Use of a butyrolactone compound in the preparation of a medicament for treating renal clear cell carcinoma

Abstract

The application discloses a kind of butyrolactone compound in preparation treatment renal clear cell carcinoma drug purposes, characteristic is the compound structural formula as shown in I, the purposes of the compound in preparation anti renal clear cell carcinoma tumor drug and in preparation promote renal clear cell carcinoma sorafenib treatment sensitivity drug, its preparation method is by carrying out fermentation culture to marine fungus, obtains fermentation, then the fermentation is soaked with ethyl acetate and is extracted to obtain crude extract, again the crude extract is sequentially separated by normal phase medium pressure column chromatography, reversed phase medium pressure column chromatography and semi-preparative reversed phase high performance liquid chromatography separation and purification is obtained, advantage is that renal clear cell carcinoma can be significantly enhanced to sorafenib treatment sensitivity, can effectively inhibit tumor growth and delay the emergence of drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceutical technology, specifically relating to the use of a butyrolactone compound in the preparation of a drug for treating clear cell renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system, with clear cell renal cell carcinoma (ccRCC) accounting for approximately 70-80%. Its pathogenesis mainly involves the abnormal activation of the hypoxia-inducible factor (HIF) pathway caused by the inactivation of the Von Hippel-Lindau (VHL) gene, which in turn leads to the overexpression of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), thereby promoting tumor angiogenesis and progression.

[0003] For localized renal cell carcinoma, surgical resection is the primary curative treatment. However, approximately 30% of patients already have metastases at initial diagnosis, and the postoperative recurrence rate is high. With a deeper understanding of the molecular mechanisms of renal cell carcinoma, the treatment of advanced renal cell carcinoma has entered the era of targeted and immunotherapy. Sorafenib, as the world's first approved oral multi-target kinase inhibitor for metastatic renal cell carcinoma (mRCC), exerts its anti-tumor and anti-angiogenic effects by targeting multiple kinases such as RAF, VEGFR, and PDGFR, marking a milestone in the history of targeted therapy for renal cell carcinoma. Even in the era of immunotherapy, sorafenib continues to play an important role in clinical treatment in many regions worldwide due to its proven efficacy, manageable toxicity profile, and broad drug accessibility.

[0004] However, similar to most targeted therapies, sorafenib faces a significant challenge in clinical application due to drug resistance. Most patients develop acquired resistance after 6-15 months of treatment, leading to disease progression. Studies have shown that the mechanisms of sorafenib resistance are extremely complex, involving multiple factors such as activation of compensatory signaling pathways within tumor cells (e.g., PI3K / Akt, STAT3), tumor microenvironment remodeling, epigenetic modifications, and tumor stem cell enrichment. Current main strategies for combating resistance include switching to targeted therapies with other mechanisms of action or immunotherapy, but the efficacy and duration of subsequent treatment are often limited. In contrast, combination therapy strategies—especially the search for synergistic drugs that can enhance the initial efficacy of sorafenib (sensitization) or reverse resistance—are considered a more promising direction for breakthroughs.

[0005] Marine microorganisms, due to their unique living environment, can produce novel and diverse secondary metabolites, making them an important resource for natural product chemistry and new drug development. Butyrolactones are a class of natural products with a five-membered unsaturated lactone ring as their core structure, widely found in the metabolites of marine fungi such as Aspergillus. Studies have shown that these compounds possess various pharmacological activities, including anti-inflammatory, antioxidant, and neuroprotective effects, with low toxicity, demonstrating promising applications in drug development. Currently, there are no reported studies on the application of butyrolactones in improving the sensitivity of clear cell renal cell carcinoma cells to sorafenib treatment. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide the use of butyrolactone compounds that enhance the sensitivity of clear cell renal cell carcinoma to sorafenib treatment in the preparation of drugs for treating clear cell renal cell carcinoma.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: This invention provides a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment, the structural formula of which is shown in Figure I: ,I.

[0008] The present invention also provides a method for preparing the above-mentioned butyrolactone compounds that enhance the sensitivity of clear cell renal cell carcinoma to sorafenib treatment, comprising the following steps: Step 1: Fermentation Production Streaking of Aspergillus pyrolyticus with accession number CCTCC NO: M2014086 onto PDA solid medium plates and incubating upside down in an incubator at 25-30 ℃ for 2-4 days, then picking single colonies and inoculating them into PDB liquid medium, and incubating on a shaker at 25-30 ℃ and 150-250 rpm / min for 3 days to obtain seed culture. The seed culture was added to rice medium at a volume percentage of 10-20% and incubated at 25-30 ℃ for 25-30 days to obtain fermentation product. Step 2: Extraction of crude extract Add an equal volume of ethyl acetate to the fermentation product obtained in step 1, and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract. Step 3: Isolation and preparation of compounds The crude extract obtained in step 2 was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel powder. Normal-phase medium-pressure column chromatography was performed using a 1:1 petroleum ether-ethyl acetate solution as the eluent for gradient elution, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography using a 30-100% methanol-water mixture as the eluent for linear gradient elution. The eluent fractions were collected, arranged in descending order of polarity, and combined to obtain 6 fractions. The fourth fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 1:1 acetonitrile-water mixture as the mobile phase to obtain a monomeric compound, the structure of which is shown in Figure I. ,I.

[0009] Furthermore, the PDA solid culture medium preparation method in step 1 is as follows: 6 g of potato extract powder, 20 g of glucose and 20 g of agar are added to 1000 mL of distilled water to prepare the culture medium; the PDB liquid culture medium preparation method is as follows: 6 g of potato extract powder and 20 g of glucose are dissolved in 1000 mL of water to prepare the culture medium; the rice culture medium preparation method is as follows: 90 g of rice, 3 g of sea salt and 110 mL of water.

[0010] Furthermore, in step 3, the methanol volume percentage in the reversed-phase medium-pressure column chromatography linear gradient elution ranges from 30% to 100%, the elution time is 180 min, and the flow rate is 30 mL / min.

[0011] Furthermore, the flow rate for compound separation and preparation by semi-preparative reversed-phase high-performance liquid chromatography described in step 3 is 2.0 mL / min.

[0012] The present invention also provides the use of the above-mentioned butyrolactone compounds in the preparation of anti-renal clear cell carcinoma tumor drugs.

[0013] This invention also provides the use of the above-mentioned butyrolactone compounds in the preparation of formulations that inhibit the proliferation of clear cell renal tumor cells. Butyrolactone compounds inhibit tumor growth by inhibiting the proliferation of clear cell renal cancer cells.

[0014] The present invention also provides the use of the above-mentioned butyrolactone compounds in the preparation of medicaments for improving the sensitivity of clear cell renal cell carcinoma to sorafenib treatment.

[0015] The present invention also provides the use of the above-mentioned butyrolactone compound in the preparation of a drug for regulating the SPOP-GLYR1-Cer axis.

[0016] The present invention also provides a pharmaceutical composition for treating clear cell renal cell carcinoma, comprising an effective amount of a butyrolactone compound and sorafenib, and a pharmaceutically acceptable carrier.

[0017] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses the use of a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment in the preparation of drugs for treating clear cell renal cell carcinoma. The method involves fermenting marine fungi into rice culture to obtain a fermentation product, then extracting the fermentation product with ethyl acetate to obtain a crude extract. This extract is then purified sequentially by forward medium-pressure column chromatography, reverse medium-pressure column chromatography, and semi-preparative high-performance liquid chromatography to obtain the monomeric compound 2-O-methylbutyrolactone. This compound exhibits activity that significantly enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment. Mechanistic studies have found that when this compound is used in combination with sorafenib, it demonstrates a strong synergistic antitumor effect in in vivo models and 3D-printed tumor models derived from clinical tissues, effectively inhibiting tumor growth and delaying the onset of drug resistance. This compound can be used in the preparation of combination therapies to improve sorafenib tolerance in clear cell renal cell carcinoma.

[0018] The above-mentioned Aspergillus pyrolysis ( Aspergillus ustus The strain is DJ003, with accession number CCTCC NO:M2014086. It was deposited at the China Center for Type Culture Collection on March 14, 2014, at Wuhan University, Wuhan, China. Attached Figure Description

[0019] Figure 1 High-resolution mass spectra of the compounds of this invention; Figure 2 The proton NMR spectrum of the compound of this invention; Figure 3 The carbon NMR spectrum of the compound of this invention; Figure 4 A schematic diagram of 2-O-methylbutyrolactone on 786O cell xenografts in nude mice; Figure 5 The weight statistics of 2-O-methylbutyrolactone on 786O cell xenografts in nude mice; Figure 6 Statistical analysis of the volume dynamics of 2-O-methylbutyrolactone-induced xenografts in 786O cells of nude mice; Figure 7 Immunohistochemical Ki67 staining of 786O cell xenografts in nude mice with 2-O-methylbutyrolactone; Figure 8 Statistical analysis of Ki67 immunohistochemical staining results of 2-O-methylbutyrolactone on 786O cell xenografts in nude mice; Figure 9These are 6 samples for drug sensitivity testing of 3D printed bodies; Figure 10 Statistical analysis of drug susceptibility testing results for 6 cases of 3D printed bodies; Figure 11 The effect of 2-O-methylbutyrolactone on SPOP protein in ccRCC cell lines (786O and Caki-1); Figure 12 This is a schematic diagram showing the results of the immunoprecipitation of SPOP and GLYR1 proteins. Figure 13 A schematic diagram illustrating the effect of gradient SPOP protein on GLYR1 protein; Figure 14 This is a schematic diagram illustrating the effect of gradient SPOP protein on the ubiquitination of GLYR1 protein. Figure 15 This is a schematic diagram of the targeted lipid metabolomics results after GLYR1 overexpression. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0021] Example 1: The structural formula of a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment is shown in Figure I: ,I.

[0022] Example 2, the preparation method of the butyrolactone compounds shown in Example 1, the specific steps are as follows: Step 1: Fermentation Production Aspergillus pyroximate with accession number CCTCC NO: M2014086 was used. Aspergillus ustus) Streak the culture medium on PDA solid medium (prepared by adding 6 g potato starch, 20 g glucose, and 20 g agar to 1000 mL distilled water) and incubate upside down in a 28 ℃ incubator for 3 days. Then, pick a single colony and inoculate it into PDB liquid medium (prepared by dissolving 6 g potato starch and 20 g glucose in 1000 mL water). Incubate the medium on a shaker at 28 ℃ and 200 rpm for 3 days to obtain the seed culture. Put 100 g of rice culture medium (90 g rice, 3 g sea salt, and 110 mL water) into a 1 L Erlenmeyer flask and autoclave it at 121 ℃ for 15 minutes. Finally, inoculate each flask with 15 mL of seed culture and incubate it at 28 ℃ for 30 days to obtain the fermentation product. Step 2: Extraction of crude extract Add an equal volume of ethyl acetate to the fermentation product obtained in step 1, and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract. Step 3: Isolation and preparation of compounds The crude extract obtained in step 2 was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel powder. Normal-phase medium-pressure column chromatography was performed using a 1:1 petroleum ether-ethyl acetate solution as the eluent for gradient elution, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography using a 30-100% methanol-water solution (180 min, flow rate 30 mL / min) as the eluent for linear gradient elution. The eluent fractions were collected, arranged in descending order of polarity, and combined to obtain 6 fractions. The fourth fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using a 1:1 acetonitrile-water mixture as the mobile phase at a flow rate of 2 mL / min to obtain a monomeric compound, the structure of which is shown in Figure I. ,I.

[0023] Example 3: Structural analysis of butyrolactone compound I prepared in Example 2 above.

[0024] Compound I of the present invention is a colorless oil, such as Figure 1 As shown, its molecular formula was determined to be C by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). 25 H 26 O7, the measured quasi-molecular ion peak m / z 439.1757 [M+H] + (The calculated value is 439.1770), corresponding to 13 degrees of unsaturation. Based on comprehensive analysis of nuclear magnetic resonance spectroscopy data and comparison with literature, its structure is determined as follows: Figure 2 of 11H NMR spectroscopy revealed the following characteristic groups in the structure of this compound: a 1,4-disubstituted benzene ring with aromatic proton signals of δH 7.42 (2H, d, J=8.7 Hz) and 6.90 (2H, d, J=8.7 Hz); a 1,3,4-trisubstituted benzene ring with aromatic proton signals of δH 6.49 (1H, dd, J=8.3, 2.2 Hz), 6.37 (1H, d, J=2.2 Hz), and 6.55 (1H, d, J=8.3 Hz); a methylene group with δH 3.38 (1H, d, J=14.6 Hz) and 3.32 (1H, d, J=14.6 Hz); two methoxy groups with δH 3.75 (3H, s) and 3.58 (3H, s); and an isoprene group [δH 5.01 (1H, t, J = 7.3 Hz), 3.03 (2H, d, J = 7.32Hz), 1.61 (3H, s) and 1.53 (3H, s)). Figure 3 of 13 The 166.6-C10 NMR spectrum yielded 25 carbon signals, including two carbonyl carbons (δC 169.1 and 166.6), nine quaternary carbons (δC 159.3, 154.0, 140.0, 137.2, 131.5, 126.8, 123.0, 119.7, and 84.6), nine methine carbons (δC 130.9, 129.7×2, 128.7, 122.5, 116.1×2, and 114.3), two methylene carbons (δC 38.5 and 27.7), and four methyl carbons (δC 58.3, 53.8, 25.6, and 17.6). δC 166.6 is the characteristic signal of the conjugated γ-lactone carbonyl group, and δC 84.6 is the characteristic signal of the quaternary carbon on the lactone ring. The above spectral data are basically consistent with the 2-O-methylbutyrolactone reported in the literature, so the compound is identified as 2-O-methylbutyrolactone, with the Chinese name Butyrolactone Compound I.

[0025] Given that the structure of compound I of this invention contains multiple chiral centers, in order to determine its absolute configuration, we adopted the method of optical rotation comparison and compared it with a previously reported butyrolactone compound with a similar structure (application number CN202410950677.5).

[0026] Determination of optical rotation: The specific optical rotation of compound I of the present invention was measured to be +47°.

[0027] Comparison with known compounds: The optical rotation values ​​of the compounds of the present invention were compared with those of previously reported butyrolactone compounds (whose absolute configurations are known) having the same or similar skeletons. The specific rotation value of the known compound is +37.6°.

[0028] Configuration Inference: Since the compounds of this invention are highly similar to known compounds in their core skeleton (butyrolactone ring, key substituents), and their specific rotation values ​​have the same sign (both positive) and are on similar orders of magnitude (47 vs 37.6), this indicates that they likely have the same or highly similar chiral center configuration. Considering that the absolute configurations of known compounds have been established, we infer that the absolute configuration of compound I of this invention is consistent with that of known compounds.

[0029] Table 1. Compound I 1 H and 13 C NMR data (DMSO- d 6)

[0030] Note 1: s - singlet, d - doublet, t - triplet Note 2: 1 H was obtained by 600 MHz NMR; 13 C was obtained by 150 MHz NMR.

[0031] Example 4: Butyrolactone compound I inhibits tumor growth by suppressing the proliferation of clear cell renal cancer cells. The specific steps are as follows: 1. Preparation of cells and animals Cells: Human clear cell renal carcinoma (ccRCC) cell line 786O was routinely passaged in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments. Animals: SPF-grade BALB / c nu / nu nude mice (4 weeks old). After one week of acclimatization, they were randomly divided into a solvent control group and a 2-O-methylbutyrolactone treatment group, with 5 mice in each group. Hulling conditions: temperature 22-25℃, humidity 50-60%, 12-hour light / dark cycle.

[0032] 2. Construction of a 786O cell subcutaneous xenograft tumor model in nude mice Cell suspension preparation: 786O cells in logarithmic growth phase were digested with 0.25% trypsin, washed twice with sterile PBS, and resuspended in serum-free DMEM medium. Cells were counted using a hemocytometer, and the cell density was adjusted to 2 × 10⁻⁶ cells / mL. 7 cells / mL; Preparation of cell-Matrigel mixture: The cell suspension was mixed with pre-cooled (4°C) Matrigel at a volume ratio of 1:1 to obtain a final cell density of 1×10⁻⁶. 7 The mixture was prepared at 100 cells / mL. The entire process was performed on ice to prevent matrix gel solidification. Subcutaneous inoculation: Using a pre-cooled 1mL syringe, draw up the mixture and subcutaneously inject 100μL of the mixture, containing 1×10⁻⁶ ppm, into the dorsal side of the right forelimb of each nude mouse. 6 7860 cells were injected. The mice's condition and tumor formation at the injection site were observed daily after inoculation.

[0033] 3. Drug preparation and administration regimen 1) Drug preparation: Preparation of stock solution: Accurately weigh 2-O-methylbutyrolactone, dissolve it in DMSO, prepare a 10 mM stock solution, dispense into portions, and store at -20°C protected from light; Preparation of working solution: Dilute the stock solution before each administration. The final DMSO concentration should not exceed 0.1%. Dosage calculation: Interspecies dose conversion was performed based on the effective in vitro concentration (50 nM) using the body surface area normalization method. Preliminary experiments determined the equivalent dose in nude mice to be 3 mg / kg / day.

[0034] 2) Dosing regimen: Start time: Administer medication on day 3 after tumor cell inoculation; Administration method and cycle: Intraperitoneal injection was used, once daily, for a total of 13 days until the end of the experiment (day 15).

[0035] 4. Tumor growth monitoring Monitoring frequency: Tumor size was measured every 3 days, starting from the first administration date (day 3 post-vaccination); Measurement and Calculation: Measure the longest diameter (a) and the shortest diameter (b) perpendicular to the tumor using a digital caliper. Calculate using the formula V = 1 / 2 × a × b. 2 Calculate the tumor volume. Record the data and plot the tumor growth curve.

[0036] 5. Experimental endpoint and sample collection End point: The experiment ended on day 15 after tumor cell inoculation (i.e., after the last administration). Euthanasia and Tissue Collection: Nude mice were euthanized by cervical dislocation, and subcutaneous tumor tissue was completely removed; Weighing: Immediately weigh each tumor piece using a precision electronic balance; Subpackaging: Weighed tissues are immersed in 4% paraformaldehyde for 24-48 hours for paraffin embedding; Morphological recording: Fresh tumor tissue was placed on a background with rulers and photographed to visually compare the size of the two groups of tumors.

[0037] 6. Ki67 Immunohistochemical Experiment Paraffin section preparation: The tumors obtained after the subcutaneous tumorigenesis experiment were removed, embedded in paraffin, and sectioned. The paraffin sections were then baked in a 65°C oven for 2 hours. Immunohistochemical staining: The slides were dewaxed and rehydrated sequentially in xylene-anhydrous ethanol-95% ethanol-75% ethanol; antigen retrieval was then performed using EDTA solution in an autoclave; followed by blocking with 3% hydrogen peroxide solution for endogenous peroxidase; permeabilization with 0.5% Triton solution; then blocking with 10% donkey serum; overnight incubation with Ki-67 antibody; incubation with secondary antibody the next day; followed by staining with a DAB chromogenic kit; then staining of cell nuclei with modified hematoxylin; then dehydration and clearing were performed sequentially using 75% ethanol-95% ethanol-anhydrous ethanol-xylene; finally, after the slides had dried slightly, they were mounted with neutral resin. Quantitative analysis: Random areas were selected from different samples under an optical microscope (×200x). ImageJ software was used to count Ki67-positive (brown nuclei) cells and the total number of cells, and the Ki67 positivity rate (%) was calculated.

[0038] 7. Statistical Analysis All measurement data are expressed as mean ± standard deviation. Unpaired two-tailed t-tests were used to compare tumor weight, volume, and Ki67 positivity rates between the two groups using GraphPad Prism 10.1.2 software. A p-value < 0.05 was considered statistically significant.

[0039] This experiment systematically investigated the inhibitory effect of 2-O-methylbutyrolactone (butyrolactone compound I) on the growth of clear cell renal cell carcinoma (ccRCC) by constructing a 786O cell xenograft model in nude mice. The following conclusions were drawn: 1) Tumor growth inhibition: Compared with the DMSO control group, the growth rate of xenografts in nude mice was significantly reduced after treatment with 2-O-methylbutyrolactone. Figure 6 Ultimately, the tumor weight decreased significantly. Figure 4 , 5 This indicates that compound I can effectively inhibit the growth of ccRCC xenografts. 2) Cell proliferation inhibition: Immunohistochemical Ki67 staining results showed that the proportion of Ki67-positive cells in the compound I treatment group was significantly reduced ( Figure 7 , 8 This indicates that compound I can inhibit the proliferation activity of ccRCC cells, thereby hindering the growth process of tumors.

[0040] In summary, 2-O-methylbutyrolactone (butyrolactone compound I) can play an anti-tumor role by inhibiting the proliferation of renal clear cell carcinoma cells, providing potential drug candidates for the treatment of ccRCC.

[0041] Example 5: Analysis of the enhanced drug sensitivity of butyrolactone compound I to sorafenib in the treatment of renal clear cell carcinoma.

[0042] This experiment was carried out in cooperation with Herschangdu Biotechnology Co., Ltd. and completed using its clinical sample pretreatment and 3D bioprinting technology platform.

[0043] Sample processing and cell acquisition: The renal clear cell carcinoma (ccRCC) tissue resected surgically was made into a primary tumor cell suspension according to the standard process (trimming, washing, digestion, filtration, red blood cell lysis).

[0044] Construction of 3D bioprinted body: The primary tumor cells were mixed with GelMA-30 photosensitive hydrogel in proportion to make bioink. Through the company's sample pretreatment system (Pre-Maker1), the bioink was printed into a preset three-dimensional structure and cured using a light curing instrument to construct a 3D printed body model of renal cancer for drug testing.

[0045] Drug sensitivity detection: The 3D printed body was transferred to a 48-well plate and treated with different concentration gradients of sorafenib alone or in combination with 2-O-methylbutyrolactone (butyrolactone compound I). After culturing for 72 hours, the cell viability was detected using the CellTiter method, and the OD value was read using an enzyme-labeled instrument. The sensitivity of the drug was determined according to the values of IC 50 (half inhibitory concentration), Css (steady-state blood drug concentration), and Cmax (blood drug concentration) following the following criteria: When IC 50 < Css / Cmax, it is determined to be sensitive; when IC 50 > Css / Cmax and the lower bound of the 95% confidence interval is less than Css / Cmax, it is determined to be intermediate; when IC 50 > Css / Cmax and the lower bound of the 95% confidence interval is greater than Css / Cmax, it is determined to be resistant.

[0046] Data analysis: Calculate the cell survival rate of each group, use GraphPad Prism software to fit the dose-response curve, and calculate the half inhibitory concentration (IC 50 ) of sorafenib alone and in combination with 2-O-methylbutyrolactone, and compare them.

[0047] Figure 9The presentation showcased photographs of 3D-printed samples from six renal cell carcinoma patients, their three-dimensional structural morphology under a microscope, and HE staining results. HE staining revealed uniform cell distribution within the printed samples, with morphology consistent with the original tumor tissue, indicating that the clinical samples were qualified and providing a more clinically relevant in vitro platform for drug sensitivity testing. Figure 10 Statistical analysis was conducted on the drug susceptibility test results of 6 samples. The results showed that the IC50 of the combination therapy group was... 50 The values ​​were significantly lower than those in the single-drug group (P<0.0001), and the ratios of most samples were below the threshold of 7.1 mg / L, thus indicating sensitivity.

[0048] In summary, in an in vitro model simulating the three-dimensional structure of tumors, butyrolactone compound I can effectively reduce the IC50 of sorafenib. 50 The study showed that the drug significantly improved the sensitivity of clear cell renal cell carcinoma to sorafenib. This provides new experimental evidence for exploring the combination therapy of sorafenib with butyrolactone compound I in clinical practice to overcome potential drug resistance and improve treatment efficacy.

[0049] Example 6: Butyrolactone compound I exerts its active effect by influencing the SPOP-GLYR1-Cer axis.

[0050] This embodiment aims to explore the key molecular mechanism by which butyrolactone compound I (2-O-methylbutyrolactone) regulates ccRCC. We proposed and verified the "SPOP-GLYR1-Cer" regulatory axis, in which compound I upregulates SPOP protein, promotes its interaction with GLYR1 protein and mediates GLYR1 ubiquitination and degradation, thereby altering downstream ceramide (Cer) metabolism and ultimately exerting its antitumor activity.

[0051] 1. Regulation of SPOP protein expression by 2-O-methylbutyrolactone: Cell treatment: Human clear cell renal cancer cell lines (786O, Caki-1) were seeded into 6-well plates. After adhesion, the medium was replaced with medium containing different concentration gradients (0, 20, 40, 60, 80 nM) of 2-O-methylbutyrolactone and treated for 48 hours.

[0052] Protein extraction and Western blotting: Cells were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by the BCA method. Equal amounts of protein were loaded onto a sample, separated by SDS-PAGE electrophoresis, and transferred to a PVDF membrane. Immunoblotting was performed using anti-SPOP antibody (1:5000) and internal control GAPDH antibody (1:50000). The relative expression level of SPOP protein was analyzed after development.

[0053] The results are as follows Figure 11As shown, in 786O and Caki-1 cells, treatment with 2-O-methylbutyrolactone significantly and dose-dependently increased the expression level of SPOP protein, indicating that it has a positive regulatory effect on SPOP.

[0054] 2. Verification of the interaction between SPOP and GLYR1 protein Co-Immunoprecipitation (Co-IP): Cells were transfected with a plasmid expressing the SFB tag GLYR1 (SFB-GLYR1). Thirty-six hours later, immunoprecipitation was performed using anti-FLAG antibody (for GLYR1). The resulting complex was separated by SDS-PAGE electrophoresis and detected by Western blotting using anti-SPOP antibody (for SPOP) to verify the direct binding of SPOP to GLYR1. Results are shown below. Figure 12 As shown, the Co-IP results confirmed that SFB-GLYR1 and Myc-SPOP specifically bind in cells, verifying the direct interaction between the two.

[0055] Effect of SPOP on GLYR1 protein stability: Myc-SPOP (0, 400, 800 ng) was overexpressed in cells at gradients. After 36 hours, the protein was extracted and the protein level of GLYR1 was detected by Western blot (primary antibody: anti-FLAG) to analyze the effect of SPOP on GLYR1 stability. Results are as follows: Figure 13 As shown, with the increase of Myc-SPOP overexpression, the protein level of GLYR1 decreased in a dose-dependent manner, indicating that SPOP can negatively regulate the protein stability of GLYR1.

[0056] 3. Detection of SPOP-mediated GLYR1 ubiquitination modification After co-transfecting cells with the Myc-SPOP, SFB-GLYR1, and HA-Ub (HA-Ub) plasmids according to the illustrated protocol, cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours. Immunoprecipitation with anti-FLAG antibody was performed to enrich GLYR1 and its bound ubiquitinated protein complex. Subsequently, Western blotting with anti-HA antibody was used to analyze the ubiquitination modification level of GLYR1.

[0057] The results are as follows Figure 14 As shown, in cells co-transfected with Myc-SPOP, SFB-GLYR1 and HA-Ub plasmids, a significant HA-Ub signal was detected on immunoprecipitated GLYR1 cells, and this signal was enhanced when SPOP was co-expressed, proving that SPOP can promote the ubiquitination modification of GLYR1, which is likely the mechanism by which it mediates the degradation of GLYR1 via the ubiquitin-proteasome pathway.

[0058] 4. Effects of GLYR1 overexpression on lipid metabolomics Sample preparation: A stable 786O cell line overexpressing GLYR1 (GLYR1-OE) and an empty vector control group (Vector) were constructed. Cells in the logarithmic growth phase were collected, washed with PBS to obtain cell pellets, flash-frozen in liquid nitrogen, and sent to Maiwei Metabolism (Wuhan Maiwei Metabolism Biotechnology Co., Ltd.) for targeted lipidomics analysis.

[0059] Sample analysis included sample pretreatment (lipid extraction), qualitative and quantitative analysis based on the UPLC-MS / MS platform, and multivariate statistical analysis. The study focused on changes in sphingolipid metabolites such as ceramides (Cer).

[0060] Data Analysis: We focused on the changes in the ceramide metabolite profile induced by GLYR1 overexpression. Results are presented in formats such as volcano plots to identify significantly different ceramide metabolites. Results are as follows: Figure 15 As shown in the lipidomics volcano plot (based on externally sent detection data), the levels of several specific ceramide molecules (such as Cer(d18:2 / 26:0), Cer(t18:0 / 23:1), etc.) changed significantly in GLYR1-overexpressing cells. This indicates that the expression level of GLYR1 directly affects the metabolic homeostasis of intracellular ceramides.

[0061] In summary, this experiment systematically elucidated the action pathway of butyrolactone compound I from multiple levels, including protein expression, protein-protein interaction, post-translational modification, and downstream metabolites. Specifically, 2-O-methylbutyrolactone → upregulates SPOP protein expression → SPOP binds to GLYR1 and enhances its ubiquitination and degradation → downregulates GLYR1 protein levels → alters the ceramide (Cer) metabolic profile. The elucidation of this "SPOP-GLYR1-Cer" regulatory axis provides new theoretical evidence for understanding the molecular mechanism of butyrolactone compound I's anti-clear cell renal cell carcinoma action.

[0062] All data presented in the above experiments have undergone comprehensive checking, verification, and organization to ensure that the data are as complete, accurate, and error-free as possible. Excel software was used for initial data organization; subsequent statistical graphs were presented using GraphPad Prism 10.1.2. A p-value < 0.05 was considered statistically significant, while a p-value > 0.05 was considered not statistically significant.

[0063] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. The use of a butyrolactone compound in the preparation of a drug for treating clear cell renal cell carcinoma, characterized in that, The structural formula of this compound is shown in Figure I: ，I。 2. Use of the butyrolactone compound of claim 1 in the preparation of an agent that inhibits the proliferation of renal clear carcinoma tumor cells.

3. Use of the butyrolactone compound of claim 1 in the preparation of a medicament for enhancing the sensitivity of clear cell renal cell carcinoma to sorafenib treatment.

4. The use of the butyrolactone compound of claim 1 in the preparation of a medicament for regulating the SPOP-GLYR1-Cer axis.

5. A pharmaceutical composition for treating clear cell renal cell carcinoma, characterized in that, It contains an effective amount of butyrolactone compound and sorafenib, as well as a pharmaceutically acceptable carrier.

6. A method for preparing a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment, characterized in that... Includes the following steps: Step 1: Fermentation Production Streaking of Aspergillus pyrolyticus with accession number CCTCC NO: M2014086 on a plate of PDA solid medium, incubating upside down in an incubator, picking a single colony and inoculating it into PDB liquid medium to obtain seed culture, adding the seed culture to rice medium at a volume percentage of 10-20%, and then incubating to obtain fermentation product. Step 2: Extraction of crude extract Add an equal volume of ethyl acetate to the fermentation product obtained in step 1, and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract. Step 3: Isolation and preparation of compounds The crude extract obtained in step 2 was sequentially purified by normal-phase medium-pressure column chromatography, reversed-phase medium-pressure column chromatography, and semi-preparative reversed-phase high-performance liquid chromatography to obtain a monomeric compound, the structure of which is shown in Figure I. ，I。 7. A method for preparing a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment, as described in claim 6, characterized in that... Step 1 is as follows: Streaking Aspergillus pyrolyticus with accession number CCTCC NO: M2014086 onto a PDA solid medium plate, incubating it upside down in an incubator at 25-30 ℃ for 2-4 days, then picking a single colony and inoculating it into PDB liquid medium, and incubating it on a shaker at 25-30 ℃ and 150-250 rpm / min for 3 days to obtain seed culture. Add the seed culture to rice medium at a volume percentage of 10-20%, and incubate it under static conditions at 25-30 ℃ for 25-30 days to obtain fermentation product.

8. A method for preparing a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment, as described in claim 6, characterized in that... Step 3 is as follows: The crude extract obtained in Step 2 is first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel powder and subjected to normal-phase medium-pressure column chromatography. Gradient elution is performed using a 1:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent is collected. The collected eluent is then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution is performed using a methanol-water solution with a methanol volume percentage of 30-100% as the eluent, and the eluent fractions are collected and arranged in descending order of polarity to obtain 6 components. The fourth component is then purified by semi-preparative reversed-phase high-performance liquid chromatography using a 1:1 mixture of acetonitrile and water as the mobile phase to obtain a monomeric compound, the structure of which is shown in Figure I.

9. A method for preparing a butyrolactone compound that enhances the sensitivity of clear cell renal cell carcinoma to sorafenib treatment, as described in claim 8, characterized in that: In the reversed-phase medium-pressure column chromatography linear gradient elution, the methanol volume percentage ranges from 30% to 100%, the elution time is 160-200 min, and the flow rate is 25-35 mL / min.

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