Application of artesunate in preparation of PRMT1 inhibitor and / or alpha-ketoglutaric acid supplement
Artesunate addresses the shortcomings of existing PRMT1 inhibitors and α-ketoglutarate supplements in tumor immunotherapy by targeting PRMT1 and reprogramming IDH2-mediated mitochondrial metabolism, achieving significant effects in inhibiting tumor growth and enhancing anti-tumor immunity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-09
- Publication Date
- 2026-03-10
AI Technical Summary
Existing PRMT1 inhibitors and α-ketoglutarate supplements have problems such as poor activity, insufficient selectivity and high toxicity in tumor immunotherapy, and there is a lack of effective means to enhance the efficacy of anti-tumor immunotherapy.
Artesunate was used as a PRMT1 inhibitor and/or α-ketoglutarate supplement. By targeting and binding to PRMT1, it inhibited its arginine methyltransferase activity and reprogrammed IDH2-mediated mitochondrial metabolism, inducing α-ketoglutarate accumulation and promoting anti-tumor immunity.
It significantly inhibited tumor growth in various tumor-bearing mice, promoted anti-tumor immunity, increased the number of tumor-infiltrating T cells, and improved the efficacy of tumor immunotherapy, while having no significant effect on mouse body weight.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the use of artesunate in the preparation of PRMT1 inhibitors and / or α-ketoglutarate supplements. Background Technology
[0002] In recent years, tumor immunotherapy has become a mainstream approach to cancer treatment due to its significant efficacy and low toxicity. Immune checkpoint therapy (ICT), in particular, has shown remarkable efficacy in some cancers, including melanoma. However, most patients either do not respond initially or eventually relapse due to acquired tumor resistance. Therefore, there is an urgent need to explore new immunotherapeutic methods to further improve the efficacy of treatment for cancer patients.
[0003] Arginine methylation is a post-translational modification involved in regulating various biological processes, including transcription, RNA splicing, and DNA damage responses, and is associated with cancer pathogenesis and progression. Protein arginine methyltransferase 1 (PRMT1) is a major member of PRMTs in mammalian cells, catalyzing monomethylation (MMA) and asymmetric dimethylation (ADMA) of arginine residues. PRMT1 was initially identified as regulating chromatin accessibility through histone H4 methylation. Subsequently, various non-histone targets of PRMT1, such as CIITA and EZH2, were discovered. Recent studies have increasingly emphasized the crucial role of PRMT1 in regulating anti-tumor immunity. For example, PRMT1-mediated cGAS methylation inhibits the cGAS / STING / PD-L1 signaling pathway, thereby enhancing immune surveillance efficacy; PRMT1 deficiency activates the interferon pathway, enhancing the efficacy of ICT in melanoma; and PRMT1 negatively regulates p-STAT1-driven MHC I expression, thereby limiting the efficacy of PD-1 immunotherapy. These findings suggest that inhibiting PRMT1 may be a promising adjunctive strategy to enhance ICT. The above studies indicate that inhibiting PRMT1 can significantly enhance anti-tumor immunity to suppress tumor progression. However, current PRMT1 small molecule inhibitors generally suffer from poor activity, insufficient selectivity, and high toxicity. Therefore, further development of novel small molecule inhibitors targeting PRMT1 is beneficial for improving the treatment efficacy for cancer patients.
[0004] Cancer cells compete for and consume essential nutrients to meet their rapid proliferation needs or modulate the tumor immune microenvironment to achieve immune evasion. Alpha-ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid (TCA) cycle and glutamate metabolism, primarily produced by oxidative decarboxylation of isocitrate dehydrogenase 1 / 2 (IDH1 / 2) or deamination of glutamate dehydrogenase 1 (GDH1). α-KG affects tumor-related signaling pathways through multiple mechanisms and is considered a potential anticancer drug. Dietary α-KG supplementation has shown some positive effects in treating age-related diseases, including cancer. By modulating the activity of chromatin epigenetic modifying enzymes, α-KG can alter DNA and histone methylation patterns, thereby inhibiting tumor progression. For example, α-KG supplementation can induce hypomethylation of WNT target gene site DNA and histone H3K4me3, thereby inhibiting the growth of colorectal cancer. In terms of anti-tumor immunomodulation, α-KG enhances the expression of β2-microglobulin (B2M) by regulating the demethylation of H3K4me1, thereby improving the efficacy of PD-1 blockade immunotherapy in renal cell carcinoma. The mutant IDH1 (mIDH1) inhibits α-KG-dependent enzymes, reducing CD8+ T cell recruitment and interferon-γ (IFNγ) levels in cholangiocarcinoma. Nevertheless, the precise role of α-KG as an anticancer drug and its impact on tumor immunity require further investigation. Furthermore, identifying effective α-KG supplements could also facilitate the development of new potential anticancer drugs.
[0005] Artemisinin and its derivatives, derived from the traditional Chinese medicine Artemisia annua, are effective against parasitic diseases such as malaria parasites. They also possess anti-inflammatory, anti-tumor, and immunomodulatory functions, showing significant efficacy against leukemia, breast cancer, lupus erythematosus, rheumatism, and aging. Recent studies have shown that artemisinin-based drugs also exhibit good effects in inhibiting the proliferation and inducing apoptosis of triple-negative breast cancer (TNBC) cells. They may exert their effects through multiple mechanisms, particularly in the generation of reactive oxygen species (ROS) and the disruption of mitochondrial function, effectively triggering apoptosis in cancer cells. Simultaneously, they can inhibit the growth and spread of cancer cells by disrupting proliferation signaling pathways such as PI3K / AKT / mTOR in breast cancer cells, thereby preventing rapid division and metastasis. Furthermore, artemisinin-based drugs have been shown to synergize with traditional chemotherapy drugs (such as paclitaxel and doxorubicin). This synergistic effect not only improves the efficacy of chemotherapy but also reduces toxicity to normal cells and lowers side effects. Studies have also shown that pretreatment of tumor cells with heme can increase the tumor-suppressive activity of artemisinin by 100-fold, and it remains effective against multidrug-resistant tumor cells. The above studies reported the function of artemisinin derivatives as anti-tumor cytotoxic drugs by inducing oxidative stress, DNA damage, apoptosis and anti-angiogenesis. However, no studies have proposed the application of artemisinin derivatives as PRMT1 small molecule inhibitors or α-KG supplements in anti-tumor immunotherapy drugs. Summary of the Invention
[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of artesunate in the preparation of PRMT1 inhibitors and / or α-ketoglutarate supplements, and to apply them to anti-tumor immunotherapy, aiming to solve the problem of the lack of effective PRMT1 inhibitors and / or α-ketoglutarate supplements for enhancing anti-tumor immunotherapy.
[0007] The technical solution of the present invention is as follows: Firstly, it provides the application of artesunate in the preparation of PRMT1 inhibitors and / or α-KG supplements.
[0008] In a preferred embodiment, the structural formula of artesunate is: .
[0009] In a preferred embodiment, artesunate, an inhibitor of PRMT1, inhibits the arginine methyltransferase activity of PRMT1 by targeting and binding to PRMT1.
[0010] In a preferred embodiment, artesunate in the α-KG supplement targets and binds to PRMT1, inhibiting the methylation modification of IDH2, a downstream molecule of PRMT1, and reducing its expression, thereby reprogramming IDH2-mediated mitochondrial metabolism and inducing the accumulation of α-ketoglutarate.
[0011] In a preferred embodiment, the PRMT1 inhibitor and / or α-KG supplement is used to prepare an anti-tumor immunotherapy drug.
[0012] In a preferred embodiment, the tumor is selected from one or more of breast cancer, melanoma, colorectal cancer, and lung cancer.
[0013] In a preferred embodiment, the antitumor immunotherapy drug has the following effects: inhibiting tumor volume, reducing tumor weight, and increasing the number of tumor-infiltrating T cells, or one or more of these effects.
[0014] In a second aspect, an antitumor immunotherapy drug is provided, comprising: a PRMT1 inhibitor containing artesunate and / or an α-KG supplement.
[0015] Preferred technical solutions also include: pharmaceutically acceptable excipients.
[0016] In a preferred embodiment, the pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable excipients, pharmaceutically acceptable additives, and pharmaceutically acceptable adjuvants.
[0017] Beneficial Effects: This invention reveals that artesunate can target and inhibit the arginine methyltransferase activity of PRMT1, and inhibit the methylation modification of its downstream molecule IDH2 and reduce its expression, thereby inducing mitochondrial metabolic disorders in cancer cells, including the accumulation of various metabolites such as α-ketoglutarate. Therefore, this invention provides a novel application of artesunate for the preparation of PRMT1 inhibitors and / or α-KG supplements. Furthermore, this invention verifies that artesunate can significantly inhibit tumor growth in various tumor-bearing mice and promote anti-tumor immunity, and its use as a PRMT1 inhibitor and / or α-KG supplement can be used in the preparation of anti-tumor immunotherapeutic drugs. Attached Figure Description
[0018] Figure 1 Figure 1 shows the tumor growth detection results of artemisinin and its derivatives on tumor-bearing mice. Figure 2 shows the tumor anatomy of tumor-bearing mice after treatment with saline, artemisinin, dihydroartemisinin, and artesunate. Figure 3 shows the changes in tumor volume in each group. Figure 4 shows the tumor weight statistics in each group. Figure 5 shows the body weight statistics of mice in each group.
[0019] Figure 2The graph shows the tumor growth detection results of different doses of artesunate in breast cancer-bearing mice; where A is a schematic diagram of tumor anatomy after treatment with saline, 20 mg / kg artesunate, and 50 mg / kg artesunate; B is a graph of tumor volume changes in each group; C is a graph of tumor weight statistics in each group; and D is a graph of mouse body weight statistics in each group.
[0020] Figure 3 Figure 1 shows the tumor growth detection results of artesunate in colorectal cancer-bearing mice; Figure 2 shows the tumor anatomy of mice after treatment with saline and 20 mg / kg artesunate; Figure 3 shows the changes in tumor volume in each group; Figure 4 shows the tumor weight statistics in each group; Figure 5 shows the body weight statistics of mice in each group.
[0021] Figure 4 This is a graph showing the tumor growth detection results of artesunate in melanoma-bearing mice; where A is a schematic diagram of tumor anatomy after treatment with saline and 20 mg / kg artesunate in control mice; B is a graph showing the changes in tumor volume in each group; C is a graph showing the tumor weight in each group; and D is a graph showing the body weight of mice in each group.
[0022] Figure 5 The graphs show the results of detecting the number of various immune cells infiltrating mouse tumor tissues by artesunate. A represents the statistical results of CD3+ T cells and tumor-associated macrophages in the tumor tissues of breast cancer-bearing mice; B represents the statistical results of CD8+ GZMB+ T cells and CD8+ IFN-γ+ T cells in the tumor tissues of colorectal cancer-bearing mice; C represents the statistical results of CD8+ T cells in the tumor tissues of breast cancer-bearing mice; D represents the flow cytometry representation and statistical results of CD8+ GZMB+ T cells in the tumor tissues of breast cancer-bearing mice; and E represents the flow cytometry representation and statistical results of CD8+ IFN-γ+ T cells in the tumor tissues of breast cancer-bearing mice.
[0023] Figure 6 The results of the detection of the number of various immune cells infiltrating the spleen tissue of mice by artesunate are shown in Figure A; where A is the statistical result of CD8+ T cells in the tumor tissue of breast cancer-bearing mice; and B is the flow cytometry representation and statistical result of CD8+ IFN-γ+ T cells in the tumor tissue of breast cancer-bearing mice.
[0024] Figure 7This is a diagram for screening potential binding targets of artesunate; where A is a schematic diagram of the artesunate probe structure; B is a flowchart of the ABPP experiment; C is a list of potential binding targets for human and murine cancer cells; D is a gene correlation analysis diagram of PRMT1 with genes related to the immune-related signaling pathway-antigen presentation signaling pathway; E is a gene correlation analysis diagram of RPN1 and RPN2 with genes related to the immune-related signaling pathway-antigen presentation signaling pathway.
[0025] Figure 8 This is a graph showing the binding detection results of artesunate with PRMT1 and its specific sites. Specifically, A shows the interaction between artesunate and PRMT1 in MDA-MB-231 human cancer cells; B shows the interaction between artesunate and PRMT1 in 4T1 mouse cancer cells; C shows the thermostability of artesunate with PRMT1 in MDA-MB-231 human cancer cells; D shows the thermostability of artesunate with PRMT1 in 4T1 mouse cancer cells; E shows the fluorescence co-localization of artesunate and PRMT1; F shows the binding of purified PRMT1 protein to the artesunate probe; G shows the mass spectrum of the binding site of artesunate and PRMT1; H shows the molecular docking diagram of artesunate binding to PRMT1; and I shows the micro-thermophoresis animation of artesunate with wild-type PRMT1 and PRMT1 cysteine 119 mutant protein.
[0026] Figure 9 This is a graph showing the detection of PRMT1 enzyme activity and the downstream molecule IDH2 by artesunate; where A is the enzyme activity of PRMT1 arginine methyltransferase by artesunate; B is the protein expression of PRMT1 and IDH2 in MDA-MB-231 human cancer cells by artesunate; C is the protein expression of PRMT1 and IDH2 in MC38-OVA mouse cancer cells by artesunate; D is the interaction diagram of PRMT1 and IDH2 in MDA-MB-231 human cancer cells; E is the interaction diagram of PRMT1 and IDH2 in MC38-OVA mouse cancer cells; F is the methylation modification diagram of IDH2 by artesunate; and G is the methylation modification diagram of PRMT1 and IDH2 by artesunate.
[0027] Figure 10 This diagram illustrates the effects of artesunate on mitochondrial metabolism and related metabolites. A represents a schematic diagram of mitochondrial metabolism; B represents the effect of artesunate on isocitrate levels in cancer cells; C represents the effect of artesunate on succinic acid levels in cancer cells; D represents the effect of artesunate on malic acid levels in cancer cells; E represents the effect of artesunate on glutamine levels in cancer cells; F represents the effect of artesunate on α-ketoglutarate levels in the serum of tumor-bearing mice; G represents the effect of artesunate on α-ketoglutarate levels in cancer cells; and H represents the effect of artesunate on mitochondrial respiration in cancer cells.
[0028] Figure 11 The graphs show the effects of PRMT1 inhibition and α-ketoglutarate supplementation on tumor immunity: A shows the change in tumor volume in mice after PRMT1 knockdown; B shows the statistical graph of tumor weight in mice after PRMT1 knockdown; C shows the flow cytometry representation and statistical results of CD8+ GZMB+ T cells in mouse tumor tissue after PRMT1 knockdown; D shows the expression of antigen-presenting genes (key molecules regulating tumor immunity) in mouse tumor tissue after PRMT1 knockdown; E shows the flow cytometry representation and statistical results of different concentrations of α-ketoglutarate supplementation on the expression of antigen-presenting molecules (key molecules regulating tumor immunity) in MDA-MB-231 cancer cells; and F shows the flow cytometry representation and statistical results of different concentrations of α-ketoglutarate supplementation on the presentation of OVA SIINFEKL antigen peptide (key peptide regulating tumor immunity) in MC38-OVA cancer cells. Detailed Implementation
[0029] This invention provides the application of artesunate in the preparation of PRMT1 inhibitors and / or α-ketoglutarate supplements. To make the objectives, technical solutions and effects of this invention clearer and more explicit, the invention is further described in detail below.
[0030] Inhibiting PRMT1 or supplementing with α-KG can effectively activate anti-cancer immunity. However, research on small molecule inhibitors targeting PRMT1 or α-KG supplements is still relatively scarce. This invention discloses the application of artemisinin derivative artesunate in the preparation of PRMT1 inhibitors and / or α-KG supplements, and its use in the preparation of anti-tumor immunotherapy drugs. Artesunate has the following benefits: Artesunate can significantly activate anti-tumor immunity in breast cancer, colorectal cancer, and melanoma-bearing mice and inhibit tumor growth in mice. Artesunate can target PRMT1 and bind to its cysteine 119 site. Artesunate inhibits the arginine methyltransferase activity of PRMT1, thereby inhibiting the arginine methylation modification of the downstream molecule IDH2 by PRMT1, thereby reducing IDH2 expression. Artesunate reprograms IDH2-mediated mitochondrial metabolism to induce the accumulation of α-KG, a key intermediate product of mitochondrial metabolism. Artesunate has wide clinical applications, high safety, and is suitable for long-term use. Artesunate can not only be further developed as a standalone anti-tumor small molecule drug, but it can also be used as a PRMT1 inhibitor or α-KG supplement to prepare anti-tumor immunotherapy drugs, thereby further expanding clinical efficacy and having important economic and social value.
[0031] Based on this, embodiments of the present invention provide the application of artesunate in the preparation of PRMT1 inhibitors and / or α-KG supplements.
[0032] Specifically, this invention discloses artesunate, an artemisinin derivative, as a small molecule drug that can both inhibit PRMT1 and replenish α-ketoglutarate levels. This invention discovers that artesunate can target and inhibit the arginine methyltransferase activity of PRMT1, and inhibit the methylation modification of the downstream molecule IDH2 of PRMT1 and reduce its expression. Artesunate can induce mitochondrial metabolic disorders in cancer cells, including inducing the accumulation of various metabolites such as α-ketoglutarate. After inhibiting PRMT1 and promoting the accumulation of α-ketoglutarate, artesunate can significantly enhance anti-tumor immunity. Therefore, artesunate can be used to prepare PRMT1 inhibitors and / or α-KG supplements.
[0033] The structural formula of artesunate is as follows: .
[0034] In some embodiments, artesunate, an inhibitor of PRMT1, inhibits the arginine methyltransferase activity of PRMT1 by targeting and binding to PRMT1.
[0035] In some embodiments, artesunate in the α-KG supplement targets and binds to PRMT1, inhibits the methylation modification of IDH2, a downstream molecule of PRMT1, and reduces its expression, thereby reprogramming IDH2-mediated mitochondrial metabolism and inducing the accumulation of α-ketoglutarate.
[0036] In some embodiments, the PRMT1 inhibitor and / or α-KG supplement is used to prepare an antitumor immunotherapy drug. Examples of this invention demonstrate that artesunate can significantly inhibit tumor growth in various tumor-bearing mice and promote antitumor immunity.
[0037] In some embodiments, the tumor is selected from one or more of breast cancer, melanoma, colorectal cancer, and lung cancer.
[0038] In some embodiments, the efficacy of the antitumor immunotherapy drug includes one or more of the following: inhibiting tumor volume, reducing tumor weight, and increasing the number of tumor-infiltrating T cells.
[0039] This invention provides an anti-tumor immunotherapy drug, comprising: a PRMT1 inhibitor containing artesunate and / or an α-KG supplement.
[0040] In some implementations, pharmaceutically acceptable excipients are also included.
[0041] In some embodiments, the pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable excipients, pharmaceutically acceptable additives, and pharmaceutically acceptable adjuvants.
[0042] The present invention will be further described below through specific embodiments.
[0043] Example 1 This embodiment verifies that artesunate can significantly inhibit tumor growth in breast cancer, colorectal cancer, and melanoma-bearing mice, as detailed below: 1. To establish a mouse model of breast cancer and compare the anticancer effects of artemisinin and its derivatives: 6-week-old female Balb / c mice were injected with 1 × 10⁻⁶ mol / L adipose tissue. 5 Four T1 cells were collected. After tumor formation in mice, the mice were divided into four groups and injected intraperitoneally with control saline, 20 mg / kg artemisinin (ART), 20 mg / kg dihydroartemisinin (DHA), and 20 mg / kg artesunate (ARTS), respectively. The drugs were administered every two days for a total of three doses. Tumor volume and body weight were recorded every two days. Fifteen days after tumor formation, the mice were euthanized by cervical dislocation. Tumor tissue and spleen tissue were harvested, weighed, and photographed. The results are as follows: Figure 1 As shown, artesunate has the best anti-tumor effect compared to artemisinin and dihydroartemisinin. Tumor-bearing mice treated with artesunate have the smallest tumor volume and the lightest tumor weight. None of the three drugs have a significant effect on the weight of the mice.
[0044] 2. Establishing a mouse model of breast cancer to determine that artesunate can inhibit tumor growth in a dose-dependent manner: 6-week-old female Balb / c mice were injected with 1 × 10⁻⁶ oz. fat pads. 5 4T1 (breast cancer cells) cells were collected. After tumor formation in mice, they were divided into three groups and injected intraperitoneally with control saline, 20 mg / kg artesunate, and 50 mg / kg artesunate, respectively, every two days for a total of three administrations. Tumor volume and body weight were recorded every two days. Fifteen days after tumor formation, mice were euthanized by cervical dislocation. Tumor and spleen tissues were dissected, weighed, and photographed. Results are as follows: Figure 2 As shown, artesunate can inhibit tumor volume and tumor weight in mice in a dose-dependent manner, and neither high nor low doses of artesunate have a significant effect on the weight of mice.
[0045] 3. Establishment of colorectal cancer and melanoma mouse models to determine whether artesunate can inhibit tumor growth in colorectal cancer or melanoma-bearing mice: 6-week-old male C57BL / 6 mice were subcutaneously injected with 8 × 10⁸ ppm of artesunate. 5 MC38-OVA (colorectal cancer cells) or 5 × 10⁻⁶ 5B16 F10-OVA (melanoma cells) were used. After tumor formation in mice, they were divided into two groups. The control group received intraperitoneal injections of saline, while the other group received 20 mg / kg artesunate, administered every two days for a total of three doses. Tumor volume and body weight were recorded every two days. Fifteen days after tumor formation, mice were euthanized by cervical dislocation. Tumor and spleen tissues were dissected, weighed, and photographed. Results are as follows: Figure 3 and Figure 4 As shown, artesunate significantly inhibited tumor volume and tumor weight in mice bearing colorectal cancer or melanoma, and had no significant effect on the body weight of mice given 20 mg / kg of artesunate.
[0046] Example 2 This embodiment verifies that artesunate can significantly enhance the anti-tumor immunity of tumor-bearing mice, as detailed below: 1. Flow cytometry was used to detect the effect of artesunate on the number of various immune cells in mouse tumor tissues: Tumor tissues from mice in each group were collected, minced with scissors, and then digested at 37°C for 30 min (digestion solution formulation: DMEM medium containing 5% serum + 0.5% collagenase IV + 0.05% DNase). Single-cell suspensions were then collected using a 70 μm cell sieve. Cells were resuspended in FASC (PBS containing 5% serum) buffer and stained on ice in the dark for 30 min with the following antibodies: APC anti-mouse CD45, FITC anti-mouse CD3, APC / Cyanine7 anti-mouse CD4, PerCP / Cyanine5.5 anti-mouse CD8a, Brilliant Violet 421™ anti-human / mouse Granzyme B, PE anti-mouse IFN-γ, Brilliant Violet 421™ anti-mouse / human CD11b, and Brilliant Violet 650™ anti-mouse F4 / 80. Dead cells were excluded using the Zombie Aqua™ Fixable Viability Kit. Intracellular cytokine staining was performed using the Cytofix / cytooperm fixation / permeabilization kit. Results are as follows: Figure 5 As shown, artesunate can significantly increase the number of CD3+ T cells, CD8+ T cells, cytotoxic GZMB+ CD8+ T cells, IFN-γ+ CD8+ T cells, and tumor-associated macrophages in mouse tumor tissues.
[0047] 2. Flow cytometry was used to detect the effect of artesunate on the number of various immune cells in the spleen tissue of mice: Spleen tissue was collected from mice in each group, and single-cell suspensions were collected directly using a 70 μm cell sieve. The spleen was then subjected to erythrocyte lysis. The single-cell suspensions were collected and resuspended in FASC (PBS containing 5% serum) buffer, and stained with the aforementioned antibodies. The results are as follows: Figure 6 As shown, artesunate can significantly increase the number of CD8+ T cells, cytotoxic GZMB+ CD8+ T cells, and IFN-γ+ CD8+ T cells in mouse spleen tissue.
[0048] Example 3 This embodiment verifies that artesunate targets PRMT1 and binds to its cysteine 119 site, as detailed below: 1. Potential target of artesunate, PRMT1, was screened using activity-based proteomics (ABPP): Cells were divided into three groups: the "control group" was treated with DMSO; the "Probe group" was treated with 100 μM artesunate probe (artesunate containing an alkyne group, ARTS-P) for 4 h; the "Compete group" was first treated with 800 μM artesunate for 4 h, then with 100 μM artesunate probe for 4 h, followed by UV 365 irradiation for 30 min. Cells were collected and proteins were extracted. A mixture of 0.5 mmol / L biotin azide, 100 μmol / L tris(3-hydroxypropyltriazolylmethyl)amine, 1 mmol / L trichloroethyl phosphate, and 1 mmol / L copper sulfate was added to the protein lysis buffer. The mixture was reacted at room temperature for 1 h, precipitated with cold acetone, and centrifuged to obtain the protein. The protein was then purified by biotin affinity assay on streptavidin beads and identified by mass spectrometry. The results are as follows: Figure 7 As shown, through ABPP experiments, this embodiment screened out four potential target proteins with high confidence: RPN1, RPN2, PRMT1, and MYH9. Unlike RPN1 and RPN2, only PRMT1 expression showed a significant negative correlation with genes related to antigen presentation (a key process in tumor immunity).
[0049] 2. A series of biochemical experiments were conducted to verify the target molecule and identify specific binding sites: Cells were also divided into the three groups mentioned above. Pull-down Western blotting and immunofluorescence experiments were performed to detect whether artesunate interacted with and co-localized the target protein. Next, PRMT1 protein was purified and labeled in vitro to determine whether artesunate could directly label PRMT1. Then, a thermostability experiment was conducted, treating protein lysates incubated with artesunate or the control at different temperatures, followed by Western blot analysis. For the identification of binding sites: the purified PRMT1 protein was incubated with either control DMSO or artesunate, and then mass spectrometry was performed to identify the specific binding amino acid sites. A PRMT1 point mutant plasmid was constructed, and the point mutant protein was purified. Microthermophoresis was performed to detect the binding constant of the point mutant protein with artesunate, comparing it with the binding constant of the wild-type target protein to determine if the binding force was weakened, thus proving that the amino acid site is the key binding site between artesunate and the target protein. Furthermore, the binding mode of artesunate to PRMT1 can be visualized using Autodock Vina and Pymol software, and the binding amino acid sites can also be marked. The results are as follows... Figure 8 As shown, artesunate interacts with PRMT1 and exhibits co-localization. Artesunate binding enhances the thermal stability of PRMT1. ARTS-P labeling of PRMT1 is dose-dependent. Artesunate specifically binds to the cysteine 119 amino acid site of PRMT1.
[0050] Example 4 This embodiment verifies that artesunate reprograms IDH2-mediated mitochondrial metabolism and induces the accumulation of α-ketoglutarate, as detailed below: 1. Enzyme activity and protein co-precipitation (Co-IP) assays were used to detect the inhibitory effect of artesunate on PRMT1 enzyme activity and its influence on the expression of downstream molecules of PRMT1: First, the arginine methyltransferase assay was used to detect whether artesunate could inhibit PRMT1 enzyme activity. Then, various cancer cells were treated with control DNSO, 10 μM artesunate, and 20 μM artesunate, respectively, and the effects of artesunate on the protein expression of PRMT1 and IDH2 were detected. Next, a Co-IP assay was performed to detect the interaction between PRMT1 and its downstream molecule IDH2. In addition, cells were overexpressed with IDH2 and then divided into control DNSO and artesunate treatment groups, and Co-IP was used to detect ADMA modification of IDH2 protein; or divided into control DNSO + PRMT1 overexpression group and artesunate + PRMT1 overexpression group, and Co-IP was used to detect ADMA modification of IDH2 protein. Results are as follows: Figure 9As shown, artesunate significantly inhibits the arginine methyltransferase activity of PRMT1, and artesunate inhibits the ADMA modification of PRMT1 on its downstream molecule IDH2, thereby inhibiting its protein expression.
[0051] 2. The effect of artesunate on mitochondrial metabolism in cancer cells was detected using liquid chromatography-mass spectrometry (LC-MS) and cellular oxygen consumption assays: First, cells were divided into three groups: control (DNSO), 10 μM artesunate, and 20 μM artesunate. Cells were then collected and resuspended in 800 mL of ice-cold solution of 80% methanol and 20% ddH2O. The samples were vortexed and then frozen in liquid N2 for 10 min, followed by thawing on ice for 10 min. This freeze-thaw cycle was repeated twice. The samples were centrifuged at 13000 g for 15 min to form spheres of cell debris, lipids, and proteins. The supernatant was evaporated, and the resulting metabolites were resuspended. Metabolites were normalized to protein concentration and measured using a TSQ Quantis Plus (Thermo Scientific, USA) instrument. Next, the effect of artesunate on mitochondrial respiration was detected using a cellular oxygen consumption assay: Cancer cells treated with either control (DMSO) or artesunate were reseeded in 96-well plates and cultured overnight. The results were then analyzed using a mitochondrial stress assay kit. The results are shown below. Figure 10 As shown, artesunate significantly affects the levels of mitochondrial metabolism-related metabolites, including inhibiting isocitrate levels and inducing the accumulation of malic acid, succinic acid, and key mitochondrial metabolic intermediates α-ketoglutarate and glutamine. Artesunate significantly inhibits mitochondrial respiration. These results indicate that artesunate significantly induces mitochondrial metabolic disorders.
[0052] Example 5 This embodiment verifies that PRMT1 inhibition and α-ketoglutarate supplementation significantly promote anti-tumor immunity, as detailed below: 1. Constructing a PRMT1 knockdown-induced tumor-bearing mouse model and using flow cytometry and RT-qPCR to determine that PRMT1 knockdown significantly promotes anti-tumor immunity in mice: First, a PRMT1 stably knocked-down 4T1 breast cancer cell line was constructed using lentiviral technology, including Ctrl shRNA cells, shPRMT1-1 cells, and shPRMT1-2 cells. Then, 1 × 10⁵ cells of each of these three cell types were injected into the fat pads of 6-week-old female Balb / c mice. Tumor volume and body weight were recorded every three days. Fifteen days after tumor formation, mice were euthanized by cervical dislocation, and tumor tissue was dissected for flow cytometry and RT-qPCR to detect T cell infiltration and antigen-presenting-related gene expression within the tumor tissue. Results are as follows: Figure 11 As shown in the AD diagram, PRMT1 knockdown significantly inhibited tumor growth in mice and promoted anti-tumor immunity.
[0053] 2. Flow cytometry was used to detect the effect of α-ketoglutarate supplementation on the expression of cancer cell antigen-presenting molecules to determine whether α-ketoglutarate supplementation can promote anti-tumor immunity: MDA-MB-231 breast cancer cells and MC38-OVA colorectal cancer cells were treated with DMSO, 2 mM α-ketoglutarate, and 5 mM α-ketoglutarate, respectively. Flow cytometry was then used to detect the expression of antigen-presenting molecules (key molecules regulating tumor immunity) and the presentation of the OVA SIINFEKL antigen peptide (key peptide regulating tumor immunity) in cancer cells. The results are as follows: Figure 11 As shown in the middle EF, α-ketoglutarate supplementation significantly enhances the ability of cancer cell antigen presentation (a key process in regulating tumor immunity).
[0054] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
Claims
1. Use of artesunate in the preparation of a PRMT1 inhibitor and / or an alpha-KG supplement.
2. Use according to claim 1, characterized in that, The structural formula of the artesunate is: 。 3. Use according to claim 1, characterized in that, In the PRMT1 inhibitor, the artesunate targets and binds to PRMT1, thereby inhibiting the arginine methyltransferase activity of PRMT1.
4. Use according to claim 1, characterized in that, In the alpha-KG supplement, the artesunate targets and binds to PRMT1, thereby inhibiting the methylation modification of the downstream molecule IDH2 of PRMT1 and reducing the expression of IDH2, thereby reprogramming the mitochondrial metabolism mediated by IDH2 and inducing the accumulation of alpha-ketoglutarate.
5. The use according to claim 1, characterized in that, The PRMT1 inhibitor and / or the alpha-KG supplement are used in the preparation of an antitumor immunotherapy drug.
6. Use according to claim 1, characterized in that, The tumor is selected from one or more of breast cancer, melanoma, colorectal cancer, lung cancer.
7. Use according to claim 1, characterized in that, The efficacy of the antitumor immunotherapy drug includes one or more of inhibiting tumor volume, reducing tumor weight, and increasing the number of tumor-infiltrating T cells.
8. An antitumor immunotherapeutic agent, characterized by, Comprise: The PRMT1 inhibitor and / or the alpha-KG supplement containing artesunate.
9. The medicament according to claim 8, characterized in that, Also comprise: A pharmaceutically acceptable excipient.
10. The medicament according to claim 8, characterized in that, The pharmaceutically acceptable excipient includes one or more of a pharmaceutically acceptable excipient, a pharmaceutically acceptable additive, and a pharmaceutically acceptable adjuvant.