Application of chlorsanguinarine in preparation of medicine for treating PRCC-TFE3 rearrangement renal cell carcinoma

Abstract

The invention discloses an application of sanguinarine chloride (SGC) in preparation of a medicine for treating a PRCC-TFE3 rearranged renal cell carcinoma (rRCC), and belongs to the technical field of biological medicine, in particular to an application of sanguinarine chloride (SGC) in preparation of a medicine for treating a PRCC-TFE3 rearranged renal cell carcinoma (rRCC). According to the application, it is found for the first time that SGC has a specific killing effect on PRCC-TFE3 rRCC cells and has extremely low toxicity on normal kidney cells; the SGC has multiple anti-tumor action mechanisms: on one hand, the SGC is directly combined with and antagonizes a VEGFR2 receptor on a cell membrane to block autocrine signal transduction of VEGFB; on the other hand, the SGC inhibits transcription of VEGFB (vascular endothelial growth factor B) through ROS / p-STAT3 axis epigenetics; besides, the SGC can inhibit the production of lactic acid and the acylation of H3K18, and down-regulate the secretion of chemotactic factors, so that the infiltration of polymorphic myeloid-derived suppressor cells (PMN-MDSCs) in a tumor microenvironment is remarkably inhibited; in an immune sound mouse model, the SGC shows an excellent in-vivo anti-tumor effect, and a brand new targeting and immunoregulation double-effect candidate drug is provided for clinically treating the PRCC-TFE3 rRCC lacking an effective standard therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical targeted therapy and immunotherapy technology, specifically relating to the preparation of a natural small molecule compound, sanguinarine chloride (SGC), for therapeutic purposes. PRCC-TFE3 Rearranged renal cell carcinoma ( PRCC-TFE3 New uses for rRCC drugs. Background Technology

[0002] TFE3 Rearranged renal cell carcinoma ( TFE3 rRCC, also known as Xp11.2 translocation / TFE3 gene fusion-related renal cell carcinoma, belongs to the microphthalmia-associated transcription factor (MiTF) family of tumors. TFE3 rRCC is a highly aggressive type of renal cell carcinoma with an extremely poor prognosis. It accounts for 15% of renal cell carcinomas in patients under 45 years of age and a staggering 20-50% in children. Currently, more than 20 different types of renal cell carcinoma have been identified. TFE3 Fusion of partner genes, among which PRCC-TFE3 It is the most common type of fusion (Moch H, et al. The 2022 World Health Organization Classification of tumours of the urinary system and male genital organs-part A: renal, penile, and testicular tumours. Eur Urol. 2022 Nov;82(5):458-468.), exhibiting unique biological behavior and highly malignant characteristics.

[0003] at present, TFE3 rRCC is resistant to conventional therapies, and there is currently no standard treatment for this disease in clinical practice. For advanced or metastatic patients, empirical treatments targeting clear cell renal cell carcinoma (ccRCC), such as tyrosine kinase inhibitors (TKIs), mTOR inhibitors, or immune checkpoint inhibitors (ICIs), are typically used. However, due to the strong tumor heterogeneity caused by fusion genes, the clinical response rates of these conventional therapies vary greatly, and resistance is highly likely to develop. Therefore, treatments targeting specific fusion genotypes (such as...) are crucial. PRCC-TFE3 The development of novel, specific, and highly effective treatment strategies for tumors is a pressing clinical challenge that needs to be addressed.

[0004] Sanguinarine chloride (SGC) is a natural benzophenanthrene alkaloid extracted from plants such as Macleayacordata, possessing certain anti-inflammatory activity. Its parent compound, sanguinarine, is a quaternary ammonium alkaloid, while SGC is in hydrochloride form. Salt formation significantly improves its water solubility and stability, giving it superior potential for absorption and drug development in vivo (Sun W, Xu Y, Liu Z, et al. Studies on pharmacokinetic properties and intestinal absorption mechanism of sanguinarine chloride: in vivo and in situ. Toxicol Mech Methods. 2025;35(1):43-52.;Pérez Palacios AF, Medina Parra JA, Córdoba Velasco DS, et al. New Strategies to Improve Drug Solubility and Its Impact on Bioavailability: APatent Review (2015-2024). AAPS PharmSciTech. 2025;27(1):57. ;Nyamba I, Sombié CB, Yabré M, et al. Pharmaceutical approaches for enhancing solubility and oral bioavailability of poorly soluble drugs. Eur J Pharm Biopharm. 2024;204:114513). Currently, regarding the use of SGC for treatment... PRCC-TFE3 rRCC, as well as its molecular mechanisms and applications in regulating tumor lactylation modification, targeting the VEGFB-VEGFR2 axis, and remodeling the immune microenvironment of PMN-MDSCs, have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide a novel, efficient, and safe treatment. PRCC-TFE3 The drug for rRCC aims to address the lack of effective targeted and immunotherapeutic drugs for this subtype of renal cell carcinoma in existing technologies.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Sanguinarine chloride (SGC) in the preparation of therapeutic PRCC-TFE3 Applications of rRCC drugs; drug dosage forms include pharmaceutically acceptable carriers and dosage forms such as oral formulations, injections, or targeted nanodelivery systems, such as lipid nanoparticles or polymer micelle delivery systems.

[0007] The mechanism of SGC is as follows: 1) Inhibiting tumor growth through dual blockade of the VEGFR2 signaling pathway; the dual blockade includes direct antagonism of VEGFR2 receptors on the tumor cell membrane surface and inhibition via the ROS / p-STAT3 axis. VEGFB Transcription.

[0008] 2) Downregulates the accumulation of lactate in tumor cells and the lactylation modification of histone H3K9 (H3K9la), including H3K9 lactylation (H3K9la) and H3K18 lactylation (H3K18la).

[0009] 3) Remodeling the tumor immune microenvironment and significantly inhibiting the infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into tumor tissue.

[0010] 4) By inhibiting lactate-driven H3K9 lactylation, the secretion of chemokines CXCL1 and / or CXCL8 is downregulated, thereby blocking the recruitment of PMN-MDSCs.

[0011] In addition, this application also provides a treatment PRCC-TFE3 rRCCs are pharmaceutical or pharmaceutical compositions, including SGCs; the pharmaceutical / composition may also contain pharmaceutically acceptable excipients and be formulated into pharmaceutically conventional dosage forms such as tablets, capsules, tablets, solutions, suspensions, etc., for oral or non-oral administration.

[0012] Preferably, the above-mentioned drug or drug composition further includes a PMN-MDSC depleting agent (such as Ly6G neutralizing antibody 1A8, etc.) as a combination antitumor drug.

[0013] This application discloses for the first time the embodiments of the present application. PRCC-TFE3 A novel oncogenic pathway driven by a fusion gene exists in rRCC: the PRCC-TFE3 fusion protein directly promotes the transcription of a key glycolytic enzyme, leading to lactate accumulation and upregulation of lysine acetyltransferase (KAT6A), resulting in increased histone H3K9 lactylation (H3K9la), and subsequently epigenetic activation. VEGFB SGCs can directly antagonize the VEGFR2 receptor on the surface of tumor cells and inhibit the VEGFR2 signaling pathway through the ROS / p-STAT3 axis. VEGFBThe transcription of this pathway disrupts the cancer pathway.

[0014] Compared with existing technologies, this application utilizes SGC to prepare therapeutic... PRCC-TFE3 rRCC drugs have the following advantages: 1) High specificity and safety: This invention, through high-throughput screening of a library of 1823 compounds, discovered that SGC at micromolar concentrations exhibits high specificity and safety. PRCC-TFE3 rRCC cells (such as UOK120) have extremely strong lethal effects (cell viability fold change <0.5), while having very low toxicity to normal kidney cells (HEK293).

[0015] 2) Dual targeting mechanism: Unlike traditional TKIs that only competitively inhibit kinase domains, SGC blocks the VEGFR2 pathway through a unique "two-pronged" mechanism: (1) It directly binds to and antagonizes the VEGFR2 receptor on the cell membrane surface, blocking VEGFRB binding to it and downstream PI3K / AKT and MEK / ERK signaling; (2) It induces intracellular ROS accumulation and inhibits STAT3 phosphorylation, thereby inhibiting the pathway at the epigenetic level. VEGFB The transcription of VEGFB. This dual intervention strategy at the ligand and receptor levels can reduce VEGFB accessibility in multiple ways, thereby providing multi-dimensional synergistic therapeutic advantages.

[0016] 3) Remodeling the Tumor Immune Microenvironment (TIME): This invention is the first to demonstrate that SGC can significantly reduce intratumoral lactate levels and H3K18 lactylation, thereby inhibiting tumor cells from secreting chemokines CXCL1 and CXCL8, effectively blocking the infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment, and relieving their inhibition of CD8+. + The immunosuppressive effect of T cells.

[0017] 4) Excellent in vivo efficacy: SGC showed excellent tumor growth inhibition in both immunodeficient nude mice and immunocompetent C57BL / 6J mouse models. In particular, it showed superior tumor inhibition effect to other candidate compounds in mice with intact immune systems through immunomodulatory mechanisms, and has extremely high clinical translation potential. Attached Figure Description

[0018] Figure 1 High-throughput drug screening identifies SGC as an anti- PRCC-TFE3rRCC lead compound and in vitro / in vivo efficacy validation graphs; A is a volcano plot showing the effect of drugs on UOK120 cell viability (compared to the control group), with green dots representing drugs that effectively induced UOK120 cell death, n=4; B is a volcano plot showing the killing effect of drugs (green dots in Figure A) on HEK293 cells (compared to the control group), with 5 red dots representing the 5 drugs with the strongest killing effect on UOK120 cells, demonstrating their different toxicities to HEK293 cells, n=4; C shows the cell viability of UOK120 and HEK293 cells after treatment with the top 20 compounds (including the top 5 and their killing effect ratios) as determined by the CCK-8 assay, n=4; D shows the cell viability of UOK120, 786-O, and HEK293 cells after treatment with different concentrations of SGC as determined by the CCK-8 assay, n=8, showing the selective killing results of SGC on UOK120 and HEK293 cells; data in the figures are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0019] Figure 2 Tumor inhibition curves and organ indices in BALB / c nude mice bearing UOK120 tumors after treatment with five drugs that have the strongest killing effect on UOK120; In the figure, A represents the tumor inhibition curve (n=5), and B represents the organ index (n=5). Data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0020] Figure 3 Tumor inhibition curves and organ indices of RENCA-PT tumor-bearing C57BL / 6J mice after treatment with five drugs that have the strongest killing effect on UOK120; In the figure, A represents the tumor inhibition curve (n=5), and B represents the organ index (n=5). Data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0021] Figure 4The results of the study on the effects of SGC on the proteomic profile and VEGF pathway of UOK120 cells; In the diagram: A is a waterfall plot, showing the protein changes in UOK120 cells treated with the five drugs with the strongest killing effect compared to the control group. Red dots represent significantly differentially expressed upregulated proteins, and blue dots represent downregulated proteins, with n=3. B is a volcano plot, showing the protein changes in UOK120 cells treated with SGC compared to the control group. Red dots represent significantly differentially expressed upregulated proteins, and blue dots represent downregulated proteins, with n=3. C is a bubble diagram of GO enrichment analysis, showing the effect of SGC on the UOK120 protein profile, which is enriched in the VEGF-VEGFR2 signaling pathway; D is a mechanism diagram, showing the detailed effect of SGC on the VEGF-VEGFR2 pathway in UOK120 cells.

[0022] Figure 5 This is a graph showing the prediction results for molecular docking.

[0023] Figure 6 A computer simulation diagram of SGC combined with VEGFR2; In this diagram, A represents a molecular docking simulation, and B represents a two-dimensional molecular docking interaction diagram.

[0024] Figure 7 This is a molecular dynamics simulation diagram; In this diagram, A is the RMSD plot from the molecular dynamics simulation; and B is the RMSF plot from the molecular dynamics simulation.

[0025] Figure 8 The experimental results are for the combination of SGC and VEGFR2. In the figures: A is a CETSA graph showing that SGC significantly thermostable VEGFR2 protein in UOK120 cell lysates; B is the IP-LC-MS / MS quantitative analysis result, n=5; data in the figures are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0026] Figure 9 The results of the downstream signal blocking investigation after SGC combines with VEGFR2; In the figures, A is an immunoprecipitation blot image showing the competitive binding of SGCs and VEGFRB to VEGFR2; B is a protein immunoblot image showing the activation status of downstream pathways of VEGFR2; C is a cell viability map obtained from CCK-8 assays, showing that downstream signaling is blocked due to SGC binding to VEGFR2, thereby inhibiting the survival and proliferation of tumor cells (n=6). Data in the figures are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0027] Figure 10 The results of the study on the inhibition of lactic acid production by SGC; Wherein: A is a bar chart of proteomics data, showing the differences in lactate production-related proteins in the glycolysis / gluconeogenesis pathway before and after SGC treatment, with the names of the related proteins indicated on the horizontal axis, n=3; B is a graph showing the results of lactate content detection by LC-MS / MS, showing that SGC reduced lactate levels in UOK120 cells, n=6; data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0028] Figure 11 The results of the investigation on SGC's suppression of H3K9la; Wherein: A is the investigation of H3K9la expression level, where lactate in UOK120 cells promotes H3K9la, n=3; B is the investigation of H3K9la expression level by ChIP-qPCR. VEGFB The resulting image at the promoter shows that H3K9la can bind to... VEGFB At the promoter, and SGC can suppress this process, n=4; the data in the figure are represented as mean±SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0029] Figure 12 The results show the mRNA and protein expression of VEGFB; In the figure, A represents mRNA expression (n=4), and B represents protein expression (n=4). This indicates that SGC downregulates the mRNA and protein expression levels of VEGFB by inhibiting lactate production. Data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0030] Figure 13 The graph shows the results of ROS detection by flow cytometry, indicating that SGC can promote ROS production in UOK120 cells.

[0031] Figure 14 The graphs show the protein expression of p-STAT3 and the mRNA expression of VEGFB. In the figure, A represents the p-STAT3 level of UOK120 cells at different time points after treatment with 2 μM SGC; B represents the VEGFB mRNA level of UOK120 cells at different time points after treatment with 2 μM SGC (n=5); C represents the p-STAT3 level of UOK120 cells 12 h after treatment with different concentrations of SGC; and D represents the VEGFB mRNA level of UOK120 cells 12 h after treatment with different concentrations of SGC (n=5). Data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0032] Figure 15 The figure shows the results of SGC suppressing PMN-MDSC recruitment; Figure A shows the Transwell assay results of SGC treatment on the recruitment of dHL60 cells by UOK120 cells, indicating that SGC treatment inhibited the recruitment of dHL60 cells by UOK120 cells (n=5). Figure B shows the expression level of H3K18la, indicating that SGC can inhibit the expression level of H3K18la in UOK120 cells (n=6). Figure C shows the effect of SGC on the secretion of CXCL1 in UOK120 cells using ELISA, indicating that SGC can inhibit the secretion of CXCL1 in UOK120 cells (n=6). Data in the figures are expressed as mean ± SEM. * P <0.05, ** P <0.01,*** P <0.001. ns = No significant difference.

[0033] Figure 16 The results of the co-culture activity assessment of UOK120 and PMN-MDSCs; Where A represents the CCK-8 assay (n=5), and B represents the EdU staining assay (n=5); data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference.

[0034] Figure 17 The figure shows the results of the in vivo exhaustion experiment; The data in the figure are expressed as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001. ns = No significant difference. Detailed Implementation

[0035] The present invention will be described in detail below with reference to the embodiments, so as to further illustrate the technical solution of the present invention. However, these embodiments do not constitute a limitation on the protection scope of the present invention.

[0036] Cell sources involved in the examples: UOK120, a renal cell carcinoma line, is a carrier PRCC-TFE3 The fusion gene, RRID: CVCL_B099, was a gift from Professor W. Marston Linehan of the National Cancer Institute.

[0037] Human embryonic kidney cells HEK293, purchased from ATCC, RRID: CVCL_0045.

[0038] Mouse renal cell carcinoma RENCA, purchased from ATCC, RRID: CVCL_2174.

[0039] Human promyelocytic leukemia cells HL60, purchased from ATCC, RRID: CVCL_0002.

[0040] Example 1: High-throughput screening for identification of SGC and its in vitro cytotoxicity assay 1) Screening method: A custom screening library (Cat# L2000) containing 1,823 compounds was created using Selleck Chemicals. This library encompasses inhibitors targeting multiple targets through various mechanisms, including mTOR, mesenchymal-epithelial transition factor (MET), cyclin-dependent kinases (CDKs), epigenetic modifiers, and various active components of natural drugs. This application utilizes this library to target the expression of... PRCC-TFE3 The renal cell carcinoma line UOK120 with fusion gene was screened in vitro and treated for 48 hours (the final drug concentration was 10 μM).

[0041] 2) Results Analysis: Using a cell viability assay kit (MCE, HY-K0301), the top 100 compounds that significantly decreased UOK120 cell viability (Fold Change < 0.5, P < 0.05) were screened. The toxicity of these compounds was further investigated in human embryonic kidney cells HEK293 using the same kit. The results are shown below. Figure 1 As shown, A is a volcano plot of the effect of drugs on the viability of UOK120 cells, and B is a volcano plot of the killing effect of drugs (green dots in A) on HEK293 cells. The five red dots in the figure represent the five drugs with the strongest killing effect on UOK120 cells, showing their different toxicities to HEK293 cells.

[0042] 3) Selecting the drug: After treatment (UOK120 kills to the maximum extent, such as...) Figure 1 (as shown in C) and safety (HEK293 toxicity minimized, such as Figure 1 Using a dual-standard filtration process (as shown in D), sanguinarine chloride (SGC) was ultimately identified as the highest priority compound.

[0043] Example 2: The remarkable tumor-suppressing effect of SGC in an in vivo model 1) Immunodeficient nude mouse xenotransplantation model: UOK120 cells were subcutaneously inoculated into BALB / c nude mice (purchased from Nanjing Anjiekang Biotechnology Co., Ltd.). After tumor formation, the mice were administered SGC (5 mg / kg, in physiological saline containing 0.5% CMC-Na, by gavage every other day). Results were as follows... Figure 2 As shown, SGC significantly inhibited tumor volume growth without causing a significant decrease in body weight or obvious liver, spleen, and kidney toxicity in nude mice.

[0044] 2) Immunologically healthy mouse xenotransplantation model: Transfection PRCC-TFE3RENCA-PT cells (RENCA cells were constructed by transfecting a plasmid expressing the PRCC-TFE3 fusion protein with lentivirus and then inoculated into C57BL / 6J mice (purchased from Nanjing Anjiekang Biotechnology Co., Ltd.)

[0045] The lentiviral transfection method described above is a conventional method in this field. This embodiment is performed using the method disclosed in the references “Gödecke N, Hauser H, Wirth D. Stable Expression by Lentiviral Transduction of Cells. MethodsMol Biol. 2024;2810:147-159.;Qian Y, Liu Z, Liu Q, et al. Transduction of Lentiviral Vectors and ADORA3 in HEK293T Cells Modulated in Gene Expression and Alternative Splicing. Int J Mol Sci. 2025;26(9):4431.”

[0046] Test results as follows Figure 3 As shown, SGC exhibits a much stronger tumor-suppressing effect than other compounds screened in the same batch (such as EDD, CAS, SSA, etc.), demonstrating that SGC has a unique therapeutic advantage under a complete immune system.

[0047] Figure 2 China B and Figure 3 The results of the B assay all demonstrated the effect of SGC administration (5 μM concentration) on the quality of various organs in nude mice and mice, indicating that SGC at this administration concentration did not show obvious toxicity, but significantly induced apoptosis in UOK120 cells.

[0048] Example 3: Effects of SGC on the protein profile and VEGF pathway of UOK120 cells 1) Proteomic detection: UOK120 cells were treated with SGC (5 μM concentration) for 48 h, and total protein was extracted. The effect of SGC on the protein profile of UOK120 cells was investigated using proteomics analysis. The results are as follows: Figure 4As shown in Figures A and B, Figure A is a waterfall plot, showing the protein changes in UOK120 cells treated with the five most potent cytotoxic drugs compared to the control group. Red dots in the figure represent significantly differentially expressed upregulated proteins, and blue dots represent downregulated proteins. Figure B is a volcano plot, showing the protein changes in UOK120 cells treated with SGC compared to the control group. Red dots in the figure represent significantly differentially expressed upregulated proteins, and blue dots represent downregulated proteins. This result indicates that SGC has a broad impact on the proteomic profile of UOK120 cells.

[0049] 2) Bioinformatics analysis: Enrichment analysis was performed on proteins affected by SGC in UOK120 cells using Gene Ontology (GO) enrichment analysis. The results are as follows: Figure 4 As shown in Figure C, the effects of SGC on the UOK120 proteome are enriched in the VEGF-VEGFR2 signaling pathway. The VEGF-VEGFR2 pathway under the influence of SGC was mapped using proteomics data, and the resulting mechanism map is shown below. Figure 4 As shown in Figure D, SGC has a profound impact on the VEGF-VEGFR2 pathway in UOK120 cells.

[0050] The GO enrichment analysis used in this embodiment is a conventional method in the art, and the method disclosed in the reference “Chen L, Zhang YH, Wang S, Zhang Y, Huang T, Cai YD. Prediction and analysis of essential genes using the enrichments of gene ontology and KEGG pathways. PLoS One. 2017;12(9):e0184129.” is used in this embodiment.

[0051] Example 4: Molecular mechanism of SGC directly binding to VEGFR2 1) Target prediction and docking: Molecular docking prediction was performed using the website CB-Dock 2 (http: / / 183.56.231.194:8001 / cb-dock2 / php / blinddock.php#job_list_load). The prediction results are as follows: Figure 5 As shown, SGC has a high affinity for the receptor kinase VEGFR2.

[0052] Molecular docking simulations were performed using AutoDock Vina 1.1.2 software. The simulation results are as follows: Figure 6As shown, A is a molecular docking simulation diagram, showing the binding between SGC and receptor kinase VEGFR2; B is a two-dimensional molecular docking interaction diagram, showing the cooperative hydrogen bonding and hydrophobic interaction between SGC and receptor kinase VEGFR2; the results show that there is a cooperative hydrogen bonding and hydrophobic interaction between SGC and receptor kinase VEGFR2.

[0053] Molecular dynamics (MD) simulations were performed using the software GROMACS 2022.5. The simulation results are as follows: Figure 7 As shown in the figure; where A is the RMSD plot from molecular dynamics simulation, indicating that SGC-VEGFR2 maintains a stable conformation during the 50 ns simulation; B is the RMSF plot from molecular dynamics simulation, indicating that the SGC-VEGFR2 structure has good rigidity. The results show that the SGC-VEGFR2 complex remains highly stable within 50 ns (low RMSD and RMSF values), indicating a strong binding between SGC and VEGFR2, suggesting potential persistent inhibitory effects.

[0054] 2) Experimental verification: Cellular thermal displacement analysis (CETSA) results are as follows Figure 8 As shown in Figure A, SGC significantly stabilizes VEGFR2 protein in UOK120 cell lysate. At temperatures ≥46℃, the binding of SGC increases the residual amount of VEGFR2 protein to more than twice that of the control group.

[0055] The CETSA method is a conventional method in this field, and the method disclosed in the reference “Tu Y, Tan L, Tao H, Li Y, Liu H. CETSA and thermal proteome profiling strategies for target identification and drug discovery of natural products. Phytomedicine. 2023;116:154862” is used in the examples.

[0056] The results of quantitative analysis by immunoprecipitation combined with liquid chromatography-mass spectrometry (IP-LC-MS / MS) are as follows: Figure 8 As shown in Figure B, this further directly demonstrates that SGCs have a strong and specific binding to VEGFR2 on the cell membrane, with the binding amount being more than 5 times that of the control group membrane receptor.

[0057] IP-LC-MS / MS is a conventional method in the art, and the method described in the examples is as disclosed in the references “van Nuland M, Rosing H, Schellens JHM, Beijnen JH. Bioanalytical LC-MS / MS validation of therapeutic drug monitoring assays in oncology. Biomed Chromatogr. 2020;34(1):e4623. ;Adaway JE, Keevil BG. Therapeutic drug monitoring and LC-MS / MS. J ChromatogrB Analyt Technol Biomed Life Sci. 2012;883-884:33-49. ;Persson H, Preger C, Marcon E, Lengqvist J, Gräslund S. Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis. Methods Mol Biol. 2017;1575:175-187.”

[0058] 3) Downstream signal blocking: The competitive binding of SGC and VEGFRB to VEGFR2 was investigated using immunoprecipitation. The results are as follows: Figure 9 As shown in Figure A, SGC competitively displaces VEGFRB from VEGFR2. Western blotting (WB) was used to examine the activation status of the VEGFR2 downstream pathway, and the results are as follows... Figure 9 As shown in Figure B, competitive binding of SGC to VEGFR2 leads to the inhibition of intracellular PI3K / AKT and MEK / ERK phosphorylation cascade signaling.

[0059] Cell viability was assessed using the CCK-8 assay, and the results were as follows: Figure 9 As shown in Figure C, the downstream signaling caused by SGC binding to VEGFR2 is blocked, thereby inhibiting the survival and proliferation of tumor cells.

[0060] Example 5: SGC downregulates VEGFB expression by inhibiting H3K9la caused by lactate production. 1) Inhibition of lactic acid production: By analyzing proteomics data, we focused on proteins related to lactate production in the glycolysis / gluconeogenesis pathway. The results are as follows: Figure 10As shown in Figure A, SGC inhibited the expression of most enzymes involved in lactate production in UOK120 cells. The lactate content in UOK120 cells after SGC treatment was detected using LC-MS / MS, and the results are shown below. Figure 10 As shown in Figure B, SGC inhibited lactate production in UOK120 cells.

[0061] The determination of lactate content using LC-MS / MS is a routine method in this field. The method described in the examples is as disclosed in the reference "Afshar M, van Hall G. LC-MS / MS method for quantitative profiling of ketone bodies, α-keto acids, lactate, pyruvate and their stable isotopically labelled tracers in human plasma: An analytical panel for clinical metabolic kinetics and interactions. J Chromatogr B Analyt Technol Biomed Life Sci. 2023;1230:123906."

[0062] 2) H3K9la binding inhibition: The level of lactylation (H3K9la) of histone H3 at position 9 was detected using Western blotting (WB) in UOK120 cells. The results are as follows: Figure 11 As shown in Figure A, lactate in UOK120 cells promotes H3K9la.

[0063] Using chromatin immunoprecipitation combined with qPCR (ChIP-qPCR) technology, the activity of H3K9la was investigated. VEGFB The binding at the promoter and the effect of SGC on this process are shown in the following results. Figure 11 As shown in Figure B, it is proven that H3K9la can bind to VEGFB At the promoter, and SGC is able to suppress this process.

[0064] ChIP-qPCR is a conventional method in the field, and in this embodiment, the method disclosed in the reference “Kim TH, Dekker J. ChIP-Quantitative Polymerase Chain Reaction (ChIP-qPCR). Cold Spring Harb Protoc. 2018; 2018(5).” is used.

[0065] 3) VEGFB downregulation: The mRNA levels of VEGFB in SGC and lactate-treated cells were detected using real-time quantitative polymerase chain reaction (qPCR). The results are as follows: Figure 12 As shown in Figure A; the protein content of VEGFB in cells after SGC and lactate treatment was detected by Western blotting, and the results are shown in Figure A. Figure 12 As shown in Figure B. These results indicate that SGC downregulates the mRNA and protein expression levels of VEGFB by inhibiting lactate production.

[0066] Example 6: SGC Suppression via ROS / p-STAT3 Axis VEGFB Epigenetic transcription 1) ROS generation: The reactive oxygen species (ROS) content in UOK120 cells at different time points after SGC treatment was detected using flow cytometry. The results are as follows: Figure 13 As shown, SGC can promote ROS production in UOK120 cells. Flow cytometry analysis is a standard method in this field for detecting ROS content, and the method disclosed in the examples is as described in the reference "Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods MolBiol. 2010;594:57-72."

[0067] 2) p-STAT3 inhibition: Western blotting was used to detect the phosphorylation level of transcription factor STAT3 (p-STAT3) and the corresponding VEGGFB mRNA level in UOK120 cells at different time points after treatment with different concentrations of SGC. The results are as follows: Figure 14 As shown in the figure, A and B illustrate the time dependence of p-STAT3 and VEGFB on SGC treatment, while C and D illustrate the concentration dependence of p-STAT3 and VEGFB on SGC treatment.

[0068] Combined with published studies elucidating the signaling axis of "ROS downregulating VEGF transcriptional activity by inhibiting p-STAT3" (Zheng Z, Chen H, Zhao H, et al. Inhibition of JAK2 / STAT3-mediated VEGF upregulation under high glucose conditions by PEDF through a mitochondrial ROS pathway in vitro. Invest Ophthalmol Vis Sci. 2010;51(1):64-71.; Choi YK, Kim J, Lee KM, et al. Tuberatolide B Suppresses Cancer Progression by Promoting ROS-Mediated Inhibition of STAT3 Signaling. Mar Drugs. 2017;15(3):55.), this indicates that in UOK120 cells, SGC inhibits VEGF transcriptional activity through the ROS-STAT3 pathway. VEGFB Transcription.

[0069] Example 7: SGC inhibits PMN-MDSC infiltration and remodels the tumor immune microenvironment. 1) PMN-MDSC infiltration: HL60 cells were induced to differentiate into dHL60 cells by treating them with 1.3% DMSO (Shanghai Yuanye Biotechnology Co., Ltd., V87300) for 6 days, which served as a PMN-MDSCs cell model.

[0070] The induction method is a conventional method in the field, and the method disclosed in the reference "Bhakta SB, Lundgren SM, Sesti BN, et al. Neutrophil-like cells derived from the HL-60 cell-line as agenetically-tractable model for neutrophil degranulation. PLoS One. 2024;19(2):e0297758." is used in the examples.

[0071] The effect of SGC on the recruitment of dHL60 cells by UOK120 cells was investigated using Transwell technology. The results are as follows: Figure 15As shown in Figure A, SGC treatment inhibited the recruitment of dHL60 cells by UOK120 cells. Transwell is a conventional method in the art, and the method disclosed in the example was performed according to the reference "Li G, Lin J, Zhang C, et al. Microbiota metabolite butyrateconstrains neutrophil functions and ameliorates mucosal inflammation ininflammatory bowel disease. Gut Microbes. 2021;13(1):1968257."

[0072] The effect of SGC on H3K18la cells in UOK120 cells was investigated using Western blotting. The results are as follows: Figure 15 As shown in B.

[0073] The effect of SGC on the secretion of CXC chemokine ligand 1 (CXCL1) in UOK120 cells was investigated using an enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 15 As shown in C.

[0074] The above results indicate that SGC can inhibit the expression level of H3K18la and the secretion of CXCL1 in UOK120 cells. Combined with the elucidation of the signaling axis of "H3K18la upregulating CXCL1 expression to promote neutrophil recruitment" in published studies (Zhang P, et al. Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer. Nat Commun.2026;17(1):2526.), this indicates that in UOK120 cells, SGC inhibits intracellular lactate production, reduces H3K18la levels, thereby downregulating CXCL1 expression and blocking PMN-MDSC recruitment and infiltration. ELISA detection of CXCL1 secretion is a routine method in this field. In the examples, the method disclosed in the reference "Song AQ, Gao B, Fan JJ, et al. NLRP1 inflammasomecontributes to chronic stress-induced depressive-like behaviors in mice. JNeuroinflammation. 2020;17(1):178." was used.

[0075] 2) Detection of co-culture activity of UOK120 and PMN-MDSCs The effects of dHL60 co-culture and SGC treatment on UOK120 cell viability were detected using CCK-8 and EdU staining assays. The results are as follows: Figure 16 As shown in the figure, A represents the results of the CCK-8 assay, and B represents the results of the EdU staining assay. This indicates that co-culture with dHL60 promoted the proliferation of UOK120 cells, while SGC treatment inhibited this effect.

[0076] EdU staining is a standard procedure in the field, and the method described in the examples is as disclosed in the reference "Alvarez KLF, Poma-Acevedo A, Fernández-Sánchez M, Fernández-Díaz M. An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation. BMC Vet Res. 2020;16(1):230."

[0077] 3) In vivo exhaustion experiment: PMN-MDSCs in C57BL / 6J mice were depleted using Ly6G neutralizing antibody 1A8 (BioXcell, BE0075-1, a general-purpose Ly6G neutralizing antibody and neutrophil depletion agent). Administration method: 200 μg of 1A8 per mouse, diluted with PBS to a final volume of 200 μL, was administered intraperitoneally every other day for 3 weeks. This was combined with SGC treatment (5 mg / kg, using physiological saline containing 0.5% CMC-Na, administered by gavage every other day for 3 weeks). Tumor size in mice was assessed. Results are as follows: Figure 17 As shown, PMN-MDSC depletion significantly enhances the in vivo antitumor efficacy of SGC, directly demonstrating that the in vivo therapeutic effect of SGC is highly dependent on its immunosuppressive effect on PMN-MDSCs.

[0078] The depletion of neutrophils by Ly6G neutralizing antibody 1A8 in vivo is a conventional method in the art, and the method disclosed in the example is as described in the reference "Olofsen PA, Stip MC, Jansen JHM, et al. Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody. Cells. 2022;11(21):3406."

[0079] The above examples demonstrate that sanguinarine chloride (SGC) can serve as a multi-target, multi-mechanism candidate drug, acting both as a direct target to block VEGFR2 and as an epigenetic regulator and microenvironment remodeling agent to inhibit... PRCC-TFE3 The development of rRCC has provided a completely new drug option for this refractory malignant tumor.

Claims

1. Sanguisorbide chloride in the preparation of therapeutic agents PRCC-TFE3 Application of rRCC drugs.

2. The application according to claim 1, characterized in that, The drug can antagonize VEGFR2 receptors on the surface of tumor cell membranes, and simultaneously inhibit the ROS / p-STAT3 axis. VEGFB Transcription.

3. The application according to claim 2, characterized in that, The drug can downregulate the accumulation of lactate in tumor cells and the lactylation modification of histone H3.

4. The application according to claim 3, characterized in that, The lactylation modification of histone H3 includes H3K9 lactylation and H3K18 lactylation.

5. A treatment PRCC-TFE3 The rRCC drug or drug composition is characterized by... The drug or drug composition includes SGC.

6. The drug or drug composition according to claim 5, characterized in that, The drug or drug composition also includes a PMN-MDSC depleting agent.

7. The drug or drug composition according to claim 6, characterized in that, The PMN-MDSCs depletion agent is a Ly6G neutralizing antibody.

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