Application of CX6258 in preparation of medicine for treating tumors

Abstract

The invention relates to application of CX6258 in preparation of medicines for treating tumors, and belongs to the technical field of medicines. The research finds that CX6258 can inhibit the proliferation activity of tumors, acts on NIMA-related kinase 8 in a targeting manner to activate the immunoreaction induced by tumor radiotherapy, and enhances the therapeutic effect of radiotherapy on tumors such as colorectal cancer and the remote isolation effect induced by radiotherapy; meanwhile, the CX6258 can enhance the treatment effect of immunotherapy (anti-PD1) on tumors such as colorectal cancer, and the CX6258 can be used as a medicine for treating tumors, an NEK8 inhibitor, a tumor radiotherapy synergist, a tumor immunotherapy synergist and the like. The invention provides a new drug choice for treatment and radiotherapy of colorectal cancer and other tumors, and enriches the application range of CX6258.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to the application of CX6258 in the preparation of drugs for treating tumors. Background Art

[0002] Colorectal cancer (CRC), as a highly malignant tumor type, ranks among the top in global cancer incidence and mortality statistics. Approximately 22% of colorectal cancer patients are found to have advanced distant metastases at the initial diagnosis. The 5-year survival rate of patients with advanced metastatic colorectal cancer (mCRC) is only 10%, and the median overall survival is only about 2 years. The clinical treatment of metastatic colorectal cancer mainly relies on radiotherapy and chemotherapy. Research has shown that radiotherapy can not only effectively control the locally irradiated tumor, but also effectively control the tumors that are not irradiated distally, that is, the abscopal effect induced by radiotherapy. According to the 2022 NCCN guidelines for colorectal cancer, radiotherapy should be given to patients with mid- to late-stage rectal cancer, which can effectively reduce the local recurrence rate and improve the prognosis. It is reported that complete remission, major remission, and minor remission each account for 1 / 3 after radiotherapy, but about 30% of patients show radiotherapy resistance, and the incidence of the abscopal effect is also extremely low in clinical practice. Therefore, studying the key targets and potential mechanisms affecting radiotherapy resistance and the abscopal effect in colorectal cancer and finding effective intervention measures are the key points and difficulties in current colorectal cancer research.

[0003] During radiotherapy, cancer cells often inevitably evolve cell populations that are resistant to radiation exposure, and this tolerance evolution leads to a decline in the anti-tumor effect, which is one of the obstacles to tumor treatment. Tumor radiotherapy resistance can come from within tumor cells, such as changes in the genes or phenotypes of tumor cells themselves in response to radiation exposure; it can also come from the protection of tumor cells by the tumor microenvironment, that is, external resistance. A variety of factors inside and outside tumor cells are closely related to tumor cell radiotherapy resistance, mainly including: (1) Abnormal activation of DNA damage repair pathways in tumor cells, such as homologous recombination and non-homologous end joining repair, which can quickly repair DNA damage caused by radiotherapy; (2) Abnormal activation of signal pathways that promote tumor proliferation in tumor cells (such as PI3K / AKT, MAPK); (3) Abnormal activation of stress signals (such as autophagy) induced by radiotherapy in tumor cells, causing adaptive changes in tumor cells and promoting tumor cell survival; (4) Cancer Stem Cells (CSCs), as an important component of tumors, have stronger DNA damage repair and immune response evasion abilities; (5) Epigenetic factors (such as DNA methylation, histone modification, and chromatin structure) can regulate the expression of genes related to DNA damage repair and cell survival; (6) Tumor heterogeneity leads to different responses to radiation in different regions within the same tumor; (7) Downregulation of tumor cell surface antigens, activation of immune checkpoints (such as the expression of Programmed cell death ligand 1 (PD-L1)), and recruitment of immunosuppressive cells promote tumor immune escape; (8) The hypoxic environment leads to a decrease in the generation of reactive oxygen species (ROS) and changes in the tumor microenvironment, reducing the damage to tumor cells. Therefore, exploring the intrinsic molecular mechanisms of colorectal cancer radiotherapy resistance and clarifying the pathways by which tumor cells inhibit anti-tumor immunity triggered by radiotherapy are the premise and basis for establishing effective treatment strategies, eliminating tumor treatment resistance, and improving tumor treatment sensitivity, and have important clinical significance.

[0004] The abscopal effect (AE) refers to the phenomenon that during tumor radiotherapy, in addition to the significant inhibition of the irradiated target tumor, distant tumors that have not received radiotherapy also show shrinkage or disappearance. Currently, the abscopal effect caused by radiotherapy alone is very rare, and the abscopal effect is of great significance for the treatment of advanced metastatic tumors. Clinical studies have shown that the key factors for enhancing the abscopal effect of radiotherapy include irradiation mode, dose selection, treatment timing, and the synergistic effect of immunotherapy. Based on the mechanism of the abscopal effect, the factors enhancing the abscopal effect mainly include the following aspects: (1) Enhancing the tumor antigen immunogenicity of the local irradiated site: Damage-associated molecular patterns (DAMPs), including double-stranded DNA (dsDNA) fragments, high-mobility group protein B1 (HMGB1), calreticulin (CRT), ATP, and heat shock proteins, interact with cells and immune factors in the tumor microenvironment to activate cytotoxic T lymphocytes (CTLs) and promote the release of inflammatory cytokines such as IFN-γ. Radiotherapy can induce the cytoplasmic accumulation of dsDNA to activate the cGAS-STING pathway to induce the production of IFN-β, thereby activating CD8 + T cells to transform into CTLs to kill tumors. Recent studies have found that nanoscale coordination polymers can play an anti-tumor role by amplifying radiotherapy-mediated oxidative stress-induced immunogenic cell death. Exploring the intrinsic mechanism of inhibiting radiotherapy-induced immunogenic death will be an effective strategy for enhancing the abscopal effect. (2) Combined treatment with immunotherapy: Immunotherapy can enhance the antigen presentation ability of DC cells and the radiosensitivity of tumors by inhibiting immunosuppressive factors such as TGF-β; in addition, immunotherapy can relieve immune checkpoint inhibition of tumors on the non-radiotherapy side distally, activate CD8 + T cells, thereby enhancing the killing effect on distant tumors. Even with the combined radiotherapy and immunotherapy, the incidence of the abscopal effect is still low, and multiple factors affect its predictability, such as treatment regimens, radiotherapy doses, fractionation patterns, and tumor types, and immune tolerance in the tumor area still generally restricts the abscopal effect. Currently, the types of drugs available for inducing the abscopal effect of radiotherapy are relatively limited and may cause immune-related adverse reactions. Therefore, finding important targets targeting the immune microenvironment and promoting the abscopal effect of radiotherapy through the combined use of targeted therapy and immunotherapy has become the best strategy. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of CX6258 in the preparation of drugs for treating tumors.

[0006] The compound (E)-5-chloro-3-((5-(3-(4-methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)indolin-2-one, namely CX6258, has the chemical formula C 26 H 24 ClN3O3, with the CAS number 1202916-90-2, and its structural formula is shown in Formula (I):

[0007]

[0008] Previous studies reported that CX6258 is a potent, kinase-selective pan-Pim kinase inhibitor and also an inhibitor of haploid germ cell-specific nuclear protein kinase (Haspin). The present invention discovers that CX6258 can target Never-in-Mitosis A-related kinase 8 (NEK8), activate the radiotherapy-induced CD8 + T cell anti-tumor immune response, thereby sensitizing the efficacy of colorectal cancer radiotherapy and enhancing the occurrence of abscopal effect. None of these effects of CX6258 have been reported in previous studies.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect, the present invention provides the use of CX6258 in the preparation of a drug for treating tumors.

[0011] The present invention discovers that CX6258 exhibits good inhibitory proliferation activity in colorectal cancer cells. This indicates that CX6258 can be used as a potential anti-tumor small molecule drug for the treatment of colorectal cancer. Compared with existing treatment methods, CX6258 may have a more significant anti-tumor effect and can more effectively inhibit the growth and spread of colorectal cancer cells.

[0012] As a preferred embodiment of the use described in the present invention, the tumor is colorectal cancer.

[0013] In the second aspect, the present invention provides the use of CX6258 as an inhibitor of Never-in-Mitosis A-related kinase 8 (NEK8).

[0014] The present invention discovers that CX6258 can inhibit the kinase activity of NEK8 and can be used as an inhibitor of NEK8.

[0015] In the third aspect, the present invention provides the use of CX6258 in the preparation of a tumor radiotherapy sensitizer.

[0016] The present invention has found through research that CX6258 can promote the immune response induced by tumor radiotherapy, enhance the efficacy of radiotherapy for tumors such as colorectal cancer, and can be used as a radiosensitizer for tumor radiotherapy.

[0017] In a fourth aspect, the present invention provides the use of CX6258 in the preparation of a synergistic agent for tumor immunotherapy.

[0018] CX6258 can enhance the efficacy of immunotherapy (anti-PD1) for tumors such as colorectal cancer and can be used as a synergistic agent for tumor immunotherapy.

[0019] In a fifth aspect, the present invention provides the use of CX6258 in the preparation of a synergistic agent for combined tumor radiotherapy and immunotherapy.

[0020] As a preferred embodiment of the application described in the present invention, the antibody used in the immunotherapy is anti-PD1.

[0021] In a sixth aspect, the present invention provides the use of CX6258 in the preparation of an inducer for enhancing the abscopal effect of tumors.

[0022] The present invention has found through research that CX6258 can enhance the abscopal effect induced by radiotherapy and can be used as an inducer for enhancing the abscopal effect of tumors.

[0023] As a preferred embodiment of the application described in the present invention, the tumor is colorectal cancer.

[0024] CX6258 can significantly enhance the effect of tumor radiotherapy. Compared with current tumor radiotherapy methods, this drug is expected to show better therapeutic effects, can enhance the abscopal effect induced by radiotherapy, and thus improve the ability of the immune system to attack tumors.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The present invention has found through research that CX6258 can enhance the effect of tumor radiotherapy, induce the abscopal effect, and inhibit the proliferation of tumor cells. CX6258 is more safe and reliable. Within a certain dose range, this drug has low toxicity and good tolerance, which helps to reduce side effects and risks during the treatment process. The present invention provides a new drug option for the treatment of tumors such as colorectal cancer and radiotherapy, and enriches the application scope of CX6258. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the virtual screening workflow based on molecular docking in Example 1 of the present invention;

[0028] Figure 2 is the information of the top five compounds among the final small molecule compounds in Example 1 of the present invention;

[0029] Figure 3 This is the 2D and 3D structural model of the binding of CX6258 and NEK8 in Example 1 of the present invention;

[0030] Figure 4 This is the inhibitory effect of different concentrations of HSD1590 and CX6258 on colorectal cancer cells in Example 2 of the present invention;

[0031] Figure 5 This is the test result of the interaction between NEK8 protein and CX6258 in Example 3 of the present invention;

[0032] Figure 6 This is the inhibitory effect of different concentrations of CX6258 on NEK8 protein in Example 3 of the present invention;

[0033] Figure 7 This is the result of the effect of CX6258 combined with radiotherapy on tumor size in Example 4 of the present invention, where A represents the MC38 subcutaneous tumor model, B represents the CT26 subcutaneous tumor model, and C represents the MC38-shNEK8 subcutaneous tumor model;

[0034] Figure 8 This is the result of the effect of CX6258 combined with radiotherapy or anti-PD1 on tumor size in Example 4 of the present invention;

[0035] Figure 9 This is the schematic diagram of the treatment process of CX6258 combined with radiotherapy for multiple tumors in Example 4 of the present invention;

[0036] Figure 10 This is the result of the effect of CX6258 combined with radiotherapy on the size of subcutaneous tumors in multiple tumors in Example 4 of the present invention;

[0037] Figure 11 This is the result of the effect of CX6258 combined with radiotherapy on orthotopic transplanted tumors in multiple tumors in Example 4 of the present invention, where A represents the results of in vivo imaging monitoring and B represents the tumor appearance and staining results. Detailed implementation manners

[0038] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0039] Other materials, reagents, etc. used in the examples can be obtained from commercial channels without special instructions.

[0040] Example 1 Computer virtual screening of small molecule drugs for NEK8 protein

[0041] Currently, the experimentally resolved three-dimensional structure of human NEK8 protein has not been obtained, but its AlphaFold prediction model has relatively high structural confidence and reliability. Based on this, the present invention targets the ATP binding site of Human NEK8 protein (the key residues are VAL10, VAL18, and LYS33) reported in the UniProt database to conduct computer-aided virtual screening, aiming to discover small molecule compounds with high affinity for the target protein.

[0042] The virtual screening workflow based on molecular docking is as Figure 1 shown. The specific steps of the virtual screening are as follows:

[0043] 1. Protein structure preprocessing

[0044] Obtain the predicted three-dimensional structure of Human NEK8 (AlphaFold ID: AF-Q86SG6-F1) from the AlphaFold database. Use the Protein Preparation Wizard module to perform hydrogenation and energy optimization on the protein (using the OPLS2005 force field, with an RMSD threshold of ). Centering on the VAL10, VAL18, and LYS33 residues, use the ReceptorGrid Generation module to generate a grid file, and set the grid box size to

[0045] 2. Compound library preprocessing

[0046] Perform hydrogenation and energy optimization on the two-dimensional structures of the MCE 50K diversity compound library (containing 50,000 compounds) through the LigPrep module of the software to generate three-dimensional structures for subsequent virtual screening.

[0047] 3. Molecular docking screening

[0048] Use the Virtual Screening Workflow module for virtual screening. First, use the high-throughput screening (HTVS) mode of the Glide module to preliminarily screen the 50,000 preprocessed small molecule compounds, and retain the compounds ranked in the top 10%; then use the standard precision (SP) mode for the second round of screening, and retain the top 10% of the compounds again; finally, use the high-precision (XP) mode for the third round of screening to obtain the final ranking of small molecule compounds.

[0049] The information of the top five compounds among the final small molecule compounds is as Figure 2 shown. The results show that the first-ranked one is HSD1590, and its chemical formula is C 20 H18 BN3O3, with a CAS number of 2379279-96-4; the second-ranked CX6258, with a chemical formula of C 26 H 24 ClN3O3, with a CAS number of 1202916-90-2.

[0050] CX6258 can form two hydrogen bond interactions with the NEK8 protein: the carbonyl group on the indolinone acts as a hydrogen bond acceptor to form a hydrogen bond interaction with ALA83, with a distance of NH acts as a hydrogen bond donor to form a hydrogen bond interaction with ALA83, with a distance of The 2D and 3D structural models of the binding of CX6258 to NEK8 are as Figure 3 shown.

[0051] Example 2 Antitumor Activity Detection

[0052] Using HSD1590 (concentration gradients: 0, 1, 2, 5, 10, 20, 40, 80, 160, and 200 uM), CX6258 (concentration gradients: 0, 1, 10, 50, 100, 200, 500, and 1000 nM) as drugs, and human colorectal adenocarcinoma epithelial cells (DLD1), constructed radioresistant SW480 cells (IRSW480), human colorectal adenocarcinoma cells (HCT15), human colon cancer cells (LOVO), human colon cancer cells (SW480) as tumor cells, an antitumor activity experiment was carried out. The construction method of IRSW480 refers to the reference DOI: 10.1186 / s12916-024-03375-2.

[0053] The CCK8 experiment was used to detect the antitumor activity of the drugs. The specific operations are as follows:

[0054] Using a 96-well plate for plating, the cell density was controlled at 1000 cells / well, and 200 μL of culture medium was added to each well. After mixing, it was placed in a 5% CO2, 37 °C cell culture incubator for culture. After the cells adhered, drugs with a certain concentration gradient were added, and after continuing to culture for 22 h, CCK8 solution was added. 10 μl of CCK8 solution and 90 μl of DMEM culture medium (a total of 100 μl) were added to each well, and the culture was continued for 2 h. The absorbance value (OD value) of each well was measured using an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm, and the IC 50 value of the drug killing the tumor was counted.

[0055] The inhibitory effects of different concentrations of HSD1590 and CX6258 on colorectal cancer cells are as Figure 4As shown, the results show that both HSD1590 and CX6258 have inhibitory effects on colorectal cancer cells, and the killing of colorectal cancer cell lines is dose-dependent. Among them, the IC 50 values of HSD1590 against five colorectal cancer cell lines, namely DLD1, IRSW480, HCT15, LOVO, and SW480, are between 12.71 and 44.65 μM; the IC 50 values of CX6258 against the five colorectal cancer cell lines DLD1, IRSW480, HCT15, LOVO, and SW480 are between 293.3 and 487.2 nM (0.29 - 0.48 μM); the IC 50 of CX6258 is significantly lower than that of HSD1590, indicating that CX6258 has a better inhibitory effect on colorectal cancer cells and higher safety.

[0056] The inventors also conducted anti-tumor activity experiments on the compounds ranked third, fourth, and fifth in Figure 2 , and the results showed that their inhibitory effects on colorectal cancer cells were significantly lower than those of HSD1590 and even more significantly lower than those of CX6258.

[0057] Example 3 Binding Test of CX6258 to NEK8

[0058] 1. Preparation of Human NEK8 Recombinant Protein

[0059] (1) Transformation of Competent Cells and Screening of Positive Clones

[0060] Take Rosetta competent cells out of the -80°C refrigerator and place them on ice to thaw slowly. Add the pET32a-NEK8(human)-6×His plasmid (Shandong Weizhen Biotechnology Co., Ltd.) to the thawed competent cells, gently mix, and incubate on ice for 30 minutes. Then place the mixture in a 42°C water bath for heat shock treatment for 90 seconds, and immediately transfer it to ice and let it stand for 1 minute. Then add 900 μL of pre-warmed LB liquid medium at 37°C, culture it in a 37°C shaker at 158 rpm for 1 hour, centrifuge at 6000 rpm for 4 minutes, remove 900 μL of the supernatant, and evenly spread the remaining bacterial liquid on the LB solid medium containing ampicillin. Invert the plate and place it in a 37°C constant temperature incubator for 16 hours to observe the colony growth. Select positive (containing recombinant plasmid) single colonies and inoculate them into 3 mL of LB liquid medium containing ampicillin, culture them overnight at 37°C, and store them at -20°C.

[0061] (2) Small-Scale Expression and Identification of Recombinant Protein

[0062] Select positive single colonies and inoculate them into 3 mL of LB liquid medium containing ampicillin, and culture them with shaking at 37°C until the OD 600The value reached 0.6. Take a part of the culture as the uninduced control group, and add IPTG inducer with a final concentration of 1 mM to the remaining bacterial solution (induced group), and culture it with shaking at 37 °C for 3 hours. Take 150 μL of the bacterial solution from the induced group and the control group respectively, centrifuge at 12,000 g for 2 minutes, collect the bacterial cell precipitate and resuspend it with 40 μL of 1× loading buffer, and perform SDS-PAGE analysis to identify the expressed recombinant protein.

[0063] (3) Large-scale expression and lysis detection of recombinant protein

[0064] Inoculate 100 μL of the bacterial strain stored at -20 °C into 100 mL of LB liquid medium containing ampicillin, and culture it with shaking at 37 °C overnight. Inoculate 100 mL of the overnight culture into 2 L of LB liquid medium, and expand the culture at 37 °C until the OD 600 value is about 0.6, and then reduce the culture temperature to 30 °C. Add IPTG inducer with a final concentration of 0.5 mM, continue to culture it with shaking at 30 °C for 3 hours, centrifuge at 8,000 rpm for 3 minutes to collect the bacterial cells, resuspend them with 50 mL of pre-cooled NTA-0 buffer, and incubate on ice for 30 minutes. Then use an ultrasonic disruptor to lyse the bacterial cells, set the parameters as a power of 200 W, work for 3 seconds, and pause for 4 seconds, for a total of 99 cycles. Then centrifuge at 16,000 rpm at 4 °C for 50 minutes, and collect the supernatant and precipitate respectively. Take a small amount of the supernatant and precipitate for SDS-PAGE detection to identify the expressed recombinant protein, and store the remaining samples at 4 °C for later use.

[0065] (4) Purification process of recombinant protein

[0066] ① Filter the reserved supernatant through a 0.22 μm filter membrane for later use;

[0067] ② Pretreatment of Ni-NTA column: Load an appropriate amount of Ni-NTA packing material, and wash it successively with 3 column volumes of deionized water, 0.1 M EDTA solution, deionized water, and NTA-0 buffer (pH 8.0), then bind it with 5 column volumes of 0.1 M NiSO4 solution, and finally wash it with equilibration buffer (pH 4.0) and NTA-0 buffer (pH 8.0) until the pH value of the effluent is 8.0.

[0068] ③ Load the filtered supernatant at a flow rate of 1 mL / min.

[0069] ④ Wash the column with NTA-0 buffer (pH 8.0) until the Bradford detection solution does not change color.

[0070] ⑤ Perform gradient elution with elution buffers containing 20 mM, 60 mM, 200 mM, and 500 mM imidazole respectively, and collect the eluate in fractions.

[0071] ⑥Wash the column material with three column volumes of deionized water, and finally store the column material with 20% ethanol.

[0072] ⑦Perform SDS-PAGE analysis on each eluted fraction collected, and concentrate, wash, and dry the eluate containing the recombinant protein to obtain purified NEK8 recombinant protein.

[0073] 2. Surface Plasmon Resonance (SPR) experiment to determine the binding of CX6258 to NEK8 protein

[0074] (1) Protein coupling

[0075] ① Place 200 mL of 1×PBS running buffer, a distilled water bottle, and a waste liquid bottle in the left and right trays of the instrument respectively, and correctly connect the corresponding liquid inlet pipelines.

[0076] ② Hold the CM5 chip with the side printed with the label facing up. Gently push it into the instrument card slot along the direction indicated by the arrow on the chip, and then close the chip chamber door.

[0077] ③ Inject the EDC and NHS mixed solution into channel 2 of the chip at a flow rate of 10 μL / min for 420 seconds to activate the chip surface.

[0078] ④ Dilute the ligand protein (the above purified NEK8 protein) to 50 μg / mL with pH 4.0 sodium acetate buffer, and inject it into channel 2 at a flow rate of 10 μL / min. The immobilization time is 420 seconds, and record the coupling curve in real time.

[0079] ⑤ Inject the ethanolamine solution at a flow rate of 10 μL / min to block channel 2 for 420 seconds.

[0080] ⑥ As a control, repeat steps (3)-(5) for channel 1, with the difference that in step (4), use sodium acetate buffer without protein to replace the protein solution.

[0081] (2) Interaction test between NEK8 protein and CX6258

[0082] ① Solvent normalization: The experiment uses 1×PBS-P+ containing 5% DMSO as the running buffer. Replace the original buffer in the left tray of the instrument with 1×PBS-P+ containing 5% DMSO, and connect the corresponding liquid inlet pipelines. Prepare a 5% DMSO concentration calibration curve by mixing 4.5% and 5.8% DMSO stock solutions.

[0083] ② Sample analysis: The compound to be tested, CX6258, was diluted into multiple concentration gradients in a 96-well plate using DMSO and sequentially bound to the target protein (NEK8 protein) from low to high concentrations through the chip. The flow rate was set at 30 μL / min, and the action time for each concentration point was 150 seconds. After each binding, the chip was regenerated with 10 mM glycine-hydrochloric acid buffer (pH 2.0) for 5 minutes, and this process was repeated until the detection of all concentration points was completed.

[0084] The experimental data were collected by BIAcore T200 Control software (v.2.0, GE Healthcare), and the background value of the reference channel was deducted. The data were fitted to a 1:1 Langmuir binding model using BIAcore T200 Evaluation software (v.2.0, GE Healthcare) to calculate the binding constant and dissociation constant.

[0085] The test results of the interaction between NEK8 protein and CX6258 are as Figure 5 shown. The test results show that the dissociation constant KD (μM) of CX6258 and NEK8 protein is 3.38, the association rate constant ka (1 / Ms) is 4443.454, and the dissociation rate constant kd (1 / s) is 0.015018875.

[0086] 3. In vitro kinase inhibition experiment

[0087] After the in vitro purification of human NEK8 protein, the in vitro kinase activity was detected, and it was found that NEK8 could consume ATP for kinase reaction at 200 nM.

[0088] The purified NEK8 protein obtained from the above experiment was added with CX6258 drug at a certain concentration gradient for phosphorylation reaction. The concentration gradient of CX6258 was: 0, 1, 2, 5, 10, and 20 μM. The phosphorylation reaction system was: 100 μM ATP, 5 μg NEK8 protein, CX6258 (added at different concentration gradients respectively), 1× kinase buffer, made up to 50 μL with ddH2O, and reacted at 37 °C for 30 min. After the reaction, the kinase activity was detected using the Kinase-Lumi TM Ultra-strong chemiluminescence kinase activity detection kit.

[0089] The inhibitory effects of CX6258 at different concentrations on NEK8 protein are as Figure 6 shown. The results show that the IC 50 of CX6258 on the kinase activity inhibition of NEK8 protein is 3.011 μM.

[0090] The above results indicate that CX6258 is a small molecule drug for NEK8 protein.

[0091] Example 4 In Vivo Experiment on the Effect of CX6258 on the Efficacy of Radiotherapy and Abscopal Effect

[0092] Unilateral subcutaneous tumor models were constructed using mouse colon cancer cells MC38, CT26, and MC38 with NEK8 knocked out (MC38-shNEK8). The MC38-shNEK8 subcutaneous tumor model was constructed to clarify whether the radiosensitizing effect of CX6258 on colorectal cancer functions through the NEK8 target.

[0093] On day 0, MC38, MC38-shNEK8, or CT26 was injected (5×10 5 cells / mouse) subcutaneously into the back of mice to construct unilateral subcutaneous tumor models (MC38 subcutaneous tumor model, MC38-shNEK8 subcutaneous tumor model, CT26 subcutaneous tumor model).

[0094] 1. CX6258 Combined with Radiotherapy for Unilateral Subcutaneous Tumor Model

[0095] Model mice in the control group were not treated with anything.

[0096] When the tumor size of model mice in the experimental group exceeded 100mm 3 they were treated.

[0097] Mice were given CX6258 by gavage (25 mg / kg, once a day), as the inhibitor group (CX6258);

[0098] Received 15 Gy radiotherapy once, as the radiotherapy group (IR);

[0099] Received 15 Gy radiotherapy once and were given CX6258 by gavage (25 mg / kg) every day starting from the day of radiotherapy, as the inhibitor + radiotherapy group (CX6258 + IR).

[0100] The tumor size was measured every two days. When the experimental endpoint was reached, when the mice were near death or the treatment cycle ended, the tumors were dissected, weighed, and photographed.

[0101] The results of the effect of CX6258 combined with radiotherapy on tumor size are as Figure 7As shown, where A represents the MC38 subcutaneous tumor model, B represents the CT26 subcutaneous tumor model, and C represents the MC38-shNEK8 subcutaneous tumor model. The results showed that CX6258 alone could inhibit tumor growth to a certain extent, and the inhibitory effect of CX6258 combined with radiotherapy on tumor growth was significantly enhanced, indicating that the combination of CX6258 and radiotherapy could significantly improve the efficacy of radiotherapy. In the absence of NEK8, CX6258 had basically no inhibitory effect on tumor growth and did not show a synergistic effect when combined with radiotherapy, that is, the effect of CX6258 in inhibiting the growth of colorectal cancer tumors and sensitizing the efficacy of colorectal cancer radiotherapy disappeared, indicating that the effect of CX6258 in sensitizing the efficacy of colorectal cancer radiotherapy was mainly produced by targeting NEK8.

[0102] 2. CX6258 combined with radiotherapy or anti-PD1 treatment of the MC38 subcutaneous tumor model

[0103] The control group of model mice was not treated with anything.

[0104] When the tumor size of the experimental group of model mice exceeded 100 mm 3 treatment was carried out.

[0105] The mice were given CX6258 by gavage (25 mg / kg, once a day) as the chemotherapy group (CX6258).

[0106] Intraperitoneal treatment with anti-PD1 (200 mg / kg, twice a week) as the immune group (anti-PD1).

[0107] The mice were given CX6258 by gavage (25 mg / kg, once a day) + intraperitoneal treatment with anti-PD1 (200 mg / kg, twice a week) as the chemotherapy + immune group (CX6258 + anti-PD1).

[0108] Intraperitoneal treatment with anti-PD1 (200 mg / kg, twice a week) + 15 Gy radiotherapy (once) as the immune + radiotherapy group (anti-PD1 + IR).

[0109] The mice were given CX6258 by gavage (25 mg / kg, once a day) + intraperitoneal treatment with anti-PD1 (200 mg / kg, twice a week) + 15 Gy radiotherapy (once) as the chemotherapy + immune + radiotherapy group (CX6258 + anti-PD1 + IR).

[0110] The tumor size was measured every two days. When the experimental endpoint was reached, the mice were sacrificed, the tumors were dissected and separated, and weighed and photographed.

[0111] The results of the effect of CX6258 combined with radiotherapy or anti-PD1 on tumor size are asFigure 8 As shown, the results show that using CX6258 alone can inhibit tumor growth to a certain extent; the combined use of CX6258 and anti-PD1 enhances the inhibitory effect on tumor growth to a certain degree, indicating that CX6258 can also promote the immune efficacy of anti-PD1 to a certain extent; the combined use of CX6258, radiotherapy, and anti-PD1 significantly enhances the inhibitory effect on tumor growth, indicating that the triple therapy of CX6258, radiotherapy, and anti-PD1 can greatly inhibit tumor growth, suggesting that CX6258 can significantly improve the efficacy of combined radiotherapy and immunotherapy, providing a new treatment strategy for colorectal cancer treatment.

[0112] 3. CX6258 combined with radiotherapy for the treatment of a multiple tumor model of orthotopic and subcutaneous tumors of colorectal cancer

[0113] On day 0, C57BL / 6 mice were anesthetized and laparotomized. The cecum was pulled out, and 100 μL of MC38-luc cells (1×10 6 cells / mouse) were injected behind the cecal serosa, and then the abdomen was closed. After the operation, 200 μL of double antibiotics were given to prevent infection, and the status of the mice was observed daily. On day 14, MC38 cells (5×10 5 cells / mouse) were injected subcutaneously into the right back of C57BL / 6 mice. On day 24, the mice were anesthetized with pentobarbital sodium, and the subcutaneous tumor on the right side of the mice was irradiated with 15 Gy, and CX6258 (25 mg / kg) was given by gavage to the mice every day starting from the day of radiotherapy. In vivo imaging of mice was used to monitor the growth of orthotopic tumors in the cecum, and the size of subcutaneous tumors was measured every two days. When the experimental endpoint was reached, the mice were sacrificed. The tumors were dissected, weighed, and photographed. The tumor tissues of orthotopic tumors were fixed with formaldehyde fixative and then subjected to H&E staining (HE) experiments.

[0114] The schematic diagram of the treatment process of CX6258 combined with radiotherapy for multiple tumors is as Figure 9 shown.

[0115] The results of the effect of CX6258 combined with radiotherapy on the size of subcutaneous tumors in multiple tumors are as Figure 10 shown.

[0116] The results of the effect of CX6258 combined with radiotherapy on orthotopic tumors in multiple tumors are as Figure 11 shown, where A represents the results of in vivo imaging monitoring, and B represents the tumor appearance and staining results.

[0117] Figure 10 and Figure 11The results showed that, compared with the control group, CX6258 combined with radiotherapy not only significantly reduced the size of the subcutaneous tumors treated with radiotherapy, but also significantly reduced the size of the in-situ tumors on the non-irradiated side distally. This indicates that CX6258 can induce and enhance the abscopal effect of radiotherapy.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of CX6258 in the preparation of drugs for treating tumors.

2. Use of CX6258 as an inhibitor of NIMA-related kinase 8.

3. Application of CX6258 in the preparation of tumor radiotherapy enhancers.

4. Application of CX6258 in the preparation of tumor immunotherapy enhancers.

5. Application of CX6258 in the preparation of enhancers for tumor radiotherapy combined with immunotherapy.

6. Application of CX6258 in the preparation of inducers for enhancing tumor abscopal effects.

7. The use according to any one of claims 1 or 3 to 6, characterized in that: The tumor is colorectal cancer.

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