Use of small molecules interfering with the interaction between hk2-ruvbl2 proteins in the preparation of anti-tumor drugs

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a small molecule interfering with HK2-RUVBL2 protein interaction in preparation of an anti-tumor drug. The application targets and regulates the interaction between metabolic enzyme hexokinase 2 (Hexokinase 2, HK2) and RUVBL2 protein, affects the transcription of the key regulation axis-PIKK (Phosphatidylinositol 3-kinase-related kinases) family protein of DNA damage response (DNA Damage Response, DDR), thereby significantly changing the sensitivity of tumor cells to radiotherapy, and then the small molecule interfering with HK2-RUVBL2 protein interaction can be used as a radiosensitizer to achieve the anti-tumor effect, has the radiosensitization characteristics of precision and low toxicity, and has good clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of small molecules that interfere with the interaction of HK2-RUVBL2 protein in the preparation of antitumor drugs. Background Technology

[0002] Radiotherapy is one of the core methods of cancer treatment and is widely used in the clinical treatment of various solid tumors. However, the strong DNA damage repair ability of tumor cells can effectively repair radiotherapy-induced DNA damage, leading to radioresistance, which severely restricts the treatment effect and affects the patient's prognosis. Hexokinase 2 (HK2), as a key rate-limiting enzyme in the glycolysis pathway, is abnormally highly expressed in various tumors. In addition to regulating metabolism, its non-canonical function in the cell nucleus has been proven to be closely related to the malignant progression of tumors, but the relevant molecular regulatory mechanisms are still unclear.

[0003] Currently, commonly used radiosensitization strategies in clinical practice mainly focus on PARP inhibitors and PIKK family inhibitors. These drugs generally have drawbacks such as high toxicity, easy development of drug resistance, and insufficient target specificity, which limit their clinical application and make it difficult to meet the demand for highly effective and low-toxicity treatment.

[0004] Currently, targets for radiosensitizing have not been systematically developed, and there is still a lack of low-toxicity, high-efficiency, and highly specific intervention programs in clinical practice. Therefore, it is urgent to elucidate the mechanism of action of HK2 in tumor radioresistance and to develop novel radiosensitizing agents that target the interaction of this protein, in order to overcome existing technological bottlenecks, significantly improve the efficacy of tumor radiotherapy, and provide new strategies for clinical tumor treatment. Summary of the Invention

[0005] The purpose of this invention is to provide the application of small molecules that interfere with the HK2-RUVBL2 protein interaction in the preparation of antitumor drugs. This invention discovers a novel radiosensitization strategy that regulates DNA damage response by interfering with the interaction of HK2-RUVBL2. As a result, small molecules that interfere with the HK2-RUVBL2 protein interaction have low toxicity, high efficiency, and high specificity in radiosensitization, and have significant potential for clinical translation.

[0006] This invention provides the application of small molecules that interfere with the HK2-RUVBL2 protein interaction in the preparation of antitumor drugs.

[0007] Preferably, the antitumor treatment includes antitumor therapy via radiotherapy.

[0008] Preferably, the application includes the use of small molecules that interfere with HK2-RUVBL2 protein interaction as radiosensitizers.

[0009] Preferably, the application includes the use of small molecules that interfere with the HK2-RUVBL2 protein interaction as inhibitors of DNA damage response.

[0010] Preferably, the tumor includes a solid tumor.

[0011] Preferably, the solid tumor includes colorectal cancer.

[0012] Preferably, the interference with HK2-RUVBL2 protein interaction includes 5″-methoxyhexahydrocurcumin.

[0013] The present invention also provides an antitumor drug, the active ingredient of which includes a small molecule that interferes with the interaction between the HK2-RUVBL2 protein and the antitumor drug.

[0014] Preferably, the small molecule that interferes with the HK2-RUVBL2 protein interaction includes 5″-methoxyhexahydrocurcumin.

[0015] Preferably, the concentration of 5″-methoxyhexahydrocurcumin is 5~30 μM.

[0016] Beneficial effects: This invention provides the application of small molecules that interfere with the HK2-RUVBL2 protein interaction in the preparation of antitumor drugs. By targeting the interaction between HK2 and RUVBL2 proteins, this invention affects the transcription of the DDR key regulatory axis—the PIKK family proteins—significantly altering the sensitivity of tumor cells to radiotherapy. Therefore, the small molecule interfering with the HK2-RUVBL2 protein interaction can serve as a radiosensitizer to achieve an antitumor effect. Compared to the high toxicity and drug resistance problems of traditional radiosensitizers, the small molecule interfering with the HK2-RUVBL2 protein interaction described in this invention, by inhibiting the HK2-RUVBL2 interaction, can achieve precise and low-toxicity radiosensitization and has good clinical translational potential. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a diagram showing the validation results of HK2 deletion causing genomic instability in tumor cells in Example 1; data are from 3 independent experiments (n = 3), expressed as mean ± standard deviation, and significance analysis was performed using a two-sided unpaired t-test (E, F); NS, no statistically significant difference; P <0.05; P <0.01; P <0.001; Figure 2 This is a diagram showing the verification results of HK2 maintaining tumor cell genome stability through interaction with RUVBL2 in Example 2; Figure 3 This is a graph showing the results of virtual screening of small molecule compounds that inhibit the HK2-RUVBL2 interaction in Example 3; Figure 4 This is a graph showing the validation results of the interaction between HK2 and RUVBL2 in Example 4, which promoted tumor survival after IR. Data are expressed as mean ± standard deviation (n = 3). Significance analysis was performed using two-way ANOVA (B, E, H, K); NS, no statistically significant difference. P <0.05; P <0.01; P <0.001; Figure 5 This is a graph showing the verification results of the correlation between high expression of HK2 and RUVBL2 and tumor radiotherapy resistance in Example 5; Figure 6 This is a graph showing the verification results of 5"-MHHC inhibiting DNA damage repair in tumor cells in Example 6; data are expressed as mean ± standard deviation (n = 3), and significance analysis was performed using one-way ANOVA (D, E) and two-way ANOVA (G); NS, no statistically significant difference; P <0.05; P <0.01; P <0.001. Detailed Implementation

[0019] This invention provides the use of small molecules that interfere with HK2-RUVBL2 protein interactions in the preparation of antitumor drugs. In one embodiment, the antitumor activity includes antitumor treatment via radiotherapy. In another embodiment, the application of this invention includes the use of small molecules that interfere with HK2-RUVBL2 protein interactions as radiosensitizers and / or as DNA damage response inhibitors. In one embodiment, the tumor can be a solid tumor, and more particularly, colorectal cancer. In another embodiment, the antitumor activity of this invention includes inhibiting or delaying tumor growth.

[0020] As one embodiment, the interference with the HK2-RUVBL2 protein interaction includes 5″-methoxyhexahydrocurcumin. The 5″-methoxyhexahydrocurcumin described in this invention has the English name 5″-Methoxyhexahydrocurcumin (5″-MHHC), a molecular weight of 404.5 g / mol, CAS number 138870-96-9, and chemical formula C. 22 H 28 O7, InChI Key: TTXHEBLHSZBAEJ-UHFFFAOYSA-N, SMILES: COc1cc(C(=O)CC(C)C(O)C(=O)Cc2ccc(O)c(OC)c2)ccc1O, Appearance: Light yellow solid powder with a slight curcumin odor, Melting point range: 148~55℃ (preferably 151℃), Partition coefficient LogP: 2.1~2.9 (preferably 2.5), Solubility: Soluble in DMSO, ethanol and moderately polar organic solvents, low solubility in water (<1 mg / mL).

[0021] 5″-Methoxyhexahydrocurcumin is a reduced derivative of curcumin, consisting of two substituted benzene rings linked by a hexanedione bridge, exhibiting an overall non-conjugated hexahydrodiaryl ketone structure. The key differences between 5″-MHHC and its parent compound, curcumin, are: its β-diketone bridge is completely hydrogenated to a saturated alkyl chain; the 5″-methoxy (-OCH3) substitution enhances hydrophobicity and stability; and the intramolecular hydroxyl and methoxy groups can form a hydrogen bond network, contributing to maintaining steric stability. This structural modification significantly improves the compound's chemical stability, bioavailability, and binding affinity to protein targets.

[0022] The functional effects of the application described in this invention are as follows: When HK2 is absent or the HK2-RUVBL2 binding is disrupted, PIKK expression decreases significantly, γ-H2AX signaling decreases, DNA double-strand break repair efficiency decreases significantly, and cell radiosensitivity increases. Based on this, the small molecule compound 5″-methoxyhexahydrocurcumin, which was screened, can specifically disrupt the HK2-RUVBL2 binding and enhance the ubiquitination-proteasome degradation of RUVBL2. 5″-methoxyhexahydrocurcumin weakens the interaction between RUVBL2 and HK2, thereby inhibiting the function of RUVBL2 as a transcriptional coactivator of the RNA Pol II complex, thus reducing the transcriptional level of PIKK genes (ATM, ATR, DNA-PKcs, etc.). 5″-methoxyhexahydrocurcumin inhibits DNA damage repair (HR and NHEJ), and the increase in γ-H2AX foci leads to inhibition of the DDR pathway and a significant increase in the radiosensitivity of tumor cells. Animal experiments show that the combined use of 5″-MHHC and radiotherapy can significantly inhibit tumor growth and exhibit excellent radiosensitization effects.

[0023] The 5″-MHHC described in this invention is a natural product derivative DDR inhibitor. It has better chemical stability and biocompatibility than traditional PARP inhibitors, and has low toxicity, high efficiency, and strong specificity in radiosensitization, showing significant potential for clinical translation.

[0024] This invention discovers a novel regulatory mechanism for DNA damage repair: the metabolic enzyme HK2 localizes in the cell nucleus and mediates radiotherapy resistance through protein-protein interactions. Inhibition of the HK2-RUVBL2 interaction enables precise and low-toxicity radiosensitization. This mechanism represents the first systematic elucidation of non-classical HK2 nuclear function, expanding the biological role of metabolic enzymes in DDR (radiotherapy resistance). This invention provides a theoretical basis for the development of low-toxicity adjuvant drugs for radiotherapy and novel DDR target inhibitors.

[0025] This invention also provides an antitumor drug, the active ingredient of which includes a small molecule that interferes with the HK2-RUVBL2 protein interaction. As one embodiment, the small molecule interfering with the HK2-RUVBL2 protein interaction of this invention includes 5″-methoxyhexahydrocurcumin. As one embodiment, the effective concentration of the 5″-methoxyhexahydrocurcumin is 5-30 μM, more preferably 15 μM. As one embodiment, the drug of this invention further includes pharmaceutically acceptable excipients or carriers. As one embodiment, the dosage form of the drug of this invention may include, but is not limited to, solutions, injections, lipid nanoparticle formulations (LNPs), or polymer micelle formulations.

[0026] To further illustrate the present invention, the technical effects provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] In the following embodiments, the accession numbers of the genes used are as follows: HK2: Gene ID 3099, RUVBL2: Gene ID 10856, TRIP12: Gene ID 9320.

[0028] Example 1 To verify the potential role of HK2 in maintaining the stability of tumor cell genomes, this embodiment constructed stable HK2 knockdown cell models in human colorectal adenocarcinoma cell line HCT116, cervical cancer HeLa cell line, and non-small cell lung cancer A549 cell line. Specifically, control short hairpin RNA (shCtrl) or HK2-interfering short hairpin RNA (shHK2) was stably transfected into the above three cell lines, respectively. Whole cell lysates were collected, and the expression levels of DNA damage marker γ-H2AX and HK2 protein were detected by Western blotting. The specific steps are as follows: shCtrl: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 1); HK2 shRNA #1: 5'-CCAAAGACATCTCAGACATTG-3' (SEQ ID NO: 2); HK2 shRNA #2: 5'-CCAGAAGACATTAGAGCATCT-3' (SEQ ID NO: 3); Cell construction method: HEK293T cells were cultured in 10 cm culture dishes. When the cells reached 70% confluence, they were transfected with a total plasmid ratio of 10 μg target plasmid, 7.5 μg psPAX2 packaging plasmid, and 2.5 μg pMD2.G envelope plasmid. Forty-eight hours after transfection, the cell culture supernatant containing the packaged lentivirus was collected. The collected viral supernatant was filtered through a 0.45 μm filter to remove cell debris. The filtered viral supernatant was mixed with 6× viral concentrate and incubated at 4°C in the dark for at least 24 hours. After incubation, the cells were centrifuged at 3600 rpm for 1 hour at 4°C. The supernatant was discarded, and the viral pellet at the bottom was retained. The viral pellet was resuspended in 1 mL PBS, aliquoted, and stored at -80°C for later use.

[0029] When the target cells in the six-well plate reached approximately 70% confluence, the medium was replaced with fresh complete medium containing 20% ​​FBS and 1:1000 Polybrene. After infecting with virus solution for 48 h, the cells were passaged into 10 cm culture dishes, and stable expression cell lines were obtained by selection with puromycin.

[0030] Using a limiting dilution method, selected cells were seeded into 96-well plates at a density of approximately one cell per 100 μL of culture medium. Cells were cultured with puromycin for two weeks to select surviving monoclonal cells. These surviving monoclonal cells were then passaged sequentially into 24-well plates and 6 cm culture dishes for further amplification. Western blot analysis was used to detect the expression levels of the target protein within the cells, and stable monoclonal cell lines meeting experimental requirements were selected and retained.

[0031] The detection results of HCT116, HeLa and A549 cell lines are as follows: Figure 1 As shown in Figures A through C, the grown monoclonal cell populations were analyzed. Specifically, HCT116 cells showed shHK2#1 and #2 as monoclonal cells transfected with HK2 shRNA#1 and HK2 shRNA#2, respectively; HeLa cells showed shHK2#1 and #2 as monoclonal cells transfected with HK2 shRNA#1 and #2, respectively; A549 cells showed shHK2#1 and #2 as monoclonal cells transfected with HK2 shRNA#1, respectively; and shHK2#3 and #4 as monoclonal cells transfected with HK2 shRNA#2.

[0032] As can be seen, compared with wild-type cells, γ-H2AX signaling was significantly enhanced in HK2-deficient cells. Furthermore, in this embodiment, γ-H2AX immunofluorescence staining was performed on HCT116 cells stably expressing shCtrl or shHK2, and the formation of γ-H2AX in the cell nucleus was observed using high-resolution confocal microscopy (scale bar 10 μm). It was found that the γ-H2AX signal intensity was significantly increased in HK2-deficient HCT116 cells. Figure 1 (D). It can be seen that γ-H2AX is an early marker of cellular response to DSB, and its persistent presence suggests an increased level of endogenous DNA damage and a decreased repair efficiency.

[0033] The continuous accumulation of γ-H2AX observed without external damaging stress suggests that HK2 deficiency may impair the genomic stability of tumor cells. To further verify this hypothesis, this embodiment used a comet assay to detect DNA fragmentation levels in HCT116 cells with shCtrl and shHK2, and performed quantitative analysis of tail moments (scale bar: 100 μm). The results showed... Figure 1 As shown in Figure E, shHK2 represents a single clone of HCT116 cells transfected with shHK2#1. The same applies to the following figures, so further explanation is unnecessary. It is evident that compared to wild-type cells, the degree of DNA fragmentation is significantly increased in HCT116 cells lacking HK2. Micronucleus formation in HCT116 cells infused with shCtrl and shHK2 was observed using fluorescence microscopy, and the frequency of micronucleus occurrence was quantitatively analyzed (scale bar: 5 μm). The results are as follows... Figure 1 As shown in Figure F, the proportion of micronucleus-positive cells is also significantly increased. Micronucleus formation is a key indicator of chromosome breakage or segregation errors, and its presence suggests impaired genome stability during cell replication or division.

[0034] These results indicate that HK2 deficiency leads to the accumulation of endogenous DNA damage and a significant increase in genomic instability, suggesting that HK2 may play a key role in maintaining the genomic integrity of tumor cells.

[0035] Example 2 To further explore the molecular mechanisms by which HK2 promotes DDR in tumor cells and maintains genomic stability, this embodiment performed immunoprecipitation combined with mass spectrometry analysis on HK2 in the cell nucleus to identify the HK2 nuclear interacting proteome.

[0036] In this experiment, Flag-HK2 was overexpressed using the 3×FLAG CMV10 vector (with the HK2 protein-coding sequence inserted), and HA-RUVBL2 was overexpressed using pcDNA3.1 (with the RUVBL2 protein-coding sequence inserted). The overexpression plasmid construction steps are as follows: ① Double enzyme digestion of the vector plasmid: Take 2 μg of the vector plasmid, add 1 μL of endonuclease 1 (ECOR I), 1 μL of endonuclease 2 (BAMH I), and 5 μL of 10×rCutSmart buffer, and make up to 50 μL with ultrapure water. Digest at 37 ℃ for 15 min. Prepare an agarose gel containing Goldview nucleic acid dye, use 1×TAE as the electrophoresis buffer, load the digested products with the DNA marker for electrophoresis, and after 20 min of electrophoresis, cut the target vector band under UV light. ② Enzyme digestion product gel purification: Following the procedure of the agarose DNA recovery kit, add 2 volumes of Buffer GDP to the gel block and incubate at 55°C for 15 min; pass the solution through a column to adsorb DNA, wash sequentially with Buffer GDP and Buffer DW2, centrifuge at 15000 rpm to remove residual washings, and evaporate ethanol at room temperature; add 40 μL of preheated ultrapure water to the purification column filter membrane, let stand, centrifuge to elute DNA, and detect plasmid concentration using Nanodrop. ③ Primer design and synthesis: Amplification primers were designed based on the NCBI target gene sequence and the sticky ends of the vector enzyme digestion, and synthesized by Qingke Biotechnology Co., Ltd. ④ High-fidelity PCR amplification of the target fragment: Amplification was performed using FastPfu high-fidelity DNA polymerase. The 50 μL reaction system contained template DNA, upstream and downstream primers, 5×FastPfu Buffer, dNTPs, and FastPfu enzyme. The PCR program was set to 95℃ pre-denaturation for 2 min, 30 cycles (95℃ denaturation for 20 s, (Tm-5)℃ annealing for 20 s, 72℃ extension), and a final extension at 72℃ for 5 min. After electrophoresis of the PCR product, the target band was excised and purified according to the gel extraction method described above. ⑤ Recombinant plasmid transformation: 5 μL of homologous recombination product was added to Tsurbo chemicompetent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, and then incubated on ice for 5 min. Antibiotic-free LB medium was added, and the cells were thawed at 37℃ and 200 rpm for 1 h. After centrifugation and resuspending, the cells were plated on ampicillin-resistant LB agar plates and incubated overnight at 37℃. ⑥ Single-clone screening and identification: Select 4-6 single-clone colonies and inoculate them in ampicillin-resistant LB liquid medium with shaking culture for 8 h; freeze part of the bacterial culture with glycerol, extract plasmids from the remaining bacterial culture, and use strains with correct sequences and normal protein expression that are verified by sequencing for subsequent experiments.

[0037] Cell transfection procedure: Transfection was performed when cells reached 70% confluence. Under aseptic conditions, 500 μL, 200 μL, and 100 μL of Opti-MEM were used for single-well transfection in 10 cm and 6 cm culture dishes and six-well plates, respectively. The transfection mixture was prepared at a plasmid (μg):PEI (μL) ratio of 1:4. After gentle mixing, the mixture was briefly centrifuged and allowed to stand at room temperature for 20 min. Then, it was added dropwise to the cell culture medium. Fresh complete culture medium was replaced 6 h after transfection.

[0038] After overexpressing Flag-HK2 in HEK293T cells, nuclear-cytoplasmic separation experiments were performed, followed by immunoprecipitation using M2 gel beads. Silver staining was then used to visualize proteins binding to nuclear HK2. The results are as follows: Figure 2 As shown in Figure A; Western blotting analysis was performed on the separated cytoplasmic and nuclear components to verify the nuclear-cytoplasmic separation effect. α-Tubulin and histone H3 were used as separation controls. The results are shown in Figure A. Figure 2 As shown in B.

[0039] Flag-HK2 and HA-RUVBL2 were overexpressed in HEK293T cells, and an immunoprecipitation experiment was performed to verify the interaction between HK2 and RUVBL2. The results are as follows: Figure 2 As shown in C.

[0040] Example 3 To explore whether intervening in the protein-protein interaction between HK2 and RUVBL2 can inhibit tumor cell DNA damage repair and improve radiosensitivity, this embodiment performed molecular docking and virtual screening based on the HK2-RUVBL2 interaction interface to find small molecule compounds that may bind to this interaction region. Inhibitors binding to the HK2-RUVBL2 interaction interface were screened using molecular docking and virtual screening techniques. Information on the top candidate compounds is shown. The top three candidate inhibitors (BML-264, 5"-MHHC, and J34 HCl) are marked in red, and their three-dimensional structural models binding to HK2 are displayed. The models show the potential binding sites and interaction mechanisms of the three inhibitors with HK2. The results are as follows: Figure 3 As shown in A and B.

[0041] Three candidate molecules, ranking high in the screening results, were selected and validated in colorectal cancer cells: HEK293T cells were co-transfected with Flag-HK2 and Strep-RUVBL2-HA. After treatment with the first three candidate inhibitors for 12 hours, a Strep-RUVBL2 immunoprecipitation experiment was performed. Western blotting was used to detect the ubiquitination level of RUVBL2 and its binding to HK2, to verify the effect of the inhibitors on the binding of HK2 to RUVBL2 and the stability of RUVBL2. The results are as follows: Figure 3As shown in Figure C, only 5″-methoxyhexahydrocurcumin (5″-MHHC) can weaken the protein interaction between HK2 and RUVBL2 and significantly enhance the ubiquitination level of RUVBL2.

[0042] After treating HCT116 cells with the first three candidate inhibitors selected during screening for 12 hours, whole-cell lysates were extracted for Western blotting experiments to detect the expression level of RUVBL2. The results are as follows: Figure 3 As shown in Figure D, 5″-MHHC is the only small molecule compound that can effectively reduce the level of RUVBL2 protein in HCT116 cells.

[0043] Example 4 To further verify whether the HK2-RUVBL2 interaction affects the survival ability of tumor cells after radiotherapy, this embodiment uses a clonogenic assay system to evaluate the sensitivity of HCT116 cells to IR under different genetic backgrounds.

[0044] In HCT116 cells stably expressing shCtrl, shHK2, and RUVBL2 supplemented in a shHK2 background, colony formation experiments were performed after treatment with different doses of ionizing radiation. Representative images, quantitative analysis of colony formation numbers, and protein expression are presented. Results are as follows: Figure 4 As shown in Figures A and B, it can be seen that after RUVBL2 was replenished in this context, the radiotherapy survival rate of HK2-deficient cells was significantly improved, suggesting that HK2-mediated radiotherapy resistance depends on its regulation of RUVBL2 protein stability.

[0045] Colony formation experiments were performed on HCT116 cells stably expressing wild-type HK2-WT or mutant HK2-QA cells lacking RUVBL2 binding ability (HK2 QA mutants are four-site mutations of HK2 M283A, K315A, E316A, and E317A). Colony formation experiments were conducted on HCT116 cells stably expressing shHK2 and those supplemented with HK2-WT or HK2-QA in a shHK2 background after treatment with different doses of ionizing radiation. Representative images and quantitative analysis of colony formation numbers are presented. Results show... Figure 4 As shown in C~D, it can be seen that only HK2-WT can significantly restore the survival ability of HK2-deficient cells after IR, while HK2-QA mutants cannot play a similar role and show the same radiosensitivity as HK2 knockdown cells.

[0046] The above results indicate that the interaction between HK2 and RUVBL2 is crucial for maintaining the radiotherapy resistance of tumor cells.

[0047] Example 5 This embodiment collected colorectal cancer tumor samples after radiotherapy and performed NCCN Tumor Regression Grade (TRG) assessments based on postoperative pathological evaluation results. Patients were divided into a radiosensitive group (TRG 0 / 1) and a radioresistant group (TRG 2 / 3) according to the NCCN TRG criteria. Subsequently, immunohistochemical staining was performed on the tumor tissues to detect the expression levels of HK2 and RUVBL2 in different group samples.

[0048] Based on the tumor pathology of colorectal cancer patients after radiotherapy, and according to the NCCN TRG grading system, patients were classified into a radiosensitive group (NCCN TRG 0 / 1) and a radiotolerant group (NCCN TRG 2 / 3). Representative immunohistochemical staining images of HK2 and RUVBL2 in tumor tissues from both groups are presented. Results are as follows: Figure 5 As shown, the staining intensity and proportion of positive HK2 and RUVBL2 cells were significantly higher in the radiotherapy-resistant tumor samples than in the radiotherapy-sensitive group. This result indicates that high expression of HK2 and RUVBL2 is closely related to tumor insensitivity to radiotherapy.

[0049] Example 6 First, this embodiment uses Western blotting to detect changes in DNA damage response signals in HCT116 cells after IR treatment with 15 μM 5″-MHHC: After treating HCT116 cells with 5″-MHHC (15 μM) for 12 hours, samples were collected at different time points after exposure to ionizing radiation (10 Gy). The dynamic changes in DNA damage response signals were detected by Western blotting, and the results are as follows: Figure 6 As shown in Figure A, 5″-MHHC significantly inhibited the formation of γ-H2AX signaling in tumor cells after IR, blocking the early response process following DNA double-strand breaks. Further, after treating HCT116 cells with 5″-MHHC (15 μM) for 12 hours, samples were collected at different time points after ionizing radiation (10 Gy) for immunofluorescence staining to observe the interaction between γ-H2AX and BRCA1 (…). Figure 6 (B) or γ-H2AX with 53BP1 ( Figure 6 Colocalization of BRCA1 with γ-H2AX and 53BP1 with γ-H2AX (scale bar 5 μm). Immunofluorescence results showed that in cells treated with 5″-MHHC, the colocalization of BRCA1 with γ-H2AX and 53BP1 with γ-H2AX was significantly reduced after IR treatment, indicating that 5″-MHHC can inhibit the process of cellular DNA damage repair.

[0050] Subsequently, this embodiment used the HR and NHEJ reporter system to detect the effect of 5″-MHHC treatment on DNA double-strand break repair efficiency: after 12 hours of treatment with 5″-MHHC (15 μM), DR-GFP ( Figure 6 (middle D) and EJ5-GFP ( Figure 6 In HCT116 cells of the chemoradiotransferase (CRT) reporter system, after infection with I-SceI retrovirus, GFP expression was detected by real-time quantitative PCR to assess the efficiency of homologous recombination repair and non-homologous end joining repair. The results showed that 5″-MHHC could significantly reduce the activity of these two repair pathways in HCT116 cells, further confirming that the compound can inhibit the DNA damage repair function of tumor cells.

[0051] To assess its impact on the radiosensitivity of tumor cells, the survival of HCT116 cells after radiotherapy (IR) was examined using a colony formation assay. HCT116 cells were treated with 5″-MHHC (15 μM) for 12 hours, followed by colony formation assays after different doses of ionizing radiation. Representative images and statistical analysis of colony formation are presented. The results showed that 5″-MHHC treatment significantly reduced cell viability after IR. Figure 6 The presence of F and G in the compound indicates that it can enhance the sensitivity of tumor cells to radiotherapy.

[0052] To verify whether the effect of 5″-MHHC depends on the interaction between HK2 and RUVBL2, experiments were conducted using 5″-MHHC treatment in HCT116 cells with stably knocked-down HK2 and those with HK2-QA mutants added under HK2 knockdown backgrounds. In HCT116 cells stably expressing shCtrl, shHK2, or with HK2-WT or HK2-QA mutants added under shHK2 backgrounds, whole-cell lysates were treated with 5″-MHHC (15 μM) for 12 hours and then analyzed by Western blotting to detect RUVBL2 protein levels. The results are as follows: Figure 6 As shown in Figure H, it can be seen that reintroducing wild-type HK2 in a HK2 knockdown background can restore the protein expression level of RUVBL2, but this restoration effect is significantly inhibited by 5″-MHHC; while in cells with HK2 deficiency or reintroduced HK2-QA mutant, the protein level of RUVBL2 is not sensitive to 5″-MHHC treatment.

[0053] In summary, 5″-MHHC increases the sensitivity of tumor cells to radiotherapy by specifically blocking the protein interaction between HK2 and RUVBL2, thereby inhibiting RUVBL2-mediated DNA damage repair. This effect depends on the binding interface between HK2 and RUVBL2, further confirming that 5″-MHHC is a targeted inhibitor of the HK2-RUVBL2 interaction and a radiosensitizer.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of small molecules that interfere with HK2-RUVBL2 protein interaction in the preparation of antitumor drugs.

2. Use according to claim 1, characterized in that, The anti-tumor treatment includes anti-tumor therapy via radiation.

3. Use according to claim 2, characterized in that, The applications include the use of small molecules that interfere with the HK2-RUVBL2 protein interaction as radiosensitizers.

4. The application according to claim 2, characterized in that, The applications include the use of small molecules that interfere with the HK2-RUVBL2 protein interaction as inhibitors of DNA damage response.

5. The use according to claim 1, characterized in that, The tumors include solid tumors.

6. Use according to claim 5, characterized in that, The solid tumors include colorectal cancer.

7. The use according to any one of claims 1 to 6, characterized in that, The interference with the HK2-RUVBL2 protein interaction includes 5″-methoxyhexahydrocurcumin.

8. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The active ingredients include small molecules that interfere with the HK2-RUVBL2 protein interaction.

9. The antitumor drug according to claim 8, wherein The small molecules that interfere with the HK2-RUVBL2 protein interaction include 5″-methoxyhexahydrocurcumin.

10. The antitumor drug according to claim 8, wherein The concentration of the 5″-methoxyhexahydrocurcumin is 5~30 μM.

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