Application of oridonin in preparation of drugs for targeting USP13 inhibiting ferritin autophagy and treating hepatocellular carcinoma and combined use of oridonin

Abstract

This invention discloses the application and combination therapy of chrysanthemum ketone in the preparation of a drug targeting USP13 to inhibit ferritin autophagy and treat hepatocellular carcinoma. This invention is the first to demonstrate that chrysanthemum ketone can specifically bind to USP13, blocking its interaction with EP300, downregulating EP300, STAT3 acetylation, and NCOA4 expression, inhibiting NCOA4-mediated ferritin autophagy, and thus inhibiting the proliferation of hepatocellular carcinoma cells. Simultaneously, the combination of chrysanthemum ketone and sorafenib can exert a synergistic anti-tumor effect. This invention provides a novel targeted drug and combination therapy strategy for hepatocellular carcinoma with high USP13 expression, and has significant clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug and combination therapy regimen for treating hepatocellular carcinoma, which uses halofuginone as the active ingredient, targets USP13 and inhibits ferritin autophagy, and particularly relates to small molecule targeted therapy and synergistic drug therapy strategies based on the USP13-EP300-STAT3-NCOA4 signaling axis. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with consistently high incidence and mortality rates worldwide, characterized by insidious onset, rapid progression, high recurrence and metastasis rates, and poor prognosis. While first-line clinical drugs such as sorafenib and lenvatinib can prolong patient survival, most patients develop drug resistance rapidly, limiting their efficacy. Recent studies have confirmed that iron metabolism reprogramming and abnormal activation of ferritin autophagy are key mechanisms in the malignant proliferation, invasion, metastasis, and targeted therapy resistance of HCC.

[0003] Ferritinophagy, a selective autophagy mediated by NCOA4, is responsible for transporting ferritin to lysosomes for degradation and release of free iron, directly determining the level of the intracellular free iron pool (LIP). Elevated LIP levels exacerbate reactive oxygen species accumulation and promote lipid peroxidation through the Fenton reaction, enhancing tumor cell proliferation and survival, and reducing ferroptosis sensitivity, thereby driving tumor progression. Therefore, inhibiting abnormally activated ferritinophagy has become an important new direction in the treatment of liver cancer.

[0004] Ubiquitin-specific protease 13 (USP13) is an important member of the deubiquitinating enzyme family, significantly overexpressed in hepatocellular carcinoma (HCC) and closely associated with poor prognosis. USP13, through its deubiquitination activity at the Cys343 site, specifically removes the K48 ubiquitin chain of EP300, blocking EP300 degradation via the proteasome pathway, thereby stabilizing and upregulating EP300 protein levels. EP300, as a key acetyltransferase, catalyzes acetylation modification of STAT3 at K631 and K685 sites, promoting STAT3 nuclear translocation and enhancing its transcriptional activity. Activated STAT3 further binds to the NCOA4 promoter and activates its transcription, ultimately promoting NCOA4-mediated ferritin autophagy, increasing intracellular free iron levels, and forming a pro-tumor iron homeostasis microenvironment. Therefore, the USP13-EP300-STAT3-NCOA4 axis is a core signaling axis regulating ferritin autophagy and malignant phenotype in HCC, representing a highly promising therapeutic target.

[0005] Currently, there is a lack of safe, effective, and specific small-molecule inhibitors targeting USP13 in clinical practice. Existing deubiquitinase inhibitors generally suffer from poor specificity, high toxicity, and insufficient drug-likeness, making it difficult to meet clinical needs. Halofuginone is a small-molecule compound derived from natural plants that has shown anti-inflammatory and anti-fibrotic activities in some disease models. However, its applications in targeting USP13, blocking the interaction between USP13 and EP300, inhibiting ferritin autophagy, treating hepatocellular carcinoma, and synergistic effects when used in combination with sorafenib have not been reported in existing technologies.

[0006] Based on the aforementioned clinical needs and technological gaps, this invention provides a novel liver cancer treatment strategy centered on fentanyl targeting USP13, offering a new treatment option for hepatocellular carcinoma, especially for patients with abnormal iron metabolism and sorafenib resistance. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new use of styraxone to target USP13, inhibit ferritin autophagy, and treat hepatocellular carcinoma, as well as a synergistic treatment regimen of styraxone combined with sorafenib.

[0008] This invention, through numerous in vitro and in vivo experiments, has for the first time demonstrated that: Halofuginone is a specific small molecule inhibitor of USP13, which can directly bind to the USP13 protein, with a dissociation constant KD = 2.873 × 10⁻⁶. -7 M has high affinity and strong specificity; The acetophenone can significantly block the interaction between USP13 and EP300, inhibit the deubiquitination stabilization effect of USP13 on EP300, and accelerate the degradation of EP300. Statinone inhibits STAT3 nuclear translocation and transcriptional activation by downregulating EP300 and reducing acetylation levels of STAT3 at K631 and K685 sites. Glutamate further downregulates NCOA4 expression, inhibits NCOA4-mediated ferritin autophagy, reduces ferritin degradation, and lowers intracellular free iron pool (LIP) levels; Hexamethasone can significantly inhibit the proliferation of liver cancer cells and the growth of xenografts in vivo, and has a better therapeutic effect on liver cancer with high USP13 expression; The combination of fentanyl and sorafenib has a significant synergistic anti-tumor effect, which can further inhibit tumor proliferation, enhance iron metabolism disorder, and reverse sorafenib resistance.

[0009] Accordingly, the present invention provides the following technical solution: Application of halofuginone in the preparation of USP13 inhibitors.

[0010] Application of styraxone in the preparation of drugs that inhibit ferritin autophagy in liver cancer cells.

[0011] Application of styraxone in the preparation of drugs for treating hepatocellular carcinoma.

[0012] Based on the above applications, styraxone exerts its effects by blocking USP13EP300 binding, reducing STAT3 acetylation, and downregulating NCOA4.

[0013] A pharmaceutical composition for treating hepatocellular carcinoma, comprising styraxone and sorafenib.

[0014] The above-mentioned drug composition is used to synergistically inhibit liver cancer proliferation, regulate iron metabolism disorders, and reverse drug resistance.

[0015] The drug described in this invention can be prepared into any pharmaceutically acceptable dosage form, including but not limited to oral preparations, injections, lyophilized powder for injection, capsules, tablets, suspensions, etc. The drug described in this invention is suitable for various types of hepatocellular carcinoma, especially hepatocellular carcinoma with high USP13 expression, high EP300 expression, abnormal STAT3 activation, high NCOA4 expression, enhanced ferritin autophagy, and sorafenib resistance.

[0016] Compared with the prior art, the present invention has the following beneficial effects: I. First discovery of a novel USP13-targeting inhibitor: It was confirmed that styraxone is a small molecule that specifically binds to USP13, filling the gap in drugs targeting USP13 for the treatment of liver cancer; II. Complete and innovative mechanism: For the first time, the complete regulatory axis of "Changshanone → USP13 → EP300 → STAT3 acetylation → NCOA4 → ferritin autophagy" is elucidated; III. Clear therapeutic effect and sufficient in vitro and in vivo validation: The tumor-suppressing effect has been confirmed in cells, nude mouse xenografts, and genetically engineered mouse models; IV. Significant synergistic effect of combined drug use: The combination of fentanyl and sorafenib has a strong synergistic effect, can reverse drug resistance, and improve the efficacy of existing clinical drugs; V. Outstanding advantages in drug development: Osmanthus is a natural small molecule with a well-defined structure, high safety and low toxicity, making it suitable for clinical translation and long-term use; VI. It also has diagnostic stratification value: It can screen the population that will benefit from USP13 / EP300 / STAT3 / NCOA4 expression levels to achieve precision treatment. Attached Figure Description

[0017] Figure 1 shows the characterization of high expression of USP13 in hepatocellular carcinoma (HCC) and its promotion of tumorigenesis in vivo.

[0018] Figure 2 shows the characterization of USP13's maintenance of protein stability by deubiquitinizing the K48 polyubiquitin chain of EP300 through its catalytic site Cys343.

[0019] Figure 3 shows the characterization of HCC proliferation regulated by USP13 through EP300.

[0020] Figure 4 shows the characterization of EP300 regulating its nuclear localization through acetylation of STAT3, thereby activating the transcription of its downstream target gene NCOA4. Figure 5 shows the binding kinetics (BLI) of styraxone to USP13 protein.

[0021] Figure 6 is a characterization diagram of how Halofuginone targets USP13 to inhibit its interaction with EP300 and suppress HCC proliferation.

[0022] Figure 7 shows the inhibition of ferritin autophagy in liver cancer cells by fentanyl.

[0023] Figure 8 shows the synergistic inhibition of tumor growth in nude mice by fentanyl combined with sorafenib.

[0024] Figure 9 This is a schematic diagram of the control mechanism of the present invention. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments.

[0026] Example 1: USP13 is highly expressed in hepatocellular carcinoma (HCC) and promotes tumorigenesis in vivo. Western blot analysis confirmed that, compared with the control group, the level of USP13 protein in HCC tissue was significantly increased. Figure 1 A). We hypothesize that USP13 may act as a proto-oncogene to promote liver cancer development. To verify this hypothesis, we established two animal models: (1) a hydrodynamic tail vein injection model using oncogenic plasmids alone or in combination with USP13 plasmids; and (2) a C57BL / 6 mouse model with hepatocyte-specific USP13 gene knockout. Compared with mice injected with oncogenic plasmids alone, the oncogenic gene + USP13 group showed more prominent tumor nodules ( Figure 1 B). HE staining showed that the tumor lesions in this group were larger ( Figure 1 C). Combined immunofluorescence staining targeting the proliferation markers Ki67 and USP13 further confirmed that the proportion of proliferating cells was higher in the oncogene + USP13 group. Figure 1Consistent with this, in the second model, hepatocyte-specific USP13 knockout (USP13cKO) mice had fewer and smaller liver tumor nodules than wild-type control mice (Control). Figure 1 F). Ki67 proliferation marker staining and USP13-specific staining both showed that, compared with the control group, the proportion of proliferating cells and the intensity of USP13 fluorescence were decreased in USP13 cKO mice. Figure 1 GH).

[0027] Example 2: USP13 maintains its protein stability by deubiquitinizing the K48 polyubiquitin chain of EP300 through its catalytic site Cys343. Point mutants (Cys343A and Cys345A) were constructed, and USP13 and the mutants were co-transfected with EP300 and His-Ub plasmids, respectively. The results showed that Cys343A lost its deubiquitination activity on EP300, while Cys345A retained some activity, suggesting that Cys343 is a key catalytic site (Figure 2A). Further verification in SNU 449 and HEK-293T cells showed that USP13 overexpression decreased the Ub-K48 level in EP300, while Cys343A increased it, confirming that Cys343 is the functional site for USP13 catalyzing EP300 (Figures 2B-C).

[0028] Example 3: USP13 regulates HCC proliferation via EP300 USP13 knockout significantly reduced EP300 protein expression levels without significantly affecting its mRNA levels, indicating that USP13 regulates EP300 at the post-translational level. Replenishment of exogenous EP300 in USP13 knockout cells did not reversely regulate USP13 expression, confirming a unidirectional regulatory relationship between the two (Figures 3A-C). In summary, USP13 primarily regulates EP300 protein expression positively through post-translational modification. CCK-8 analysis showed that EP300 overexpression rescued the inhibition of liver cancer cell proliferation induced by USP13 silencing (Figures 3D-E). Simultaneously, immunofluorescence analysis showed that USP13 and EP300 exhibited co-localization within cells, with highly overlapping fluorescence signals, suggesting the formation of a functional complex (Figures 3F-G). To further clarify the mechanism of USP13 regulation of EP300, MHCC97H cells from different treatment groups were subcutaneously inoculated into nude mice to construct a xenograft tumor model. The results showed that, compared with the NC-SH group, the tumor size (Fig. 3H), tumor weight (Fig. 3I), and tumor volume (Fig. 3J) of the USP13#1 group were significantly reduced, and overexpression of EP300 reversed this inhibitory effect.

[0029] Example 4: EP300 regulates its nuclear localization by acetylation of STAT3, thereby activating the transcription of its downstream target gene NCOA4. We performed global gene expression profiling using SNU 449 cells transfected with EP300 knockout (EP300#1) combined with acetylglucoscopy and mass spectrometry (Figure 4A). Using P < 0.05 as the significance threshold, we screened out 8 genes with the most significant differential expression. Figure 4 B), including 4 upregulated genes and 4 downregulated genes. Acetylation screening revealed that the acetylation levels of A2B1, HCL9, PRKDC, and STAT3 were positively correlated with EP300. Further validation showed that EP300 knockout did not affect the total expression of the above proteins, but specifically reduced the acetylation level of STAT3 (Figure 4C). To investigate whether there is an interaction between EP300 and STAT3, immunoprecipitation was used for verification. The results showed that in SNU 449 cells, endogenous STAT3 was enriched by co-precipitation using anti-EP300 antibody; further, after exogenous co-transfection of EP300 and STAT3 in HEK-293T cells, immunoprecipitation using anti-EP300 antibody detected the co-precipitation of STAT3 ( Figure 4D). To investigate whether EP300 regulates STAT3 through acetylation modification, EP300 was overexpressed or knocked out in MHCC97H and SNU 449 cells, respectively, and the acetylation level of STAT3 was detected by immunoprecipitation. The results showed that overexpression of EP300 significantly enhanced STAT3 acetylation, while knockout of EP300 decreased its acetylation level (Figure 4E). Nucleocytoplasmic separation experiments further showed that overexpression of EP300 increased the nuclear Acetyl-STAT3 protein level, while knockout of EP300 reduced the nuclear accumulation of Acetyl-STAT3, suggesting that EP300 positively regulates the nuclear localization of activated STAT3 (Figures 4F-G).

[0030] Immunofluorescence colocalization analysis also showed that, compared with the control group, EP300 overexpression promoted the translocation of activated STAT3 into the nucleus (Figure 4H). Figure 4 As shown in Figure I, all four predicted STAT3 binding regions (P1-P4) are located upstream of the transcription start site. Sequence analysis clarified their specific sequences and strands: P1, P2, and P3 are located on the sense strand from -941 to -949 bp (TGCTTGTAA), -1502 to -1510 bp (TGCTGGGAT), and -1549 to -1557 bp (TCCTTAGAA), respectively; P4 is located on the antisense strand from -598 to -606 bp (AGCCTAGAA). All of these predicted sequences highly match the classic 9 bp common binding motif of STAT3. To verify the binding of STAT3 to the NCOA4 promoter, chromatin immunoprecipitation (ChIP) was used. After enriching the bound DNA with anti-STAT3 antibody, the enrichment of fragments in each region was detected. The results showed that STAT3 specifically bound to the NCOA4-1 (P1) and NCOA4-2 (P2) sites in the NCOA4 promoter region, with more significant enrichment at P1; while no significant binding was observed at the NCOA4-3 (P3) and NCOA4-4 (P4) sites, and their signal levels were not significantly different from those of the IgG control group. Figure 4 J). To further verify the effect of STAT3 binding to the NCOA4 promoter on its transcriptional activity, HEK-293T cells were co-transfected with STAT3 and reporter plasmids containing different NCOA4 promoter fragments. Dual-luciferase reporter assays showed that STAT3 significantly enhanced the NCOA4 promoter activity at the P1 site (J). Figure 4 K).

[0031] Example 5. Halofuginone binding kinetics (BLI) of USP13 protein. Our subsequent goal was to find small molecule drugs that selectively bind to USP13. We used Alpha-Fold3 to predict the crystal structure of the USP13 protein. For ligand processing, we used 2D SDF structure files from eight compound libraries, processed them using the LigPrep module in Schrödinger's algorithm, and generated all 3D chiral conformations. After ADME / T evaluation and using the SiteMap module to predict the optimal binding site for the USP13 protein, we used three docking methods with progressively increasing precision (HTVS, SP, and XP) to screen for ligands with the lowest binding free energy. Finally, MM-GBSA analysis was performed to screen for a potential ligand with high binding stability—Changshanone. To determine the binding ability of Changshanone to USP13, we performed BLI analysis using a protein-protein interaction analyzer. The results showed that Changshanone can directly bind to the USP13 protein, with a dissociation constant (KD) of 2.873 × 10⁻⁶. -7 M indicates that the two have a good binding affinity, providing a molecular basis for styraxone targeting USP13.

[0032] Example 6: Halofuginone targets USP13, inhibiting its interaction with EP300 and suppressing HCC proliferation. To determine the IC50 of styraxone on hepatocellular carcinoma cells, CCK-8 assay results showed that cell viability gradually decreased with increasing styraxone concentration, and the calculated IC50 value was 13.67 μM (Figure 6A). To explore the effect of styraxone on USP13, MHCC97H (Figure 6B) and SNU 449 (Figure 6C) cells were treated with different concentrations of styraxone, and USP13 protein levels were detected by Western blot. The results showed that the inhibitory effect of styraxone on USP13 was dose-dependent. To further explore whether the inhibitory effect of styraxone on USP13 affected its interaction with EP300, we treated MHCC97H (Figure 6D) and SNU 449 (Figure 6E) cells with different concentrations (0, 8, 32 μM) of styraxone, and detected changes in the binding of USP13 to EP300. The results showed that the interaction between the two gradually weakened with increasing drug concentration, confirming that styraxone can directly block the binding of USP13 to EP300. Subsequently, to clarify the effect of styraxone on the proliferation of liver cancer cells, we regulated the expression level of USP13 and detected cell viability using the CCK-8 assay. The results showed that in MHCC97H (Fig. 6F) and SNU 449 (Fig. 6G) cells, overexpression of USP13 significantly promoted cell proliferation, while styraxone treatment inhibited this promoting effect; at the same time, knockout of USP13 (USP13#1) further enhanced the proliferation-inhibiting effect of styraxone.

[0033] Example 7: Halofuginone inhibits ferritin autophagy in liver cancer cells (graph) To further clarify the regulatory role of the USP13-EP300-STAT3 signaling axis in HCC, the expression of related proteins was detected by Western blot after treatment with Halofuginone in MHCC97H cells. The results showed that the expression of USP13, EP300, Acetyl-STAT3, NCOA4, TFR1, and LC3 decreased after Halofuginone treatment, while FTH1 and SQSTM1 were upregulated, suggesting that Halofuginone regulates ferritin autophagy in HCC cells by inhibiting the EP300-STAT3-NCOA4 axis.

[0034] Example 8: Halofuginone combined with sorafenib synergistically inhibits tumor growth in nude mice. To explore the in vivo anti-hepatocellular carcinoma effect of styraxone, we established a hepatocellular carcinoma xenograft model using BALB / c-nu / nu male nude mice subcutaneously injected with MHCC97H cells (Figure 8A). Participants were randomly divided into a control group and a styraxone group (5 mg / kg). -1 ), Sorafenib group (40 mg·kg) -1 The drugs were administered orally once every 3 days for 28 consecutive days, including the control group and the combination therapy group. The results showed that tumor growth was inhibited in all drug groups compared with the control group, with the combination therapy group showing the most significant tumor-suppressing effect (Figure 8B-D). Based on tumor size and volume data, the combination drug index (CI) calculated using the Chou-Talalay method was less than 1, suggesting that styraxone and sorafenib have a synergistic anti-tumor effect.

[0035] Example 9 A schematic diagram of the regulation mechanism of this invention is shown below. Figure 9 As shown: The application of halofuginone in the preparation of a drug that targets USP13 to inhibit ferritin autophagy and treat hepatocellular carcinoma.

Claims

1. Application of halofuginone in the preparation of USP13 specific inhibitors.

2. The application according to claim 1, characterized in that, The styraxone directly binds to the USP13 protein and blocks its interaction with EP300.

3. Application of hyacinone in the preparation of drugs that inhibit ferritin autophagy in liver cancer cells.

4. The application according to claim 3, characterized in that, The ferritin autophagy is NCOA4-mediated ferritin autophagy.

5. Application of styraxone in the preparation of drugs for treating hepatocellular carcinoma.

6. The application according to claim 5, characterized in that, The styraxone exerts its effects by inhibiting USP13-EP300 binding, reducing STAT3 acetylation, and downregulating NCOA4.

7. The application according to claim 5, characterized in that, The hepatocellular carcinoma mentioned is a hepatocellular carcinoma that highly expresses USP13, EP300, or NCOA4.

8. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that, It contains styraxone and sorafenib, as well as a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The chrysanthemum ketone and sorafenib have a synergistic antitumor effect.

10. The pharmaceutical composition according to claim 8, characterized in that, The composition is used to synergistically inhibit liver cancer proliferation, inhibit ferritin autophagy, or reverse sorafenib resistance.

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