Use of solanigrine as a trim44 inhibitor in the preparation of anti-colorectal cancer drugs

Abstract

The application discloses application of solasodine as a TRIM44 inhibitor in preparation of an anti-colorectal cancer drug. The application first discovers that solasodine can be used as a specific inhibitor of TRIM44, binds to five amino acid residues G363, T365, T501, G504 and A505 of the TRIM44 protein to form a hydrogen bond, inhibits the deubiquitinating enzyme activity of TRIM44, and then up-regulates the ubiquitination level of GPX4 mediated by TRIM44, promotes the degradation of GPX4 protein, and induces ferroptosis of colorectal cancer cells. The drug takes solasodine as an active ingredient, can be prepared into an injection, a tablet, a capsule or a granule, is applied through intraperitoneal injection, intravenous injection, oral administration or local administration and the like, and is particularly suitable for colorectal cancer patients with high expression of TRIM44. The application provides a new targeted treatment strategy and drug selection for treatment of colorectal cancer, has the advantages of wide source, low toxicity and strong specificity, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of solanine as a TRIM44 inhibitor in the preparation of anti-colorectal cancer drugs. Background Technology

[0002] Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Statistics show that approximately 1.93 million new cases of CRC were diagnosed globally in 2020, with most patients diagnosed at an advanced stage with distant metastasis, resulting in a five-year survival rate of only about 20%. Currently, clinical treatments for CRC primarily include surgical resection, radiotherapy, chemotherapy, and targeted therapy. However, existing treatment regimens still face bottlenecks such as low response rates, significant side effects, easy acquisition of drug resistance, and high dependence on specific gene mutation states. Therefore, in-depth exploration of the molecular mechanisms of CRC development and progression, and the search for new specific therapeutic targets and highly effective, low-toxicity anti-tumor drugs, are urgent needs in current clinical research.

[0003] Ferroptosis, an iron-dependent, novel programmed cell death mechanism caused by lipid peroxidation, has become a hot topic in anti-tumor research in recent years. Glutathione peroxidase 4 (GPX4) is a core regulator of ferroptosis, inhibiting it by scavenging intracellular reactive oxygen species (ROS). Studies have shown that GPX4 is highly expressed in various malignant tumors, promoting tumor cell survival, proliferation, and metastasis by inhibiting ferroptosis. However, existing small molecule inhibitors of GPX4 (such as RSL3 and ML-162) generally suffer from poor water solubility, low in vivo stability, and significant off-target effects, limiting their clinical translation. Therefore, developing novel ferroptosis inducers that can indirectly regulate GPX4 stability, have high specificity, and possess independent intellectual property rights is of great significance.

[0004] Black nightshade (Solanum nigrum L.) is a traditional Chinese medicine used for clearing heat and detoxifying, often used to treat cancers and sores. Solanine is one of the main active steroidal alkaloids in black nightshade, and previous studies have shown it to have certain anti-tumor activity. However, the exact molecular targets and downstream regulatory mechanisms of solanine in colorectal cancer remain unclear, particularly its association with the ubiquitination modification system and ferroptosis regulatory network. Furthermore, trigonometric motif protein 44 (TRIM44), a unique member of the TRIM family, possesses a unique zinc finger ubiquitin protease domain (ZF-UBP), exhibiting deubiquitinating enzyme activity. Although existing literature suggests that TRIM44 is highly expressed in various solid tumors and associated with poor prognosis, whether TRIM44 affects ferroptosis in colorectal cancer by regulating GPX4 stability, and whether solanine can target TRIM44 to exert a therapeutic effect, are currently undisclosed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of solanine as a TRIM44 inhibitor in the preparation of anti-colorectal cancer drugs. This invention offers a new treatment strategy and drug selection for colorectal cancer, with broad prospects for clinical application.

[0006] The technical solution of this invention: the application of solanine as a TRIM44 inhibitor in the preparation of anti-colorectal cancer drugs.

[0007] In the above-described application, the solanine binds to at least one amino acid residue at sites G363, T365, T501, G504, and A505 of the TRIM44 protein.

[0008] In the aforementioned application, the solanine binds to five amino acid residues of the TRIM44 protein, namely G363, T365, T501, G504, and A505, to form hydrogen bonds.

[0009] In the aforementioned application, the drug inhibits TRIM44 activity through solanine, upregulates TRIM44-mediated GPX4 ubiquitination levels, and promotes ferroptosis in colorectal cancer cells.

[0010] In the aforementioned applications, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0011] In the aforementioned applications, the dosage form of the drug is injection, tablet, capsule, or granule.

[0012] In the aforementioned application, the colorectal cancer referred to is colorectal cancer with high TRIM44 expression.

[0013] In the aforementioned applications, the drug is administered via intraperitoneal injection, intravenous injection, oral administration, or local administration.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. This invention reveals for the first time the specific mechanism by which TRIM44 is highly expressed in colorectal cancer, and how it regulates GPX4 ubiquitination levels and stabilizes GPX4 protein expression through its deubiquitinating enzyme activity, thereby inhibiting ferroptosis in colorectal cancer cells and promoting the development and progression of colorectal cancer. Therefore, TRIM44 can serve as a molecular marker for the diagnosis and prognosis of colorectal cancer, as well as a novel target for drug intervention.

[0016] 2. This invention is the first to discover that solanine can act as a specific inhibitor of TRIM44. By targeting and binding to the active pocket of the TRIM44 protein (specifically involving five amino acid residues: G363, T365, T501, G504, and A505), it inhibits the deubiquitination activity of TRIM44, thereby upregulating the ubiquitination level of GPX4, promoting GPX4 protein degradation, and inducing ferroptosis in colorectal cancer cells. This mechanism provides a novel theoretical basis for the antitumor application of solanine.

[0017] 3. This invention is the first to develop solanine as a specific small molecule inhibitor of TRIM44, filling a gap in this field. As a naturally sourced bioactive small molecule, solanine has the advantages of wide availability and low toxicity, showing promising prospects for drug development.

[0018] 4. The drug of this invention uses solanine as the active ingredient and can be prepared into various dosage forms such as injections, tablets, capsules, or granules, and can be administered through various routes such as intraperitoneal injection, intravenous injection, oral administration, or local administration. For colorectal cancer patients with high TRIM44 expression, the targeted therapy strategy provided by this invention is expected to improve the specificity and effectiveness of treatment, reduce toxic side effects, and overcome the drug resistance problem of existing treatments. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of TRIM44 expression in a public database and survival analysis of colorectal cancer patients. (A) shows the expression of TRIM44 in pan-cancer in the public database; (B) shows the expression of TRIM44 in colorectal cancer and adjacent normal tissues in the public database; and (C) shows the survival difference analysis between patients with high and low TRIM44 expression in colorectal cancer patients in the public database.

[0020] Figure 2. Schematic diagram of TRIM44 immunohistochemical staining and clinicopathological association and survival analysis of clinical samples. (D) is a representative image of TRIM44 expression in colorectal cancer tumors and adjacent non-cancerous tissues analyzed by immunohistochemical methods. (E) is an analysis of the survival difference between patients with high and low TRIM44 expression in 99 colorectal cancer patients. (F) is an analysis of the relationship between TRIM44 expression score and T stage. (G) is an analysis of the relationship between TRIM44 expression score and N stage. (H) is an analysis of the relationship between TRIM44 expression score and TNM stage.

[0021] Figure 3 The diagram shows the results of Western Blot validation of TRIM44 expression in human colorectal cancer cell lines. (A) shows the expression of TRIM44 in colorectal cancer cell lines and normal colonic epithelial cells; (B) shows the results of Western Blot validation of TRIM44 knockdown in colorectal cancer cell lines.

[0022] Figure 4 A schematic diagram showing the expression level of GPX4 protein in HCT-8 cells after TRIM44 knockdown in Western Blot.

[0023] Figure 5 A schematic diagram showing the mRNA expression level of TRIM44 after knockdown of TRIM44 in HCT-8 cells using qPCR.

[0024] Figure 6 A schematic diagram showing the mRNA expression level of GPX4 in HCT-8 cells after TRIM44 knockdown, as demonstrated by qPCR.

[0025] Figure 7 This is a schematic diagram illustrating the detection of the binding level of GPX4 and ubiquitin molecules after TRIM44 knockdown using GPX4 magnetic bead antibody in an immunoprecipitation assay.

[0026] Figure 8 A schematic diagram of Western Blot results showing the GPX4 expression level after treatment with the proteasome inhibitor MG132 to knock down TRIM44.

[0027] Figure 9 A schematic diagram illustrating the effect of TRIM44 knockdown on the proliferation ability of colorectal cancer cells in a cloning experiment.

[0028] Figure 10 This is a schematic diagram illustrating the effect of TRIM44 knockdown on the migration ability of colorectal cancer cells in a scratch assay.

[0029] Figure 11 This is a schematic diagram illustrating the effect of TRIM44 knockdown on the invasive and metastatic ability of colorectal cancer cells as shown in a Transwell assay.

[0030] Figure 12 A schematic diagram illustrating the representative size of subcutaneous colon tumors in tumor-bearing mice caused by TRIM44 knockdown;

[0031] Figure 13 This is a schematic diagram of the tumor volume statistics.

[0032] Figure 14 This is a schematic diagram of the tumor quality statistics.

[0033] Figure 15 A schematic diagram of the volcano plot of the TRIM44 knockdown cell proteomic profile results;

[0034] Figure 16 This is a schematic diagram of the KEGG pathway enrichment analysis results;

[0035] Figure 17 A schematic diagram showing the overall conformation of solanine bound to TRIM44 for virtual docking;

[0036] Figure 18 This is a magnified view of the binding site.

[0037] Figure 19 A schematic diagram of Western Blot results showing the expression of TRIM44 and GPX4 proteins in colorectal cancer cells after treatment with solanine.

[0038] Figure 20 A schematic diagram illustrating the ability of solanine to inhibit the proliferation of colorectal cancer cells in a cell cloning experiment.

[0039] Figure 21 This is a schematic diagram illustrating the ability of solanine to inhibit the invasion and metastasis of colorectal cancer cells in a Transwell assay.

[0040] Figure 22 This is a schematic diagram illustrating the ability of solanine to inhibit the migration of colorectal cancer cells using a scratch assay.

[0041] Figure 23 This is a representative image from an in vivo experiment demonstrating that solanine inhibits the growth of subcutaneous tumors in colorectal cancer cells;

[0042] Figure 24 The goal was to monitor and statistically analyze the trends in tumor volume changes in four groups of mice over 35 days.

[0043] Figure 25 The results show the tumor quality statistics after 5 weeks of drug administration. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0045] Example 1: Validation of TRIM44 expression in colorectal cancer tissues

[0046] 1. Clinical Sample Collection

[0047] Surgical resection specimens were collected from 99 patients with colorectal cancer admitted to Hangzhou Normal University Affiliated Hospital and Zhejiang Provincial Hospital of Traditional Chinese Medicine between January 2013 and December 2017. These included colorectal cancer tissue samples and corresponding adjacent normal tissue samples (≥5 cm from the cancerous margin). All patients had not received radiotherapy, chemotherapy, or targeted therapy prior to surgery and were pathologically diagnosed with colorectal cancer. Clinicopathological data, including age, sex, T stage, N stage, and TNM stage, were collected. The collection and use of all samples were approved by the hospital ethics committee, and informed consent was obtained from the patients.

[0048] 2. Immunohistochemical detection

[0049] Collected tissue samples were fixed with 4% paraformaldehyde, then routinely dehydrated and embedded in paraffin to prepare 4μm thick serial sections. After dewaxing in xylene and hydration with graded ethanol, the sections underwent antigen retrieval using citrate buffer (pH 6.0), followed by blocking of endogenous peroxidase with 3% hydrogen peroxide and non-specific binding sites with 5% goat serum. Rabbit anti-human TRIM44 primary antibody (working concentration 1:200) was added and incubated overnight at 4°C. The next day, HRP-labeled goat anti-rabbit secondary antibody (working concentration 1:1000) was added and incubated at room temperature for 1 hour. DAB staining was performed, followed by hematoxylin counterstaining, dehydration, mounting, and observation and photography under a microscope.

[0050] The immunohistochemical staining results were scored using a double-blind method by two pathologists. Staining intensity scoring criteria: 0 (negative), 1 (weakly positive), 2 (moderately positive), 3 (strongly positive); positive cell percentage scoring criteria: 0 (<5%), 1 (5%–25%), 2 (26%–50%), 3 (51%–75%), 4 (>75%). The total score was the product of the staining intensity score and the positive cell percentage score; a total score ≥4 indicated high expression, and <4 indicated low expression.

[0051] 3. Results

[0052] Public database analysis shows that TRIM44 is highly expressed in various cancers, and its expression in colorectal cancer is significantly higher than that in adjacent normal tissues. Patients with high TRIM44 expression have a significantly lower prognosis than those with low expression. Figure 1 and Figure 2 As shown. Figure 1In the table, (A) shows the expression of TRIM44 in pan-cancer data from a public database; (B) shows the expression of TRIM44 in colorectal cancer and adjacent normal tissues from a public database; and (C) shows the survival difference between patients with high and low TRIM44 expression in colorectal cancer patients from a public database. Immunohistochemical results showed that the positive expression rate of TRIM44 in 99 colorectal cancer tissues was 74.7% (74 / 99), including 27 strongly positive (+++), 34 moderately positive (++), 13 weakly positive (+), and 25 negative (-); while the positive expression rate of TRIM44 in 99 adjacent normal tissues was only 50.5% (50 / 99), including 0 strongly positive, 10 moderately positive, 35 weakly positive, and 49 negative. Figure 2 In the table, (D) shows representative images of TRIM44 expression in colorectal cancer tumors and adjacent normal tissues analyzed by immunohistochemistry; (E) shows the survival difference between patients with high and low TRIM44 expression in 99 colorectal cancer patients; (F) shows the relationship between TRIM44 expression score and T stage; (G) shows the relationship between TRIM44 expression score and N stage; and (H) shows the relationship between TRIM44 expression score and TNM stage. Survival analysis showed that the 5-year survival rate of patients in the high TRIM44 expression group was 36%, significantly lower than the 71% of patients in the low TRIM44 expression group (P<0.05). Therefore, TRIM44 expression was significantly correlated with T stage, N stage, and TNM stage (P<0.05). Table 1 shows the comparison of the positive rate of TRIM44 expression in normal tissues and colorectal cancer tissues. The expression level of TRIM44 in colorectal cancer tissues was significantly higher than that in adjacent normal tissues (P<0.05).

[0053] Table 1

[0054]

[0055] The above results indicate that TRIM44 is highly expressed in colorectal cancer and is closely related to poor prognosis in patients.

[0056] Example 2: Validation of the mechanism by which TRIM44 regulates ferroptosis in colorectal cancer cells

[0057] 1. Cell lines and cell culture

[0058] Human colorectal cancer cell lines HCT-8, SW-480, HT-29, LOVO, T84, DLD-1, HCT-116, SW-620, and human normal colonic epithelial cells CCD-841 were all purchased from the American Type Culture Collection (ATCC). All cells were cultured routinely in RPMI-1640 or DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator, and passaged every 2-3 days.

[0059] 2. Construction of lentiviral vectors and establishment of stable cell lines

[0060] A specific shRNA targeting sequence was designed for the human TRIM44 gene, along with a negative control sequence (shNC). The shRNA sequence was cloned into the pLKO.1 lentiviral vector, and after confirmation by sequencing, it was co-transfected with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G into HEK-293T cells to package lentivirus. The viral supernatant was collected, filtered through a 0.45 μm filter, and stored at -80°C for later use.

[0061] HCT-8 and SW-480 cells in logarithmic growth phase were infected with TRIM44 knockdown lentivirus (shTRIM44) or negative control lentivirus (shNC) at an MOI of 10, with 8 μg / mL polybrene added to enhance infection efficiency. After 24 hours of infection, the culture medium was replaced with fresh medium, and after 48 hours, 2 μg / mL puromycin was added for selection. Single-clonal cell lines were selected and expanded after 2 weeks of selection, and the TRIM44 knockdown efficiency was verified by Western blotting. Simultaneously, a stable TRIM44 overexpression cell line (TRIM44) was constructed using the same method. OE ) and the corresponding empty vector control cell line (Vector).

[0062] 3. Experimental grouping and drug intervention

[0063] The stable cell lines constructed above were divided into the following groups:

[0064] Group A: shNC control cells + DMSO (equivalent dose);

[0065] Group B: shTRIM44 cells + DMSO (equivalent dose);

[0066] Group C: shTRIM44 cells + ferroptosis inhibitor Fer-1 (5 μM);

[0067] Group D: shTRIM44 cells + ROS scavenger N-acetyl-L-cysteine ​​(NAC, 20 mM).

[0068] Group E: TRIM44 OE Cells + DMSO (equivalent dose);

[0069] Group F: TRIM44 OE Cells + GPX4 inhibitor ML-162 (2 μM).

[0070] After 24 hours of the above treatment, each group of cells was subjected to further testing.

[0071] 4. Western blot detection of protein expression

[0072] Cells from each group were collected and lysed on ice for 30 minutes using RIPA lysis buffer containing protease and phosphatase inhibitors. The cells were then centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method. An equal volume of protein (30 μg) was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with rabbit anti-human TRIM44 antibody (1:1000), rabbit anti-human GPX4 antibody (1:1000), and rabbit anti-human β-actin antibody (1:5000), respectively. The next day, the membrane was incubated with HRP-labeled goat anti-rabbit secondary antibody (1:5000) at room temperature for 1 hour, developed using ECL chemiluminescence, and the band gray values ​​were analyzed using a gel imaging system.

[0073] 5. Real-time quantitative PCR (RT-qPCR) detection of mRNA expression

[0074] Total RNA was extracted from cells in each group using TRIzol reagent and reverse transcribed into cDNA using a reverse transcription kit. Using cDNA as a template, real-time quantitative PCR was performed using the SYBR Green assay. GAPDH was used as an internal control, and the relative expression level was calculated using the Livak method.

[0075] 6. Detection of intracellular reactive oxygen species (ROS) levels

[0076] Cells from each group were collected, washed twice with PBS, and then incubated in serum-free medium containing 10 μM DCFH-DA fluorescent probe at 37°C for 20 minutes in the dark. After incubation, the cells were washed three times with serum-free medium to remove any probes that had not entered the cells. Fluorescence intensity was detected using flow cytometry (excitation wavelength 488 nm, emission wavelength 525 nm). 10,000 cells were collected from each group, and the average fluorescence intensity was used to represent the ROS level.

[0077] 7. Detection of GPX4 ubiquitination levels by co-immunoprecipitation (Co-IP)

[0078] Cells from each group were collected and lysed on ice for 30 minutes using non-denaturing lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40). The cells were then centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. An equal volume of protein lysis buffer (500 μg) was added, along with rabbit anti-human GPX4 antibody (2 μg) and Protein A / G agarose beads. The mixture was incubated overnight at 4°C with rotation. The next day, the agarose beads were washed three times with lysis buffer, and 2×SDS loading buffer was added. The immune complexes were eluted by heating at 95°C for 10 minutes. Western blotting was performed using anti-ubiquitin antibody (Ub), and a control was detected using GPX4 antibody.

[0079] 8. Cell function experiments

[0080] (1) CCK-8 proliferation experiment: Cells in each group were inoculated at 5×10⁻⁶ cells / mL. 3 The cells were seeded at a density of 1000 cells / well in 96-well plates, with 5 replicates per group. 24 hours after drug intervention, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. The absorbance was measured at 450 nm using a microplate reader.

[0081] (2) Colony formation experiment: Each group of cells was seeded into 6-well plates at a density of 500 cells / well and cultured for 14 days, with the culture medium changed every 3 days. After colony formation, the cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 15 minutes, washed and dried, photographed and counted (≥50 cells were counted as one colony).

[0082] (3) Scratch test: Cells in each group were scratched at 5×10⁻⁶ mm. 5 Cells were seeded at a density of 100% per well in 6-well plates. When the cells reached 100% confluence, a straight line was evenly drawn on the bottom of the plate using a 200 μL pipette tip. After washing with PBS to remove detached cells, serum-free medium containing the appropriate drug was added. Images were taken under an inverted microscope at 0 and 24 hours, and the wound healing area was measured using ImageJ software to calculate cell migration rate.

[0083] (4) Transwell migration and invasion assays: Migration assays used uncoated Transwell chambers (8 μm pore size), while invasion assays used pre-coated Transwell chambers (1:8 dilution). Cells from each group were sputtered at 1×10⁻⁶ cells / mL. 5 One (transfer experiment) or 2 × 10 5Cells were seeded at a density of 100 cells / day (invasion assay) in the upper chamber (containing serum-free medium), and the lower chamber was filled with complete medium containing 10% FBS as a chemotactic agent. After 24 hours of culture, cells that had not penetrated the membrane in the upper chamber were gently wiped away with a cotton swab. Cells that had penetrated the membrane were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 15 minutes, photographed and counted under an inverted microscope.

[0084] 9. Results

[0085] Figure 3 To verify the expression of TRIM44 in human colorectal cancer cell lines using Western blotting, (A) shows the expression of TRIM44 in colorectal cancer cell lines and normal colonic epithelial cells; (B) shows the Western blotting results of TRIM44 knockdown in colorectal cancer cell lines. Western blotting results showed that the expression of TRIM44 in human colorectal cancer cell lines was significantly higher than that in normal colonic epithelial cells CCD-841. Compared with the shNC control group, the expression level of GPX4 protein in the shTRIM44 group was significantly decreased; while the expression level of TRIM44... OE The expression level of GPX4 protein in the group cells was significantly increased. Figure 4 Western blotting was used to visualize the expression level of GPX4 protein in HCT-8 cells after TRIM44 knockdown. RT-qPCR results showed that the mRNA level of TRIM44 changed accordingly after knockdown or overexpression of TRIM44, but the mRNA level of GPX4 did not change significantly, indicating that the regulation of GPX4 by TRIM44 occurs at the protein level rather than the transcriptional level. Figure 5 To demonstrate the mRNA expression level of TRIM44 after knockdown of TRIM44 in HCT-8 cells using qPCR; Figure 6 qPCR was used to show the mRNA expression level of GPX4 in HCT-8 cells after TRIM44 knockdown. Flow cytometry results showed that the ROS level in the shTRIM44 group was significantly higher than that in the shNC control group, and Fer-1 and NAC could reverse this effect.

[0086] Immunoprecipitation results showed that GPX4 ubiquitination levels were significantly increased after TRIM44 knockdown. Figure 7 To detect the binding level of GPX4 to ubiquitin molecules after TRIM44 knockdown using GPX4 magnetic bead antibody in an immunoprecipitation assay, GPX4 magnetic bead antibody was used. Pretreatment of shTRIM44 cells with the proteasome inhibitor MG132 significantly alleviated GPX4 degradation. Figure 8The results of Western blot analysis on GPX4 expression levels after TRIM44 was knocked down using the proteasome inhibitor MG132 are shown. These results indicate that TRIM44 stabilizes GPX4 protein expression by removing the ubiquitin chain of GPX4 through its deubiquitinating enzyme activity, thereby inhibiting ferroptosis in colorectal cancer cells.

[0087] Cellular function experiments showed that knocking down TRIM44 significantly inhibited the proliferation, colony formation, migration, and invasion of colorectal cancer cells. Figure 9 To demonstrate the effect of TRIM44 knockdown on the proliferative capacity of colorectal cancer cells in clonal experiments; Figure 10 To demonstrate the effect of TRIM44 knockdown on the migration ability of colorectal cancer cells using a scratch assay; Figure 11 Transwell assays showed the effect of TRIM44 knockdown on the invasive and metastatic ability of colorectal cancer cells. In vivo tumorigenesis experiments in nude mice also showed that TRIM44 knockdown significantly reduced tumor volume and mass. Figure 12 A representative image showing the effect of TRIM44 knockdown on the size of subcutaneous colon tumors in tumor-bearing mice; Figure 13 This is a statistical result of tumor volume; Figure 14 This represents the statistical results for tumor quality. Proteomics analysis revealed that oxidative stress-related signaling pathways were significantly upregulated after TRIM44 knockdown, with ferroptosis being one of the most significant regulatory pathways, and GPX4 being one of the most significantly differentially expressed genes. Figure 15 Volcano plot of cellular proteomic profiles for TRIM44 knockdown; Figure 16 The results are from the KEGG pathway enrichment analysis. Fer-1 and NAC can partially reverse the inhibition of cell proliferation and migration / invasion induced by TRIM44 knockdown. These results confirm that TRIM44 inhibits ferroptosis in colorectal cancer cells and promotes the development and progression of colorectal cancer by inhibiting GPX4 ubiquitination and degradation and stabilizing GPX4 protein expression.

[0088] Example 3: Validation of the efficacy of solanine in inhibiting ferroptosis in colorectal cancer cells by targeting TRIM44.

[0089] 1. Preparation of Solanine

[0090] Solasonine (CAS No. 19121-58-5, purity ≥98%) was purchased from a biotechnology company. A 10 mM stock solution was prepared by dissolving solasonine in dimethyl sulfoxide (DMSO), aliquoted, and stored at -20°C protected from light. Before use, it was diluted with cell culture medium to the required concentration, ensuring the final DMSO concentration did not exceed 0.1%.

[0091] 2. Establishment of stable GPX4 overexpression cell lines

[0092] Using the same method as in Example 2, GPX4 overexpression (GPX4) was constructed. OE The HCT-8 and SW-480 stable cell lines and their corresponding empty vector control cell lines (Vector).

[0093] 3. Experimental grouping and drug intervention

[0094] The stable cell lines constructed above were divided into the following groups:

[0095] Group A: Vector control cells + DMSO (equivalent dose);

[0096] Group B: Vector control cells + solanine (1 μM).

[0097] Group C: Vector control cells + solanine (1 μM) + Fer-1 (5 μM);

[0098] Group D: Vector control cells + solanine (1 μM) + NAC (20 mM);

[0099] Group E: GPX4 OE Cells + DMSO (equivalent dose);

[0100] Group F: GPX4 OE Cells + Solanine (1 μM).

[0101] After 24 hours of the above treatment, the cells in each group were subjected to Western blotting to detect the expression of TRIM44 and GPX4 proteins, flow cytometry to detect ROS levels, immunoprecipitation to detect GPX4 ubiquitination levels, and CCK-8, colony formation, scratch assay, and Transwell migration and invasion assays to detect changes in cell function, as described in Example 2.

[0102] 4. Results

[0103] Molecular docking results showed that solanine binds to five amino acid residues of the TRIM44 protein (G363, T365, T501, G504, and A505) to form five hydrogen bonds, effectively and directly binding to the active pocket of TRIM44. Figure 17 To visualize the overall conformation of solanine bound to TRIM44 in a virtual docking experiment; Figure 18This is a magnified view of the binding site. Western blot results showed that treatment with solanine (1 μM) significantly reduced the protein expression level of GPX4 in colorectal cancer cells; simultaneously, the protein level of TRIM44 was also downregulated. Combined with molecular docking and binding site verification results, this phenomenon indicates that solanine inhibits the deubiquitination enzyme activity of TRIM44 through physical binding, which may affect the protein stability of TRIM44 itself or promote its degradation, ultimately leading to increased ubiquitination and degradation of its substrate GPX4. Figure 19 Western blot results showing the expression of TRIM44 and GPX4 proteins in colorectal cancer cells after treatment with solanine. Cell function experiments showed that solanine significantly inhibited the proliferation, migration, and invasion of colorectal cancer cells. Figure 20 Cell cloning experiments showed that solanine can inhibit the proliferation of colorectal cancer cells; Figure 21 Transwell assays showed that solanine can inhibit the invasion and metastasis of colorectal cancer cells; Figure 22 Scratch assays showed that solanine inhibited the migration of colorectal cancer cells. Immunoprecipitation results showed that solanine treatment significantly increased GPX4 ubiquitination levels, indicating that solanine promotes GPX4 ubiquitination degradation by inhibiting the deubiquitination enzyme activity of TRIM44. Flow cytometry analysis showed that solanine treatment significantly increased ROS levels in colorectal cancer cells, while Fer-1 and NAC effectively reversed the solanine-induced ROS increase.

[0104] In GPX4-overexpressing cells (GPX4 OE In groups F and B, the inhibition of cell proliferation, migration and invasion, and the increase in ROS induced by solanine were significantly reversed, indicating that the anti-colorectal cancer effect of solanine mainly depends on its downregulation of GPX4. Fer-1 and NAC also reversed the inhibitory effect of solanine on colorectal cancer cells (groups C, D, and B). In vivo nude mouse tumorigenesis experiments further showed that solanine can significantly inhibit the growth of subcutaneous tumors of colorectal cancer cells. Figure 23 This is a representative image from an in vivo experiment demonstrating that solanine inhibits the growth of subcutaneous tumors in colorectal cancer cells; Figure 24 This is a statistical result of tumor volume; Figure 25 This is a statistical result of tumor quality.

[0105] The above results indicate that solanine exerts its anti-colorectal cancer effect by inhibiting TRIM44 activity, upregulating TRIM44-mediated GPX4 ubiquitination, promoting GPX4 protein degradation, and thereby inducing ferroptosis in colorectal cancer cells.

[0106] Example 4: Verification of the binding site of solanine to TRIM44 protein

[0107] 1. Homology modeling of the three-dimensional structure of TRIM44 protein

[0108] This embodiment uses a homology modeling method to construct a three-dimensional structural model of the human TRIM44 protein. The specific steps are as follows:

[0109] (1) Template search and selection: The amino acid sequence of human TRIM44 protein (UniProt accession number: Q96DX7, full length 344 amino acids) was used as the query sequence. The BLASTP algorithm was used to search the Protein Data Bank (PDB) database to find known three-dimensional structural proteins with high homology to the TRIM44 sequence as templates. After screening, the protein structure with PDB number 7xt2 was selected as the primary template (sequence identity 21.53%), and PDB number 4tn3 was selected as the secondary template (sequence identity 22.30%). Both are proteins containing zinc finger domains.

[0110] (2) Sequence alignment: Clustal Omega software was used to perform multiple sequence alignment between the TRIM44 target sequence and the template protein sequence to determine the correspondence between the target sequence and the template sequence and ensure that the sequence alignment of the modeling region was accurate.

[0111] (3) Model construction: Based on the sequence alignment results, Modeller v10.4 homology modeling software was used to map the target protein sequence onto the three-dimensional structural framework of the template protein to generate an initial three-dimensional structural model. The initial model was optimized through energy minimization and molecular dynamics simulations to finally obtain the three-dimensional structural model of the TRIM44 protein.

[0112] (4) Model Evaluation: The conformational rationality of the constructed TRIM44 three-dimensional structural model was evaluated using Ramachandran diagrams to check whether the dihedral angles of the main chain of each amino acid residue in the model were within the allowable region; at the same time, the QMEAN scoring system of SWISS-MODEL was used to evaluate the overall quality of the model. The model evaluation results showed that the constructed TRIM44 three-dimensional structural model has good conformational rationality and structural stability, and can be used for subsequent molecular docking research.

[0113] 2. Molecular docking of macadamia oleracea and TRIM44

[0114] (1) Ligand structure preparation: The three-dimensional structure file (SDF format) of solasonine (CAS No. 19121-58-5) was obtained from the PubChem database and converted to PDB format using OpenBabel software. The MMFF94 force field was used to optimize the ligand structure by minimizing energy, and the lowest energy conformation was obtained as the ligand structure for molecular docking.

[0115] (2) Receptor structure preparation: The three-dimensional structure model of TRIM44 obtained by homology modeling was used as the receptor. The receptor structure was preprocessed using AutoDockTools v1.5.6 software, including removing water molecules, adding polar hydrogen atoms, and calculating Gasteiger charge. The potential active pocket region on the surface of TRIM44 protein was predicted by the CASTp server to determine the grid center coordinates and grid size for molecular docking.

[0116] (3) Molecular docking: Molecular docking calculations for macadamia oleifera and TRIM44 were performed using AutoDock Vina v1.2.0 molecular docking software. The docking grid box was set to cover the entire potential active pocket region of the TRIM44 protein, and the docking parameters were set as follows: exhaustiveness=8, num_modes=10, energy_range=3. The molecular docking program was run to obtain multiple possible binding conformations of macadamia oleifera and TRIM44 protein.

[0117] (4) Binding Mode Analysis: Based on the docking free energy (∆G) ranked from low to high, the conformation with the lowest binding energy (i.e., the strongest binding affinity) was selected as the optimal binding mode. PyMOL v2.5 software was used to perform three-dimensional visualization analysis of the optimal docking conformation to identify non-covalent interactions such as hydrogen bonds and hydrophobic interactions between solanine and the amino acid residues of TRIM44 protein. The results showed that solanine binds to five amino acid residues of TRIM44 protein—G363, T365, T501, G504, and A505—forming five hydrogen bonds.

[0118] 3. Construction of TRIM44 point mutant plasmid

[0119] Based on the human TRIM44 gene sequence, point mutation primers targeting five amino acid residues (G363, T365, T501, G504, and A505) were designed. Overlap extension PCR was used to construct TRIM44-G363A, TRIM44-T365A, TRIM44-T501A, TRIM44-G504A, and TRIM44-A505G point mutants, respectively. Simultaneously, a wild-type TRIM44 (TRIM44-WT) overexpression plasmid was constructed. All plasmids were sequenced to confirm the correct mutation sites.

[0120] 4. Establishment of stable cell lines

[0121] Using the same method as in Example 2, TRIM44-WT and each point mutant plasmid were packaged into lentiviruses and used to infect HCT-8 and SW-480 cells. After screening with puromycin, cell lines that stably express wild-type or mutant TRIM44 were obtained.

[0122] 5. Cellular thermal displacement assay (CETSA)

[0123] The stable cell lines were collected and resuspended in PBS, then divided into two groups: one group was treated with solanine (final concentration 20 μM), and the other group was treated with an equal volume of DMSO as a control. Both groups were incubated at room temperature for 30 minutes. The cell suspension was then divided into several aliquots and heated at 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 3 minutes each, followed by rapid cooling on ice for 5 minutes. Cells were lysed by a freeze-thaw cycle three times, centrifuged at 4℃ and 20,000×g for 20 minutes, and the supernatant was collected for Western blotting. The residual amount of TRIM44 protein in the supernatant at different temperatures was detected using TRIM44 antibody. The binding of solanine to TRIM44 was determined by comparing the thermostability of TRIM44 protein between the solanine-treated group and the control group.

[0124] 6. Surface Plasmon Resonance (SPR) Analysis

[0125] SPR (Solanine Reactivity System) was performed using a Biacore T200 biomolecular interaction analyzer. Purified wild-type TRIM44 protein and point-mutated TRIM44 proteins were immobilized on a CM5 chip using an amino-coupling method. Solanine was diluted with running buffer to different concentrations (0, 0.1, 0.5, 1, 2, 5, 10, 20 μM) and sequentially flowed through the chip surface at a flow rate of 30 μL / min, with a binding time of 120 seconds and a dissociation time of 300 seconds. Binding kinetic parameters were calculated using Biacore T200 evaluation software.

[0126] 7. Solanine gradient concentration intervention experiment

[0127] Cells expressing wild-type and point mutant TRIM44 were treated with different concentrations of solanine (0, 1, 2, 4 μM) for 24 hours. Changes in TRIM44 and GPX4 protein expression, ROS levels, and cell proliferation, migration, and invasion abilities were detected according to the method described in Example 2.

[0128] 8. Results

[0129] Molecular docking results showed that solanine binds to five amino acid residues (G363, T365, T501, G504, and A505) of the TRIM44 protein, forming five hydrogen bonds. CETSA experiments showed that solanine treatment significantly improved the thermostability of wild-type TRIM44 protein (manifested as an increase in residual protein after heating), indicating that solanine can directly bind to TRIM44 protein. SPR experiments further confirmed the direct binding of solanine to wild-type TRIM44 protein. Notably, in point mutant cells (G363A, T365A, T501A, G504A, and A505G), the effect of solanine on improving the thermostability of TRIM44 protein was significantly weakened, and SPR detection showed a significantly reduced affinity of solanine for each point mutant TRIM44 protein. Western blot results showed that in cells expressing wild-type TRIM44, solanine downregulated the expression of TRIM44 and GPX4 proteins in a concentration-dependent manner; however, in point mutant cells, the downregulation effect of solanine on TRIM44 and GPX4 proteins was significantly weakened. Similarly, the inhibitory effects of solanine on the proliferation, migration, and invasion of point mutant cells, as well as its induction of ROS, were significantly weaker than in wild-type cells.

[0130] The above results indicate that solanine binds specifically to the active pocket of TRIM44 by forming hydrogen bonds with five amino acid residues (G363, T365, T501, G504, and A505) of the TRIM44 protein, thereby inhibiting the deubiquitinating enzyme activity of TRIM44.

[0131] Example 5: In vivo efficacy verification of solanine against colorectal cancer

[0132] 1. Establishment of a xenograft tumor model for colorectal cancer

[0133] Male BALB / c nude mice aged 4-6 weeks were housed in an SPF-grade animal facility. HCT-8-luc cells stably expressing luciferase tags (Vector control or GPX4) were then used. OE 5 × 10⁶ tumors per mouse were subcutaneously injected into the left axilla of nude mice. When the subcutaneous tumors grew to about 1 cm³, the tumor tissue was carefully separated with sterile scissors and cut into 1-2 mm³ tissue blocks. The tumor tissue blocks were then attached to the serosa of the cecum of the mice using bio-glue to establish an in situ colorectal cancer model.

[0134] Take another batch of nude mice, and use 3×10 6 HCT-8-luc cells (Vector control or GPX4) OEA colorectal cancer peritoneal metastasis model was established by slow injection into the peritoneal cavity of nude mice. Seven to ten days post-surgery, in vivo bioluminescence imaging was used to observe the tumor within the peritoneal cavity, indicating successful model establishment.

[0135] 2. Experimental grouping and dosing regimen

[0136] Nude mice from the in situ model and the peritoneal transfer model were randomly divided into the following groups, with 6 mice in each group:

[0137] Group A: Vector model + saline (intraperitoneal injection, once daily);

[0138] Group B: Vector model + solanine (2.5 mg / kg, intraperitoneal injection, once daily).

[0139] Group C: Vector model + solanine (2.5 mg / kg, intraperitoneal injection, once daily) + Fer-1 (5 mg / kg, intraperitoneal injection, once daily).

[0140] Group D: Vector model + solanine (2.5 mg / kg, intraperitoneal injection, once daily) + NAC (100 mg / kg, intraperitoneal injection, once daily).

[0141] Group E: GPX4 OE Model + DMSO (equivalent dose, intraperitoneal injection, once daily);

[0142] Group F: GPX4 OE Model + Solanine (2.5 mg / kg, intraperitoneal injection, once daily).

[0143] Each group received the medication continuously for 5 weeks.

[0144] 3. Observation Indicators

[0145] (1) Tumor growth monitoring: The intensity of tumor fluorescence signal in each group of mice was detected weekly using a small animal in vivo biological fluorescence imaging instrument to dynamically monitor tumor growth.

[0146] (2) Tumor size and mass: After administration, the mice were euthanized, the tumor in situ was dissected, the long diameter and short diameter of the tumor were measured, the tumor volume (V = long diameter × short diameter² / 2) was calculated, and the tumor mass was weighed.

[0147] (3) Counting of metastatic lesions: After dissecting the mice, carefully examine the metastatic lesions in organs such as the liver, lungs, and abdominal cavity, count them and measure their size.

[0148] (4) Histological examination: Tumor tissue and metastatic lesions were collected. One part was fixed with 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was used to detect the expression of TRIM44 and GPX4. The other part was used to prepare tissue homogenate. ROS level was detected using a ROS detection kit. The protein expression of TRIM44 and GPX4 was detected using Western Blot.

[0149] 4. Results

[0150] Biofluorescence imaging results showed that the tumor fluorescence signal intensity in mice treated with solanine (Group B) was significantly lower than that in the saline control group (Group A), indicating that solanine can significantly inhibit in situ growth and peritoneal metastasis of colorectal cancer. Tumor volume and mass measurements were consistent with the fluorescence imaging results; the tumor volume and mass in the solanine treatment group were significantly smaller than those in the control group.

[0151] In the groups treated with solanine in combination with Fer-1 or NAC (Groups C and D), the inhibitory effect of solanine on tumor growth was significantly reversed, further confirming that the anti-colorectal cancer effect of solanine is achieved by inducing ferroptosis. In GPX4 OE In the model groups (group E and group F), GPX4 overexpression significantly weakened the antitumor effect of solanine (group F vs group B), indicating that the anticancer effect of solanine depends on its downregulation of GPX4.

[0152] Immunohistochemical and Western blot results showed that the expression levels of TRIM44 and GPX4 in tumor tissues of the solanine treatment group were significantly lower than those of the control group, while the ROS level was significantly higher. These in vivo experimental results further confirm that solanine inhibits the occurrence and development of colorectal cancer by targeting and inhibiting TRIM44 activity, upregulating GPX4 ubiquitination levels, promoting GPX4 protein degradation, and inducing ferroptosis in colorectal cancer cells.

[0153] Example 6: Preparation of pharmaceutical formulation

[0154] 1. Preparation of Injectables

[0155] Take 10 g of solanine raw material, add an appropriate amount of water for injection, add polysorbate-80 (0.1%) as a solubilizer, stir to dissolve, adjust the pH to 5.5-6.5 with 0.1 mol / L hydrochloric acid or sodium hydroxide solution, add water for injection to make up to 1000 mL, filter through a 0.22 μm microporous membrane for sterilization, dispense into ampoules, each 2 mL (containing 20 mg of solanine), freeze-dry to prepare lyophilized powder for injection, and reconstitute with water for injection before use.

[0156] 2. Tablet preparation

[0157] Take 50 g of solanine raw material, mix it evenly with 100 g of starch, 80 g of microcrystalline cellulose and 20 g of sodium carboxymethyl starch, prepare a soft material with an appropriate amount of 70% ethanol, granulate it through a 20-mesh sieve, dry it at 60℃, add 2 g of magnesium stearate after granulation, mix evenly, compress into tablets, each tablet contains 50 mg of solanine.

[0158] 3. Preparation of capsules

[0159] Take 50 g of solanine raw material, mix it evenly with 120 g of starch and 80 g of lactose, pass it through an 80-mesh sieve, and fill it into hard capsule shells. Each capsule contains 50 mg of solanine.

[0160] 4. Preparation of granules

[0161] Take 50 g of macadamia alkaloid raw material, mix it evenly with 800 g of sucrose powder and 150 g of dextrin, prepare a soft material with an appropriate amount of 80% ethanol, granulate it through a 14-mesh sieve, dry it at 60℃, and package it after granulation. Each bag contains 50 mg of macadamia alkaloid.

[0162] Example 7: Route of administration and method of administration

[0163] The drug described in this invention can be administered via various routes, including intraperitoneal injection, intravenous injection, oral administration, or local administration.

[0164] For intraperitoneal or intravenous administration, the recommended dose of solanine is 1-10 mg / kg body weight, once daily, for 4-6 weeks as one course of treatment. For oral administration, the recommended dose is 2-20 mg / kg body weight, once or twice daily, for 4-6 weeks as one course of treatment. The specific dosage and duration of treatment may be adjusted by the clinician based on factors such as the patient's condition, weight, age, and TRIM44 expression level.

[0165] For colorectal cancer patients with high TRIM44 expression, targeted therapy using the drugs described in this invention is recommended as a first-line treatment. The expression level of TRIM44 in the patient's tumor tissue can be detected by methods such as immunohistochemistry or RT-qPCR. Patients with high TRIM44 expression can achieve better therapeutic effects using the drugs of this invention.

[0166] The above description is merely a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. Application of solanine as a TRIM44 inhibitor in the preparation of anti-colorectal cancer drugs.

2. The application according to claim 1, characterized in that, The solanine binds to at least one amino acid residue at sites G363, T365, T501, G504, and A505 of the TRIM44 protein.

3. The application according to claim 2, characterized in that, The solanine binds to five amino acid residues of the TRIM44 protein—G363, T365, T501, G504, and A505—to form hydrogen bonds.

4. The application according to claim 1, characterized in that, The drug inhibits TRIM44 activity through solanine, upregulates TRIM44-mediated GPX4 ubiquitination levels, and promotes ferroptosis in colorectal cancer cells.

5. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

6. The application according to claim 5, characterized in that, The dosage form of the drug is injection, tablet, capsule or granule.

7. The application according to claim 1, characterized in that, The colorectal cancer mentioned is a colorectal cancer with high TRIM44 expression.

8. The application according to claim 1, characterized in that, The drug can be administered via intraperitoneal injection, intravenous injection, oral administration, or local administration.

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