Use of amcinonide or a pharmaceutical composition thereof for preparing an antitumor drug

Abstract

The application relates to the application of amcinonide or a pharmaceutical composition thereof in the preparation of an antitumor drug, and finds that amcinonide can enhance the protein interaction of P62-MYCN as a P62-MYCN interaction enhancer by combining with the 11th leucine of the autophagy receptor P62, promote the autophagy degradation of MYCN, and finally inhibit the proliferation and metastasis of MYCN amplification type neuroblastoma. The application provides a new pharmacological strategy for directly intervening in the MYCN protein stability to treat neuroblastoma, and especially finds that amcinonide can degrade MYCN by enhancing the autophagy pathway, thereby expanding the new use of amcinonide in the tumor treatment field.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of ancinonide or its pharmaceutical compositions in the preparation of antitumor drugs. Background Technology

[0002] Neuroblastoma is the most common extracranial solid tumor in children, and MYCN gene amplification is closely related to tumor progression. Current treatments have limited effectiveness for high-risk patients and have a high recurrence rate, necessitating the development of novel targeted therapies.

[0003] MYCNs are not only transcription factors, but can also influence tumor growth by regulating metabolic pathways. Currently, there are no effective drugs that can directly regulate the stability of MYCNs, especially small molecule compounds that degrade MYCNs through the autophagy pathway.

[0004] P62 is an autophagy aptamer protein that recognizes and delivers MYCN to autophagosomes for degradation. Studies have found that lactylation of P62 weakens its binding to MYCN, leading to MYCN accumulation and the formation of a positive feedback loop of "MYCN–UDP-GlcNAc–OGT–EP300–P62", which promotes tumor progression.

[0005] Ancinonide (AMN) is a glucocorticoid drug that has been approved by the FDA for clinical treatment. To date, there are no reports of it promoting MYCN autophagy and degradation and inhibiting neuroblastoma by regulating the p62-MYCN interaction. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a novel use of ancinonide (AMN) in the preparation of medicaments for the treatment of neuroblastoma and other MYCN-dependent tumors. Its mechanism of action involves the interaction of P62 and MYCN to promote the autophagic degradation of MYCN.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] Application of ancinonide in the preparation of antitumor drugs.

[0009] The use of an ancinonide pharmaceutical composition in the preparation of an antitumor drug, said pharmaceutical composition comprising an ancinonide composition with an autophagy inducer or a MYCN pathway inhibitor, said MYCN pathway inhibitor including an OGT inhibitor and an EP300 inhibitor.

[0010] Furthermore, the aniline also includes its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.

[0011] Furthermore, the antitumor drugs include drugs for treating neuroblastoma.

[0012] Furthermore, the antitumor drugs include those for treating MYCN-dependent or P62 / MYCN interaction dysregulation-related tumors.

[0013] Furthermore, antrinide inhibits tumors by binding to the p62 protein to enhance its interaction with the MYCN protein, thereby promoting the autophagic degradation of MYCN.

[0014] The beneficial effects of this invention are as follows:

[0015] 1. This invention reveals for the first time that antrinone enhances the P62-MYCN interaction and promotes the autophagic degradation of MYCN by directly binding to the P-62 L11 site, providing a new strategy for targeting MYCN stability;

[0016] 2. In vitro experiments showed that ancinonide can dose-dependently reduce MYCN protein levels and inhibit neuroblastoma cell proliferation, migration, and colony formation; ancinonide can significantly inhibit tumor growth and lung metastasis, and prolong survival.

[0017] 3. This invention has advantages in clinical translation. Ancinonide is a marketed drug with clear human safety data, which can accelerate clinical translation. Furthermore, the combination of ancinonide and OGT or EP300 inhibitors can produce a synergistic anti-tumor effect. Attached Figure Description

[0018] Figure 1 This diagram illustrates the interaction between P62 and MYCN and the effect of P62 on the stability of MYCN protein under glucose deprivation. Figure A shows the IP experiment confirming the interaction between MYCN and P62; Figure B shows the secondary mass spectrum of the P62-specific peptide of the MYCN immunoprecipitation complex; and Figure C shows the regulatory effect of glucose deprivation on the stability of MYCN protein at different time points after knockdown of P62 expression.

[0019] Figure 2 This diagram illustrates the effect of six candidate compounds on the interaction between MYCN and P62 in a bimolecular fluorescence complementation experiment. Figure A shows confocal microscopy images after treatment with DMSO and the six candidate compounds; blue fluorescence represents DAPI labeling the cell nucleus, and yellow fluorescence reflects the interaction between MYCN and P62. MERGE is the merged image of both. Scale bar: 10 μm. Figure B is a bar chart showing the quantitative analysis of the relative BiFC fluorescence intensity of each group of cells after treatment with DMSO and the six candidate compounds. * indicates p < 0.05.

[0020] Figure 3The diagram illustrates the effects of six candidate compounds on the transcriptional activity of MYCN, where the horizontal axis represents DMSO and the six candidate compounds, and the vertical axis represents the relative activity of MYCN. * indicates p < 0.05.

[0021] Figure 4 This diagram illustrates the identification and mass spectrometry verification results of P62 in the MYCN immunoprecipitation complex. Figure A shows the SDS-PAGE Coomassie Brilliant Blue staining pattern of SK-N-BE cell lysates after MYCN immunoprecipitation and ancinonide cross-linking fixation. The Input lane represents the cell lysate loading control, and the "-" and "+" lanes represent the immunoprecipitation products that are uncross-linked and ancinonide-cross-linked, respectively. Figure B shows the secondary mass spectrometry verification spectrum of the P62-specific peptide. The upper part shows the identified P62 characteristic peptide sequence, and the lower part shows the secondary mass spectrometry fragment ion spectrum of this peptide. The orange peak corresponds to type Y fragment ions, and the green peak corresponds to type B fragment ions.

[0022] Figure 5 The image shows the results of HDOCK molecular docking simulation to predict the binding site of ancinonide and P62 protein; where: the light blue ribbon structure represents the three-dimensional spatial conformation of P62 protein, the yellow structure represents the small molecule compound ancinonide, which is embedded in a hydrophobic binding pocket formed on the surface of P62 protein, and the red box area is the core region where the key interaction between ancinonide and P62 protein occurs.

[0023] Figure 6 The results of in vitro protein-protein interaction verification of the key site of ancinonide binding to P62 and its stabilizing effect on the MYCN-P62 complex are shown in the figure. The top section shows the expression of wild-type P62 (WT) and the L11A mutant P62 (L11A) via MBP tag fusion. The specific mutation of the L11A mutant P62 is the mutation of leucine (Leu) at position 11 of the wild-type P62 sequence to alanine (Ala). This was used to detect the effect of different treatments on the MYCN-P62 complex. The bottom section shows the expression of MYCN via His tag fusion, detecting the effect of ancinonide treatment and cross-linking on the MYCN-P62 complex.

[0024] Figure 7 Schematic diagram showing the results of intracellular immunoprecipitation experiments verifying that an acetonide treatment enhances p62-MYCN interaction and downregulates MYCN protein levels;

[0025] Figure 8 The regulatory role of P62 on AMN-induced MYCN-P62 interaction and MYCN protein stability is shown in Figure A, where: Figure A shows the effect of P62 knockdown on ancinonide-induced MYCN-P62 interaction; Figure B shows the effects of AMN and the autophagy inhibitor chloroquine on MYCN protein stability.

[0026] Figure 9 Schematic diagrams illustrating how ancinonide treatment inhibits the invasive and colony-forming abilities of MYCN amplified neuroblastoma cells in cell invasion and colony-forming assays; where: the top shows the cell invasion assay and its quantitative analysis, * indicates p < 0.05; the bottom shows the colony-forming assay and its quantitative analysis, * indicates p < 0.05;

[0027] Figure 10 Figure A shows the results of ancinonide inhibiting neuroblastoma growth and angiogenesis in vivo. Figure A shows the inhibitory effect of ancinonide on the growth of neuroblastoma xenografts in nude mice. The left side is a bioluminescence imaging image, the middle side is the growth curve of tumor volume over time, and the right side is the tumor weight statistics at the experimental endpoint. Figure B shows the MKI67 and PECAM1 staining and quantitative analysis of tumor tissue. * indicates p < 0.05.

[0028] Figure 11 This is a schematic diagram showing the effect of ancinonide downregulating the protein expression of MYCN in neuroblastoma tissue in vivo.

[0029] Figure 12 This is a schematic diagram showing the results of ancinonide inhibiting lung metastasis and prolonging the survival of tumor-bearing mice in vivo; where: Figure A shows the tumor metastases in the lungs of mice observed by bioluminescence imaging and their quantitative analysis; Figure B shows HE staining of mouse lung tissue; Figure C shows the effect of ancinonide on the survival of tumor-bearing mice, P=0.00359;

[0030] Figure 13 This is a schematic diagram showing the histological safety assessment results of ancilonide in major organs of mice.

[0031] Figure 14 This is a schematic diagram showing the effect of the combined use of ancinonide and an OGT / EP300 inhibitor on the growth activity of neuroblastoma cells. * indicates p < 0.05. Detailed Implementation

[0032] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0033] 1. The p62 protein mediates the autophagy-lysosomal degradation of MYCN through its interaction with MYCN.

[0034] First, Western blot experiments showed that there may be an interaction between MYCN and P62. Figure 1 (Figure A in the image). Subsequently, MYCN protein was enriched by immunoprecipitation, and its bound peptides were analyzed by mass spectrometry, which detected specific peptides of P62 protein. Figure 1(See Figure B in the original text). This result indicates a possible interaction between MYCN and P62. Since P62 is an autophagy aptamer, its function is to specifically recognize, bind to, and guide proteins into the autophagy pathway, thereby promoting the final degradation of these proteins. To verify whether P62 promotes autophagic degradation by binding to MYCN, neuroblastoma SK-N-DZ cells were used as a model, with a control group (sh-Scb) and a P62 knockdown group (sh-P62). sh-Scb served as a negative control to exclude the influence of viral transfection or shRNA expression itself on cell phenotype, while sh-P62 specifically reduced the expression level of P62 protein. Subsequently, a glucose deprivation model was constructed, and both groups of cells were treated with glucose deprivation for 0, 4, 8, and 12 hours to induce autophagy. The results showed that the MYCN band in sh-Scb gradually weakened with prolonged glucose deprivation, indicating that glucose deprivation normally induces MYCN degradation. In the sh-P62 group, at treatment time of 0 hours, the expression level of MYCN was higher than that of the control group; with the extension of glucose deprivation time, the degradation rate of MYCN slowed down significantly. Figure 1 (See Figure C in the diagram). The results above indicate that P62 can promote the autophagic degradation of MYCN by binding to MYCN.

[0035] 2. Ancinonide mediates the autophagy-lysosomal degradation of MYCN by binding to the L11 site of the p62 protein.

[0036] To screen for drugs that target and inhibit MYCN expression, a systematic analysis of candidate compounds was performed. Specifically, dual fluorescence complementation assays confirmed that, among the six candidate compounds, ancinonide significantly enhanced the interaction between p62 protein and MYCN protein. Figure 2 Subsequently, MYCN transcriptional activity inhibition experiments were conducted on six candidate compounds. The results showed that ancinonide had the most significant inhibitory effect on MYCN transcriptional activity compared to other compounds. Figure 3 ).

[0037] To clarify the target of ancinonide, a pull-down experiment was conducted using ancinonide-conjugated beads, and protein identification was performed using mass spectrometry. The results showed that ancinonide can directly bind to the p62 protein. Figure 4 (See Figures A-B). Furthermore, HDOCK molecular docking simulations predicted that the binding site of ancinonide to the P62 protein is located near the 11th leucine residue of the P62 protein (…). Figure 5In vitro protein-protein interaction experiments revealed that the binding of ancinonide to P62 depends on its 11th leucine residue (L11). The L11A point mutation not only completely disrupts the binding of ancinonide to P62 but also eliminates the interaction between P62 and MYCN. Figure 6 Further immunoprecipitation analysis showed that ancinonide treatment enhanced the interaction between p62 and MYCN, accompanied by a decrease in MYCN protein levels. Figure 7 Subsequent dose-response experiments confirmed that ancinonide enhanced p62-MYCN binding in a dose-dependent manner, and that MYCN protein expression levels were correspondingly reduced. Figure 8 (See Figure A in the diagram). Notably, the autophagy inhibitor chloroquine was able to reverse the downregulation of MYCN protein expression levels mediated by ancinonide (…). Figure 8 (Figure B in the figure) This suggests that the process by which ancinonide regulates the expression level of MYCN protein depends on the autophagy pathway.

[0038] 3. Effects of Ancinonide on the proliferation and invasion of MYCN amplified neuroblastoma cells

[0039] (1) Cell invasion experiment

[0040] SK-N-DZ and SK-N-BE(2) cells were transfected with either the control sh-Scb sequence (sh-Scb group) or shRNA targeting P62 (sh-P62#1 group) to establish stable knockdown cell lines. Subsequently, cells from each group were cultured at 5 × 10⁻⁶ cells per cell line. 4Cells were seeded at a density of [number] cells / well in the upper chamber of a 24-well Transwell substrate. After full cell adhesion, the medium was changed by adding an equal volume of DMSO solution or serum-free medium containing 1.5 μM amcinonide to the upper chamber, and adding an equal volume of DMSO solution or medium containing 1.5 μM amcinonide and 10% FBS (no different from complete medium) to the lower chamber. The amcinonide stock solution concentration was 1 mM, and the volume was 0.3 μL. The cells were then cultured for another 24 hours. After culture, the medium was discarded, and cells that had migrated to the lower membrane surface were fixed with 4% paraformaldehyde or methanol and stained with 0.1% crystal violet solution. Finally, the number of cells that had penetrated the membrane was counted in multiple randomly selected fields under a microscope to quantitatively analyze cell invasion ability. The results showed that in the sh-Scb group of both SK-N-DZ and SK-N-BE(2) cell lines, the number of invasive cells in the ancinonide-treated group was significantly reduced compared to the DMSO-treated group, demonstrating that ancinonide can significantly inhibit the invasive ability of neuroblastoma cells. Furthermore, compared to the sh-Scb+DMSO group, the number of invasive cells in the sh-P62#1+DMSO group was significantly increased, indicating that knocking down the P62 gene itself enhances the invasive ability of neuroblastoma cells. The number of invasive cells in the sh-P62#1+ancinonide group was significantly reduced compared to the sh-p62#1+DMSO group, indicating that ancinonide can effectively reverse the enhanced cell invasive ability caused by P62 knockdown. Figure 9 ).

[0041] (2) Colony formation experiment

[0042] Stable knockdown cell lines were established by transfecting SK-N-DZ and SK-N-BE(2) cells with either the control sh-Scb sequence (sh-Scb group) or shRNA targeting P62 (sh-P62#1 group). Cells from each group were then seeded at a density of 500 cells / dish in 6 cm culture dishes. After cell attachment, complete culture medium containing either DMSO or 1.5 μM amcinonide was added to the dishes via medium change. Cells were cultured statically in an incubator for 10–14 days. After visible colony formation, the cells were fixed, stained, and counted. The results showed that in the sh-Scb group of both SK-N-DZ and SK-N-BE(2) cell lines, the number of colonies formed in the amcinonide-treated group was significantly lower than that in the DMSO group, demonstrating that amcinonide can inhibit the colony formation ability of neuroblastoma cells. Furthermore, compared to the sh-Scb+DMSO group, the number of cell colonies formed in the sh-P62#1+DMSO group was significantly increased, indicating that knocking down the P62 gene itself enhances the colony-forming ability of neuroblastoma cells. Conversely, the number of colonies formed in the sh-P62#1+amycinonide group was significantly reduced compared to the sh-p62#1+DMSO group, indicating that amycinonide can effectively reverse the enhanced colony-forming ability caused by P62 knockdown. Figure 9 ).

[0043] 4. Ancinonide inhibits the growth and angiogenesis of MYCN amplified neuroblastoma in vivo.

[0044] Male BALB / c mice aged 6-8 weeks were cultured in an SPF environment with a normal diet for one week, with a body weight of approximately 20 g. Subsequently, to evaluate the in vivo antitumor effect of AMN, a mouse subcutaneous xenograft model of MYCN amplified neuroblastoma was constructed. SK-N-BE(2) cells in the logarithmic growth phase were resuspended in PBS, and each mouse was subcutaneously injected with 100 μL of PBS containing 5 × 10⁻⁶ cells in one axilla. 6 A suspension of cells was used to construct a subcutaneous xenograft model. When the tumor volume grew to approximately 100 mm³, the tumor-bearing mice were randomly divided into two groups of five each, and treatment with the drug was initiated continuously for four weeks. Specific experimental groupings are as follows:

[0045] (1) PBS control group: 100 μL of PBS was injected into each mouse intraperitoneally once a day.

[0046] (2) AMN treatment group: Each mouse was injected intraperitoneally with 100 μL of an acetonide solution at a concentration of 50 mg / kg once a day.

[0047] On day 28 after the start of drug treatment (i.e., at the end of the 4-week treatment), tumor lesions were detected in real time using a small animal in vivo fluorescence imaging system. In vivo imaging results showed that compared with the PBS control group, the fluorescence signal intensity of tumor lesions in mice treated with ancinonide was significantly reduced, indicating a significant reduction in tumor burden. Figure 10 (See Figure A in the figure). During the drug treatment period, the tumor volume of mice was measured every week. After 4 weeks, the mice were sacrificed, the tumors were removed, and their weight was measured. The results showed that compared with the PBS group, the tumor volume and weight of mice in the ancinonide group were significantly reduced. Figure 10 Figure A in the diagram). Immunohistochemical staining and quantitative analysis showed that, compared with the PBS group, the expression levels of Ki67 and PECA1 in the ancinonide group were significantly reduced (see Figure A in the diagram). Figure 10 Figure B in the diagram). Furthermore, Western blot analysis of the collected tumor tissue revealed that ancinonide also downregulated MYCN protein expression in tumor tissue in vivo. Figure 11 The above results indicate that ancinonide can effectively inhibit the growth and angiogenesis of neuroblastoma in vivo.

[0048] 5. Ancinonide inhibits lung metastases of MYCN amplified neuroblastoma in vivo.

[0049] To investigate the effect of ancinonide on distant lung metastases of neuroblastoma, a MYCN amplified neuroblastoma lung metastasis model was constructed. First, SK-N-BE(2) cells in the logarithmic growth phase were selected, digested with trypsin, neutralized with complete culture medium, and collected by centrifugation. The cells were resuspended in pre-chilled sterile PBS and placed on ice for cell counting. The cell concentration was adjusted to 1 × 10⁻⁶. 6 Cells / mL, 150 μL per mouse, i.e., 1.5 × 10⁻⁶ cells / mL. 5 Each mouse was injected with 150 μL of single-cell suspension using an insulin injector. After air was expelled, the entire cell suspension was slowly and evenly injected. After tumor cell inoculation, mice were randomly divided into two groups of five each and began treatment for four consecutive weeks. Specific experimental groupings are as follows:

[0050] (1) PBS control group: 100 μL of PBS was injected into each mouse intraperitoneally once a day.

[0051] (2) AMN treatment group: Each mouse was injected intraperitoneally with 100 μL of an acetonide solution at a concentration of 50 mg / kg once a day.

[0052] Four weeks later, in vivo imaging was performed. Small animal in vivo fluorescence imaging showed that, compared with the PBS control group, the tumor signal intensity in the lungs of mice treated with ancinonide was significantly reduced. Figure 12 Figure A in the diagram). Further lung tissue section analysis four weeks later revealed numerous widely distributed metastatic lesions in the lungs of the PBS group. Figure 12 (See Figure B in the original text). The number of metastatic lesions was significantly reduced in the ancinonide group, confirming that ancinonide effectively inhibits lung metastasis of neuroblastoma. These results demonstrate that AMN can effectively inhibit lung metastasis of neuroblastoma. Furthermore, the survival rate of mice in the ancinonide group was significantly higher than that in the PBS group (P=0.0359), indicating that AMN treatment can prolong the survival of tumor-bearing mice. Figure 12 (Figure C in the diagram).

[0053] 6. Safety evaluation of AMN treatment for MYCN amplified neuroblastoma

[0054] To assess the in vivo safety of AMN, lung metastasis model mice were sacrificed after 4 weeks, and HE-stained sections of the heart, liver, and kidneys of mice in the AMN-treated group were analyzed. The results showed that, compared with the PBS group, no significant pathological changes were observed in the morphology of any organ tissue in the AMN group. Figure 13 This indicates that AMN has no significant acute toxicity to the heart, liver, and kidneys, demonstrating its good in vivo safety.

[0055] 7. Analysis of the combined effects of ancinonide and OGT / EP300 inhibitors

[0056] 1. Experimental Design:

[0057] Cell groups: single-drug ancinonide (2.5 µM), single-drug OGT-IN (5 µM), single-drug C646 (2 µM), ancinonide + OGT-IN, ancinonide + C646, treated for 48 h.

[0058] 2. MTT assay for synergistic effect:

[0059] The Chou-Talalay method was used to calculate the co-indices (CI): Ansinide + OGT-IN: CI = 0.68 (<1, co-integrative); Ansinide + C646: CI = 0.72 (<1, co-integrative). Figure 14 Therefore, this indicates that the effect of combination therapy is better than that of monotherapy.

[0060] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0061] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0062] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined in this invention may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention.

Claims

1. Application of ancinonide in the preparation of antitumor drugs.

2. The use of an ancinonide pharmaceutical composition in the preparation of antitumor drugs, characterized in that, The pharmaceutical composition comprises a combination of an antrinone with an autophagy inducer or a MYCN pathway inhibitor, wherein the MYCN pathway inhibitor includes an OGT inhibitor and an EP300 inhibitor.

3. The application according to any one of claims 1 or 2, characterized in that, The aniline also includes its pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

4. The application according to any one of claims 1 or 2, characterized in that, The antitumor drugs include drugs for treating neuroblastoma.

5. The application according to any one of claims 1 or 2, characterized in that, The antitumor drugs include those used to treat tumors that are MYCN-dependent or associated with P62 / MYCN interaction dysregulation.

6. The application according to any one of claims 1 or 2, characterized in that, Ancinonide inhibits tumors by binding to the P62 protein to enhance its interaction with the MYCN protein, thereby promoting the autophagic degradation of MYCN.

Images