmid of pRSV-Rev (Addgene #12251), pRSV-Rev (Addgene #12253), and Pmd2.G (Addgene #12259) was used, with the mass ratio of the three packaging plasmids to the target plasmid (TRIM24 knockout plasmid) being 1:1:1:1.5.
[0051] (500ng:500ng:500ng; 1.5ug)) plasmid; after transfection overnight, remove the supernatant and add 1640 medium without antibiotics;
[0052] (3) Collect cell supernatant twice, after 24h and 48h. At this time, the supernatant already contains the virus. Mix the supernatant collected twice.
[0053] (4) Centrifuge the collected supernatant to remove cell precipitate and filter it with a 0.45 nm filter membrane;
[0054] (5) Add hexammonium bromide to the filtered supernatant at a ratio of 1:500 and then add it to the colon cancer cells that have been pre-coated for infection;
[0055] (6) 48 hours after infection, remove the virus-containing culture medium and replace it with normal complete culture medium to continue culturing;
[0056] (7) When the cell density reaches about 50%, add puromycin to make the total concentration 1 μg / mL for screening;
[0057] (8) Continuously observe cell death and stop administering the drug when cell death ceases.
[0058] (9) Collect the selected stable TRIM24 knockout cell lines for subsequent experiments.
[0059] The successfully constructed TRIM24 knockout cells and control cells were subjected to cell proliferation experiments. Figure 4 The study found that knocking out TRIM24 rendered mulberry senna resistant to cancer, indicating that TRIM24 is indeed the target site for mulberry senna to exert its anti-cancer effect in colorectal cancer. The cell proliferation experiment steps are as follows:
[0060] Figure 5 Experimental steps:
[0061] a) The cells were seeded into 96-well plates at a density of 3000 cells / well; the lindane was dissolved in DMSO (dimethyl sulfoxide) to prepare a stock solution with a concentration of 10 mM, and then the stock solution was diluted with 1640 cell culture medium (purchased from Life Technologies, Gaithersburg, MD, USA) to prepare working solutions of different concentrations.
[0062] b) After cell adhesion, cells were treated with different concentrations (10, 20 μM) of drug for 24 h, 48 h, or 72 h;
[0063] c) Remove the culture medium from the 96-well plate, wash twice with PBS, add culture medium containing 10% WST-1, and incubate at 37°C for 2 hours;
[0064] d) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell proliferation inhibition rate.
[0065] 4. To further investigate the binding mode between linalool and TRIM24 protein, Discovery Studio software was used to perform computational simulations of the interaction between linalool and TRIM24, and potential binding sites were identified. The results are as follows: Figure 6 As shown, it is predicted that linalool can bind to the TRIM24 protein. These results confirm that linalool can act as an inhibitor of the TRIM24 protein. This suggests that inhibiting TRIM24 by linalool is an effective treatment for colorectal cancer and has broad application value in the clinical treatment of colorectal cancer.
Claims
1. Application of linalool in the preparation of TRIM24 protein inhibitors.
2. Application of linalool in the preparation of drugs with anticancer effects.
3. The application according to claim 2, characterized in that, The cancer mentioned is colorectal cancer; Preferably, the linalool, as a TRIM24 protein inhibitor, exerts its anti-cancer effect by binding to the TRIM24 protein.
4. Application of linalool in reducing tumor cell resistance to antitumor drugs.
5. The application according to claim 4, characterized in that, Application of linalool in reducing tumor cell resistance to antitumor drugs induced by TRIM24 overexpression.
6. The application according to claim 4, characterized in that, Application of linalool as a TRIM24 protein inhibitor in reducing the resistance of tumor cells to antitumor drugs caused by high TRIM24 expression.
7. The application according to any one of claims 1 to 6, characterized in that, The protein inhibitor or drug described includes a therapeutically effective amount of linalool and a pharmaceutically acceptable carrier.
8. The application according to any one of claims 1 to 6, characterized in that, The protein inhibitor or drug may be formulated into oral or injectable preparations.
9. The application according to claim 8, characterized in that, The oral preparations include capsules, tablets, granules, powders, pills, drop pills, sustained-release preparations, oral liquids, mixtures, and syrups.
10. The application according to claim 8, characterized in that, The oral preparations include liquid injections, powders for injection, and tablets for injection.