A novel quinoline-based dkc1 inhibitor and uses thereof

Abstract

The application relates to the technical field of medicines, in particular to a novel quinoline DKC1 inhibitor and application thereof. 23 H 26 ClN3O, a structure formula is shown as formula I. The compound can be directly and efficiently combined with an ASP125 site of DKC1 protein which is an important component of a telomerase, and effectively inhibit the pseudouridine synthase activity. Functional experiments show that the compound can significantly inhibit the proliferation ability of various DKC1 high-expression tumor cells by targeting inhibition of DKC1, and can produce better anti-tumor ability in combination with an ATR inhibitor, and exhibits potential value as a novel targeted therapeutic drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a novel quinoline DKC1 inhibitor and its application. Background Technology

[0002] The Dyskerin pseudouridine synthase 1 (DKC1) gene encodes Dyskerin, which possesses pseudouridine synthase function. It binds to and catalyzes the isomerization of uridine (U) from ribosomal RNA to pseudouridine (Ψ), and is also an important component of the telomerase complex. Pyrazofurin (PF), a pyrimidine nucleoside analog, has been shown to inhibit DKC1 pseudouridine synthase activity. However, despite clinical trials in various tumors, including colorectal cancer, its high toxicity and poor clinical efficacy prevent it from being considered an effective drug. Therefore, there is currently a lack of effective and clinically applicable DKC1 inhibitors.

[0003] Quinoline, with the molecular formula C9H7N, also known as azinaphthalene or benzopyridine, is a colorless liquid or flaky solid with a strong, pungent odor. It is hygroscopic and gradually turns brown in sunlight. Quinoline is considered one of the representative aromatic heterocyclic compounds. Quinoline and its derivatives possess various biological activities and play an important role in the development of antitumor drugs. They exhibit good antitumor effects through different mechanisms of action, such as cell cycle arrest, inhibition of angiogenesis, and disruption of cell migration.

[0004] The ASP125 residue of the DKC1 protein is the core active site for its pseudouridine monophosphate synthase catalysis, and this site is crucial for maintaining the biological activity of DKC1. Studies have shown that when telomeres are severely shortened or dysfunctional, cells recognize them as double-strand DNA breaks, triggering the DNA damage response (DDR) mechanism. In this mechanism, the ataxiatelangiectasia and Rad3-related kinase (ATR), a mutant gene involved in ataxilatelanosis, acts as a core kinase in sensing replication stress and single-strand DNA damage, playing a leading role in cell cycle checkpoint activation and maintaining genome stability. DKC1 participates in this regulatory system through its active site ASP125, influencing ATR pathway signaling and thus becoming a key node connecting telomere / ribosome function and DDR. Summary of the Invention

[0005] To address the above problems, this invention provides a novel quinoline DKC1 inhibitor and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a quinoline compound, the chemical formula of which is C. 23 H 26 ClN3O, the structural formula is shown in Formula I: Formula I.

[0007] This invention also provides the application of the quinoline compounds described in the above technical solution in the preparation of DKC1 inhibitors.

[0008] The present invention also provides the application of the quinoline compounds described in the above technical solutions in the preparation of reagents or drugs that inhibit the activity of DKC1 pseudouridine synthase.

[0009] This invention also provides the application of the quinoline compounds described above in the preparation of reagents or drugs that bind to DKC1 protein.

[0010] The present invention also provides the application of the quinoline compounds described in the above technical solution in the preparation of drugs for treating tumors with high DKC1 expression.

[0011] Preferably, the tumors associated with high DKC1 expression include one or more of liver cancer, gastric adenocarcinoma, lung adenocarcinoma, colon cancer, and breast cancer.

[0012] The present invention also provides a combination drug composition for treating tumors associated with high DKC1 expression, comprising the quinoline compounds and ATR inhibitors described in the above technical solutions.

[0013] Preferably, the molar ratio of the quinoline compound to the ATR inhibitor is 1~20:1~2.

[0014] Preferably, the ATR inhibitor includes VE-822.

[0015] Preferably, the tumors associated with high DKC1 expression include one or more of liver cancer, gastric adenocarcinoma, lung adenocarcinoma, colon cancer, and breast cancer.

[0016] The beneficial effects of this invention are: This invention utilizes a self-designed and established highly efficient screening system to successfully screen and synthesize a quinoline compound with a novel structural backbone. This structure is not reported among known DKC1-targeting molecules, representing a novel structural entity. This compound can directly and efficiently bind to the ASP125 site of the DKC1 protein, a key component of telomerase, and effectively inhibit its pseudouridine synthase activity. Functional experiments show that this compound can significantly inhibit the proliferation of various DKC1-overexpressing tumor cells by targeting and inhibiting DKC1, and can produce better anti-tumor activity when used in combination with ATR inhibitors, demonstrating its potential value as a novel targeted therapeutic drug. 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 introduced below.

[0018] Figure 1 Information on four small molecule compounds that inhibit the activity of DKC1 protease (A), structural diagram of DL51 (B), hydrogen bonds formed between DL51 and Gly223 and ASP125 residues of DKC1 protein (C, D), and flowchart of virtual screening (E). Figure 2 The results show the effects of DL51 on the proliferation of HepG2 cells (A), HCCLM9 cells (B), SGC-7901 cells (C), A549 cells (D), H1299 cells (E), HCT116 cells (F), MCF-7 cells (G), and GES-1 cells (H), as well as the effects of PF on the proliferation of A549 cells (I) and HCT116 cells (J). Figure 3 The results of RNA Dot Blot experiments and quantitative analysis using ImageJ software were used to obtain concentration trend graphs and IC50 values ​​of enzyme activity. Among them, the results of the detection of the effect of DL51 on DKC1 pseudouridine synthase activity in HepG2 cells (A), SGC-7901 cells (B), A549 cells (C), HCT116 cells (D) and H1299 cells (E), and the results of the detection of the effect of PF on DKC1 pseudouridine synthase activity in HCT116 cells (F) were presented. Figure 4 The results of in vitro interaction experiments between DL51 and DKC1 are shown; where A is the Western Blot diagram of DKC1-MBP protein and MBP protein, B is the ITC titration curve of the interaction between DL51 and DKC1, and C is the ITC titration curve of the interaction between DL51 and MBP. Figure 5The graph shows the analysis of polyribosomes in HepG2 cells after the addition of 2 μM DL51. The black curve represents the Control group, and the red curve represents the 2 μM DL51 group. Figure 6 The following data were analyzed using Graphpad Prism software: (A) Effect of DL51 and VE-822 combined on H1299 cell proliferation; (B) Sensitization effect of VE-822 on DL51; (C) Sensitization effect of DL51 and VE-822 combined on SGC-7901 cell proliferation; (D) Sensitization effect of VE-822 on DL51; (E) Sensitization effect of DL51 on VE-822; (F) Sensitization effect of DL51 and VE-822 combined on HCT116 cell proliferation; (H) Sensitization effect of VE-822 on DL51; (I) Sensitization effect of DL51 on VE-822 combined on H1299 cell apoptosis; (J) Sensitization effect of 2 μM DL51 and 0.5 μM VE-822 combined on H1299 cell apoptosis. Detailed Implementation

[0019] This invention provides a quinoline compound, the chemical formula of which is C. 23 H 26 ClN3O, the structural formula is shown in Formula I: Formula I.

[0020] This invention also provides the application of the quinoline compounds described in the above technical solution in the preparation of DKC1 inhibitors.

[0021] This invention also provides the application of the quinoline compounds described in the above-mentioned technical solutions in the preparation of reagents or drugs that inhibit DKC1 pseudouridine synthase activity. This invention does not specifically limit the types of reagents or drugs; those skilled in the art can use conventional methods.

[0022] This invention also provides the application of the quinoline compounds described in the above-described technical solutions in the preparation of reagents or drugs that bind to DKC1 protein. This invention does not specifically limit the types of reagents or drugs; those skilled in the art can use conventional methods.

[0023] This invention also provides the application of the quinoline compounds described in the above-mentioned technical solutions in the preparation of drugs for treating DKC1-overexpressing tumors. This invention does not specifically limit the type of drug; those skilled in the art can use conventional methods. In this invention, the DKC1-overexpressing tumors preferably include one or more of liver cancer, gastric adenocarcinoma, lung adenocarcinoma, colon cancer, and breast cancer.

[0024] This invention also provides a combination drug composition for treating DKC1-overexpression-related tumors, comprising the quinoline compound and an ATR inhibitor as described above. In this invention, the molar ratio of the quinoline compound to the ATR inhibitor ranges from 1 to 20:1 to 2. In this invention, the ATR inhibitor preferably includes VE-822. This invention does not specifically limit the source of VE-822; those skilled in the art can use conventional sources, such as Selleck's Berzosertib (VE-822) (CAS: 1232416-25-9). In this invention, the DKC1-overexpression-related tumors preferably include one or more of liver cancer, gastric adenocarcinoma, lung adenocarcinoma, colon cancer, and breast cancer.

[0025] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 Preparation of DL51 1. Add 2 mmol of 4-(7-chloro-4-quinolinyl)-2-methanol-phenol and 2 mmol of 1-methyl-2-(2-aminoethyl)pyrrolidine to a 250 mL single-necked flask, add 10 mL of MeOH and stir for 2 hours. After confirming the product is correct by TLC, add 5 mL of water to the single-necked flask and stir for 1 hour.

[0027] 2. After the reaction was complete, the solvent was removed by rotary evaporation. Impurities were first eluted with hydrochloric acid in a PE solution, then with a PE / DCM mixed solution (1:1 ratio), followed by elution with DCM solution. This process was repeated multiple times with a DCM / MeOH mixed solution (ratios of 80:1, 50:1, 20:1, and 10:1, respectively). The product was dried over anhydrous sodium sulfate and then rotary evaporated. The product was then dried in a vacuum oven. Yield: 40%, yellow solid.

[0028] Example 2 Virtual screening targeting the ASP125 site of the DKC1 protein This invention uses the Targetmol (L1010) library as the compound library for virtual screening. Before virtual screening, all compounds underwent energy minimization and deionization pretreatment. The docking site was set to the ASP125 residue of the DKC1 protein. Two docking conformations were selected for semi-flexible docking of each small molecule compound. Based on the docking score, the top 35 small molecule compounds in the library were selected for subsequent experimental verification. RNA dot blot experiments were used to further detect the inhibitory effect of these 35 small molecule compounds on DKC1 enzyme activity, and four small molecule compounds with strong inhibitory effects on DKC1 protein were initially screened. Figure 1 (A). Further investigation was conducted using cell proliferation and toxicity assays to assess the biological effects of these four small molecule compounds. Ultimately, the quinoline compound DL51 was selected for further research. Figure 1 (B). Molecular docking models show that DL51 forms hydrogen bonds with Gly223 and ASP125 residues of the DKC1 protein. Figure 1 (C) The overall screening process is as follows: Figure 1 As shown in D.

[0029] Example 3 Detection of the effect of DL51 on tumor cell proliferation To investigate the effect of DL51 on tumor cell proliferation, this embodiment treated different tumor cells with gradient concentrations of the drug and detected the cell proliferation level of the cells treated with the gradient concentrations, analyzing the effect of concentration gradient treatment on cell proliferation.

[0030] 1. Experimental methods, results, and analysis of the effect of DL51 treatment on cell proliferation levels. 1.1 Experimental Grouping Normal HepG2, HCCLM9, SGC-7901, A549, H1299, HCT116, MCF-7, and GES-1 cells were collected and preserved in our laboratory. HepG2 and HCCLM9 are hepatocellular carcinoma cell lines, SGC-7901 is a gastric adenocarcinoma cell line, A549 and H1299 are lung cancer cell lines, HCT116 is a colon cancer cell line, MCF-7 is a breast cancer cell line, and GES-1 is a human gastric mucosal epithelial cell line. Cells were randomly assigned in equal amounts to 6 groups: one control group and five experimental groups. Each sample was tested in triplicate.

[0031] 1.2 Experimental Methods Day 1: (1) Digest, centrifuge, resuspend and count the cells cultured one day in advance.

[0032] (2) Seed 100 μL of 1 k-3 k / well cells into a 96-well plate and culture for 24 hours at 37°C, 5% CO2 and 90% humidity.

[0033] the next day: (1) Observe the cell growth status and density under a microscope to ensure that the cells are in good growth status and that the distribution and density are uniform before conducting the experiment.

[0034] (2) Prepare drug sample solutions with different concentration gradients. Set up three replicates for each concentration, add the drug solution to the 96-well plate, and incubate at 37°C, 5% CO2, and 90% humidity for an appropriate time, such as 24, 48, or 72 hours.

[0035] The drug concentrations were as follows: DL51: HepG2 cells were treated with concentrations of 0 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. HCCLM9 cells were treated with concentrations of 0 μM, 1 μM, 2 μM, 4 μM, and 8 μM. SGC-7901 cells were treated with concentrations of 0 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. A549 cells were treated with concentrations of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM. H1299 cells were treated with concentrations of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM. The drug concentrations added to HCT116 cells were 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. The drug concentrations added to MCF-7 cells were 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. The drug concentrations added to GES-1 cells were 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. PF: The drug concentrations added to A549 cells were 0 μM, 0.5 μM, 1 μM, and 2 μM. The drug concentrations added to HCT116 cells were 0 μM, 0.25 μM, 0.5 μM, and 1 μM.

[0036] Day 3 / Day 4: (1) Thaw the CCK8 solution at room temperature and centrifuge before use.

[0037] (2) Use an inverted microscope to detect the cell growth status, and take pictures of wells with good growth status and uniform cell distribution and density.

[0038] (3) Change the medium in each well with a mixture of 10 μL of CCK-8 solution and 90 μL of complete culture medium.

[0039] (4) Incubate at 37℃, 5% CO2 and 90% humidity for 0.5-4 hours.

[0040] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well in a 96-well plate at 450 nm.

[0041] (6) Use Excel and Graphpad Prism to process the data and analyze the results.

[0042] 1.3 Experimental Results and Analysis like Figure 2 As shown in the CCK-8 assay, DL51 inhibited the proliferation of all five different types of tumor cells tested, and this effect was concentration-dependent. Notably, DL51 showed a relatively weak inhibitory effect on the growth of normal gastric epithelial cells GES-1.

[0043] The experimental data were processed and analyzed using Graphpad Prism9 to obtain concentration trend graphs and proliferation IC50 values. The results showed that the IC50 of DL51 on HepG2 cell proliferation was 1.563 μM (A), on HCCLM9 cell proliferation was 1.256 μM (B), on SGC7901 cell proliferation was 2.804 μM (C), on A549 cell proliferation was 3.5 μM (D), on H1299 cell proliferation was 3.732 μM (E), on HCT116 cell proliferation was 0.5102 μM (F), on MCF-7 cell proliferation was 4.029 μM (F), and on GES-1 cell proliferation was 3.796 μM (H). These results further indicate that DL51 can selectively inhibit tumor cell proliferation while having relatively little effect on normal cells. The known DKC1 inhibitor PF has an IC50 of 6.766 μM (I) against the proliferation of A549 cells and 0.5221 μM (J) against the proliferation of HCT116 cells. Compared with the existing DKC1 inhibitor PF, DL51 shows a similar or even lower IC50 value against tumor cells, demonstrating its potential to inhibit tumor cell proliferation.

[0044] Example 4 DL51 inhibits the enzyme activity of DKC1 in tumor cells. To detect changes in DKC1 enzyme activity levels after DL51 treatment, this embodiment measures the pseudouridine modification level in cells treated with DL51 at different concentration gradients, analyzes the effects of DL51 concentration gradient treatment on DKC1 enzyme activity levels in cells, and compares the effects of DL51 with existing DKC1 enzyme activity inhibitors by treating one type of tumor cell with PF.

[0045] The drug concentrations were as follows: DL51: HepG2 cells were treated with 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM at drug concentrations of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM at drug concentrations of 0 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM at drug concentrations of 0 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM at drug concentrations of 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM at drug concentrations of 0 μM, 0.625 μM, 1.25 μM, and 2. ... PF: The drug concentrations added to HCT116 cells were 0 μM, 1 μM, 2 μM, and 5 μM.

[0046] 1. Experimental methods, results, and analysis of the detection of intracellular pseudouridine modification levels after DL51 and PF treatment ( Figure 3 ) 1.1 Experimental Grouping Normal HepG2, SGC-7901, A549, HCT116, and H1299 cells (preserved in our laboratory) were collected. HepG2 is a hepatocellular carcinoma cell line, SGC-7901 is a gastric adenocarcinoma cell line, A549 and H1299 are lung adenocarcinoma cell lines, and HCT116 is a colon cancer cell line. Cells were randomly assigned in equal amounts to 6 groups: one control group and five experimental groups. Each sample was tested in triplicate.

[0047] 1.2 Sample Collection and Processing Cells were centrifuged at 1000 rpm for 5 minutes at room temperature (15-25 °C), then the supernatant was discarded, and the cells were washed twice with pre-chilled PBS. Each 1×102 7Add 1 mL of TRIzol reagent to the cells. Pre-chill the centrifuge to 4°C and transfer the cell lysis buffer to 1.5 mL RNase-free EP tubes. Place the EP tubes on ice and incubate for 5 minutes. Then add 200 μL of chloroform to each EP tube, mix thoroughly, and incubate on ice for 10 minutes to allow complete dissociation of the nucleoprotein complex. Centrifuge the EP tubes at 13000 rpm for 15 minutes at 4°C. During this time, take a new EP tube, add 500 μL of isopropanol, and pre-chill on ice. After centrifugation, transfer the upper aqueous phase (approximately 400 μL) to a new EP tube. Mix thoroughly and incubate on ice for 10 minutes. Centrifuge the EP tube at 13000 rpm for 10 minutes and remove the supernatant. Wash the RNA precipitate once with 1 mL of 75% ethanol and centrifuge at 12000 rpm for 5 minutes. Remove the supernatant and air-dry or vacuum-dry the RNA precipitate for 10–30 minutes. Dissolve the RNA in 30-50 μL of DEPC-treated deionized water (the amount of water added depends on the amount of RNA precipitate). Perform spectrophotometric analysis to determine the sample concentration and purity.

[0048] 1.3 Experimental Methods RNA Dot Blot Experiment: Remove the RNA sample to be tested, thaw it, and measure the RNA concentration. Perform the entire process on ice. Standardize the sample concentration and serially dilute the sample in new enzyme-free tubes, mixing thoroughly to a final concentration of 500 ng / μL. After centrifugation, heat the RNA sample at 95°C for 3 minutes to disrupt secondary structures. Immediately cool on ice afterward to prevent the reformation of secondary structures. Use the thick end of a 1 mL pipette tip to leave a circular imprint on the NC membrane to guide sample addition. Then, cut the NC membrane to the appropriate size and transfer it to a clean plastic container. Add 2 μL of RNA sample sequentially to the NC membrane, ensuring the pipette tip does not touch the membrane; the sample should diffuse naturally on the membrane. Replace the pipette tip after each addition. After the NC membrane has slightly dried at room temperature, RNA cross-linking with the membrane can proceed. Method 1: 37°C oven for 30 min. Method 2: Irradiate under a 254 nm UV lamp for 30 min–1 h. Wash the membrane with 10 mL TBST for 5 min to remove unbound RNA. Discard TBST, add 10 mL blocking buffer, and incubate at room temperature with shaking for 1 hour. Discard the blocking buffer, add blocking buffer containing primary antibody, and incubate overnight at 4°C. Wash the membrane three times with 10 mL TBST, 10 min each time. Incubate with secondary antibody at room temperature for 1 hour. Wash the membrane three times with 10 mL TBST, 10 min each time. Incubate with ECL substrate for 5 min, take pictures under a developing apparatus, and after taking pictures, place the membrane in 10 mL methylene blue staining buffer and incubate at room temperature with shaking for 30 min. Wash with ddH2O until the background is roughly clean, about 30-60 s. Acquire the methylene blue stained image using white light imaging as a loading control.

[0049] 1.4 Experimental Results and Analysis like Figure 3 As shown, RNA Dot Blot experiments revealed that DL51 treatment inhibited pseudouridine synthase activity in tumor cells. With increasing drug concentration, the pseudouridine synthase activity level in cells gradually decreased. ImageJ software was used to perform quantitative analysis on the blot results, obtaining concentration trend graphs and IC50 values. The results showed that the IC50 value of DL51 for HepG2 cells was 1.28 μM (A), for SGC-7901 cells it was 1.164 μM (B), for A549 cells it was 1.24 μM (C), for HCT116 cells it was 1.779 μM (D), and for H1299 cells it was 2.938 μM (E). In contrast, the IC50 value of PF for HCT116 cells was 0.8566 μM (F). These results indicate that the inhibitory effect of DL51 on DKC1 activity is not significantly inferior to that of the existing DKC1 inhibitor PF.

[0050] Example 5 DL51 binds to DKC1 protein in vitro To detect the in vitro binding of DL51 and DKC1 proteins, prokaryotic protein of DKC1 was extracted, and the in vitro binding of DL51 and DKC1 proteins was analyzed by isothermal titration calorimetry.

[0051] 1. Sample collection and processing 1.1 Molecular Cloning (1) The target gene is cloned into an E. coli expression vector. The choice of vector is based on the protein tag required by the experimental design. (2) After confirming that the recombinant plasmid was successfully sequenced, the plasmid was transformed into Escherichia coli BL21 competent cells for prokaryotic protein expression detection.

[0052] 1.2 Detection of prokaryotic protein expression (1) Transfer an appropriate amount of bacterial culture to 1 mL of resistant liquid LB medium and incubate on a shaker at 37°C; (2) After shaking for 6 h, take 200 μL of bacterial solution and retain it as a sample (without inducing protein sample); (3) Add 1 μL of IPTG to the remaining 800 μL of bacterial culture and place it on a shaker at 37℃ to induce expression; (4) Centrifuge the bacterial culture before and after induction at 4000 rpm for 8 min, retain the bacterial cells and add an appropriate amount of protein loading buffer, denature at 100℃ for 5 min and then perform SDS-PAGE gel electrophoresis. (5) After removing the gel with a peeler, place it in a plastic box, soak the gel in an appropriate amount of staining solution, heat it in a microwave oven to boil, and then stain it on a shaker at room temperature for 30 minutes. (6) Discard the staining solution, add an appropriate amount of decolorizing solution to cover the gel, heat in a microwave oven until boiling, and then decolorize on a flat plate shaker at room temperature for 1 hour; (7) Place the stained gel under a light and observe whether the amount of the target protein increases significantly after induction. If the expression is successfully induced, the subsequent large-scale extraction and purification of prokaryotic proteins can be carried out.

[0053] 1.3 Induced expression of prokaryotic proteins (1) Transfer an appropriate amount of bacterial culture to 20 mL of resistant LB liquid medium and incubate overnight at 37°C on a shaker; (2) Inoculate 10-20 mL of bacterial culture into 1 L of resistant LB liquid medium and culture on a shaker at 37°C and 180 rpm. (3) When the OD value of the bacterial solution reaches about 0.8-1.0, adjust the temperature to 16℃ and reduce the rotation speed to 100 rpm; (4) When the temperature drops to 16℃ and stabilizes, add IPTG (0.2 mM) to induce expression for 15 h.

[0054] 1.4 Extraction of prokaryotic proteins (1) Collect bacterial cells using a floor centrifuge and centrifuge at 5000 rpm for 10 min; (2) Add an appropriate amount of resuspension buffer to the bacterial cells and resuspend the bacterial cells using a shaking resuscitator; (3) The cells were broken up using a low-temperature ultra-high pressure continuous flow cell disruptor. The protein solution was transferred to a low-temperature high-speed centrifuge and centrifuged at 12,000 rpm for 50 min. The protein supernatant was collected, and the total protein (Ce), precipitate and protein supernatant (S) samples were collected.

[0055] 1.5 Purification of prokaryotic proteins (1) Rinse the incubation column in advance with primary water and resuspension buffer; (2) Pour the beads out of the column and incubate them with the protein supernatant at low temperature; (3) Pass the incubated system through a column and collect the flow-through protein solution (FL); (4) Rinse the column with a large amount of resuspension buffer to wash out the extra protein and collect the effluent protein solution (W0). (5) Prepare elution buffer according to the target protein tag, rinse the column multiple times with a small amount of elution buffer until the universal stain does not turn blue, and collect the protein sample; (6) Perform SDS-PAGE gel electrophoresis on each collected protein sample (Ce, precipitate, S, FL, WO, EL, etc.) and destain with Cohen stain, and observe whether the purity and amount of the eluted protein meet the experimental requirements. (7) Dialyze the qualified protein solution to reduce salt content, then centrifuge and concentrate to obtain a high concentration of target protein. Dispense the protein sample and freeze it in liquid nitrogen and store it at -80℃.

[0056] like Figure 4 As shown in Figure A, this embodiment constructs a prokaryotic expression vector for DKC1, and obtains DKC1-MBP protein with high purity through prokaryotic protein extraction and purification.

[0057] 2. Experimental Methods 2.1 ITC Experiment A microcalorimeter contains two cells, one containing water as a reference cell and the other containing the sample. The microcalorimeter must maintain both cells at identical temperatures. A thermistor detects the temperature difference between the two cells when binding occurs and feeds this information back to the heater, which compensates for the temperature difference and restores both cells to the same temperature. Sample cells and a syringe are prepared. Buffer solution is added to the sample cells, and the reference substance and analyte are added to the syringe. The sample cells and syringe are placed in the ITC instrument, and the temperature is maintained at a constant level. The reference substance is injected into the sample cells, and the baseline value of the thermal effect signal is recorded. The analyte is added to the sample cells, and the change in the thermal effect signal is recorded. Thermodynamic parameters, such as the thermodynamic equilibrium constant, enthalpy change, and entropy change, are calculated based on the measurement results.

[0058] 2.2 Experimental Results and Analysis like Figure 4 China B and Figure 4 As shown in Figure C, based on the titration curves of DL51 and DKC1-MBP proteins, it can be found that DL51 and DKC1 proteins can interact, but do not interact with the empty MBP vector.

[0059] Further analysis of the ITC experimental data and curve fitting was performed using MicroCal PEAQ-ITC Analysis software. Through concentration gradient exploration, the final experimental results showed that the optimal binding interaction was achieved when the working concentrations of DL51 and DKC1 proteins were 100 μM and 20 μM respectively, i.e., a concentration ratio of 5:1. At this point, the binding constant KD = 6.19e. -6 ±2.49e - 6 M.

[0060] The results and analysis in this section confirm the specific interaction between DL51 and DKC1 proteins, and the binding affinity was measured to be at the micromolar level. This provides direct experimental evidence for further functional studies and development of DL51 as a DKC1 inhibitor.

[0061] Example 6 Detection of the impact of DL51 on translational regulation To investigate the effect of DL51 treatment on translational regulation in tumor cells, HepG2 cells were lysed, and RNA of different components was isolated. Polyribosome analysis was performed on each component to understand the effect of DL51 on translational regulation.

[0062] 1. Sample collection and processing 1.1 Culture the HepG2 cell line, ensuring a cell count greater than 1 × 10⁻⁶. 7 The number of cells in the control group and the experimental group were the same.

[0063] 1.2 Prepare a 2 μM sample solution and add it to the cell culture dish of the experimental group. Incubate at 37℃, 5% CO2 and 90% humidity for 48 hours.

[0064] 1.3 Before collecting cells, when the cell density reaches about 70%-80%, cycloheximide (final concentration of 100 μg / mL) is added to the cell culture medium to inhibit translation for 15 minutes, thereby inhibiting intracellular protein synthesis and stabilizing the existing mRNA in the cells.

[0065] 1.4 Digest the cells with trypsin containing actinomycete ketone, then wash three times with pre-cooled PBS containing actinomycete ketone, transfer the cells to cryovials, centrifuge, remove the supernatant as thoroughly as possible, freeze in liquid nitrogen, and store at -80°C for later use.

[0066] 2 Experimental Methods 2.1 Polyribosome Analysis Experiment RIPA lysis buffer was added to the collected cell samples to lyse the cells, yielding cytoplasmic lysates. The lysates were then transferred to a gradient sucrose solution, and ultracentrifugation was used to separate RNA into different fractions, including unbound ribosomal RNA (free RNA), bound 40S and 60S ribosomal subunits, monoribosomes (80S), and varying numbers of polyribosomes. Nucleic acid concentrations were detected using a density gradient fractionation system and UV absorbance, and each fraction was collected. The RNA complex was then recovered from the sucrose solution fractions, and a ribosome map was constructed.

[0067] 2.2 Experimental Results and Analysis Results of polyribosome analysis experiments ( Figure 5 In the experimental group, the peak values ​​of the 40S and 60S subunits were higher than those in the control group, while the peak value of polyribosomes was lower than that in the control group. This indicates that ribosome assembly or translation initiation was inhibited in the experimental group, leading to the accumulation of more free subunits and a decrease in actively translating polyribosomes. This suggests that DL51 has an inhibitory effect on the translation regulation of HepG2 cells, further demonstrating that DL51 can inhibit the proliferation of tumor cells.

[0068] Example 7 Detection of the effect of combined DL51 and VE-822 on tumor cell proliferation When the DKC1 inhibitor DL51 causes telomere dysfunction and activates DDR, the ATR inhibitor VE-822 can prevent DDR from causing "synthetic lethality" in tumor cells. Therefore, we attempted to test the inhibitory effect of the combined use of DL51 and VE-822 on the proliferation of tumor cells.

[0069] To investigate the effect of combined DL51 and VE-822 on tumor cell proliferation, this embodiment treated different cell types with gradient concentrations of the drugs and measured the cell proliferation levels of the cells treated with the gradient concentrations of the drugs to analyze the effect of the combined treatment on cell proliferation.

[0070] 1. Experimental methods, results, and analysis of the effects of DL51 and VE-822 treatment on cell proliferation levels. 1.1 Experimental Grouping Normal H1299, SGC-7901, and HCT116 cells were collected and preserved in our laboratory. H1299 is a lung cancer cell line, SGC-7901 is a gastric adenocarcinoma cell line, and HCT116 is a colon cancer cell line. Cells were randomly assigned in equal amounts to 12 groups: one control group and 11 experimental groups. Five parallel experiments were performed for each sample.

[0071] 1.2 Experimental Methods Day 1: (1) Digest, centrifuge, resuspend and count the cells cultured one day in advance.

[0072] (2) Seed 100 μL of 1 k-3 k / well cells into a 96-well plate and culture for 24 hours at 37°C, 5% CO2 and 90% humidity.

[0073] the next day: (1) Observe the cell growth status and density under a microscope to ensure that the cells are in good growth status and that the distribution and density are uniform before conducting the experiment.

[0074] (2) Prepare drug sample solutions with different concentration gradients. Set up three replicates for each concentration, add the drug solution to the 96-well plate, and incubate for an appropriate time, such as 48 or 72 hours, under the conditions of 37°C, 5% CO2 and 90% humidity.

[0075] The drug concentrations were as follows: 1.25 μM DL51, 2.5 μM DL51, 5 μM DL51, 0.25 μM VE-822, 0.5 μM VE-822, 1.25 μM DL51 and 0.25 μM VE-822, 2.5 μM DL51 and 0.25 μM VE-822, 5 μM DL51 and 0.25 μM VE-822, 1.25 μM DL51 and 0.5 μM VE-822, 2.5 μM DL51 and 0.5 μM VE-822, and 5 μM DL51 and 0.5 μM VE-822 were added to H1299 cells. Add 1 μM DL51, 1.5 μM DL51, 2 μM DL51, 0.25 μM VE-822, 0.5 μM VE-822, 1 μM DL51 and 0.25 μM VE-822, 1.5 μM DL51 and 0.25 μM VE-822, 2 μM DL51 and 0.25 μM VE-822, 1 μM DL51 and 0.5 μM VE-822, 1.5 μM DL51 and 0.5 μM VE-822, and 2 μM DL51 and 0.5 μM VE-822 to SGC-7901 cells. Add the following to HCT116 cells: 0.25 μM DL51, 0.5 μM DL51, 1 μM DL51, 0.25 μM VE-822, 0.5 μM VE-822, 0.25 μM DL51 and 0.25 μM VE-822, 0.5 μM DL51 and 0.25 μM VE-822, 1 μM DL51 and 0.25 μM VE-822, 0.25 μM DL51 and 0.5 μM VE-822, 0.5 μM DL51 and 0.5 μM VE-822, and 1 μM DL51 and 0.5 μM VE-822.

[0076] Day 4 / Day 5: (1) Thaw the CCK8 solution at room temperature and centrifuge before use.

[0077] (2) Use an inverted microscope to detect the cell growth status, and take pictures of wells with good growth status and uniform cell distribution and density.

[0078] (3) Change the medium in each well with a mixture of 10 μL of CCK-8 solution and 90 μL of complete culture medium.

[0079] (4) Incubate at 37℃, 5% CO2 and 90% humidity for 0.5-4 hours.

[0080] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well in a 96-well plate at 450 nm.

[0081] (6) Use Excel and Graphpad Prism to process the data and analyze the results.

[0082] 1.3 Experimental Results and Analysis like Figure 6 As shown in Figure A, the CCK-8 assay demonstrated that the combined use of DL51 and VE-822 had a better inhibitory effect on the proliferation of H1299 cells. Figure 6 China B and Figure 6 As shown in Figure C, the experimental data were processed and analyzed using Graphpad Prism9 to obtain a concentration trend graph and the median IC50 (median inhibitory concentration) value. The results showed that 0.5 μM of the ATR inhibitor VE-822 made H1299 cells more sensitive to the DKC1 inhibitor DL51, reducing its IC50 from 3.853 μM to 2.532 μM, a reduction of half. Furthermore, 5 μM of DL51 also made H1299 cells more sensitive to VE-822, reducing its IC50 from 0.6008 μM to 0.3248 μM, also a reduction of half.

[0083] like Figure 6 As shown in Figure D, the CCK-8 assay indicates that the combined use of DL51 and VE-822 has a better inhibitory effect on the proliferation of SGC-7901 cells. Figure 6 China E and Figure 6 As shown in Figure F, the experimental data were processed and analyzed using Graphpad Prism9 to obtain the concentration trend graph and proliferation IC50 value. The results showed that 0.5 μM VE-822 made SGC-7901 cells more sensitive to DL51, reducing its IC50 from 2.057 μM to 1.31 μM, a reduction of half. Moreover, 2 μM DL51 also made SGC-7901 cells more sensitive to VE-822, reducing its IC50 from 0.6914 μM to 0.0053 μM, a reduction of 1 / 130.

[0084] like Figure 6 As shown in Figure G, the CCK-8 assay indicates that the combined use of DL51 and VE-822 has a better inhibitory effect on the proliferation of HCT116 cells. Figure 6 H and Figure 6 As shown in Figure I, the experimental data were processed and analyzed using Graphpad Prism9 to obtain a concentration trend graph and proliferation IC50 values. The results showed that 0.5 μM VE-822 made HCT116 cells more sensitive to DL51, reducing its IC50 from 1.049 μM to 0.6257 μM, a reduction of half. Moreover, 1 μM DL51 also made HCT116 cells more sensitive to VE-822, reducing its IC50 from 0.2086 μM to 0.0766 μM, a reduction of one-third.

[0085] The results further indicate that the combined use of DL51 and VE-822 has a stronger inhibitory effect on tumor cell proliferation.

[0086] 2. Experimental methods, results, and analysis of the effects of DL51 and VE-822 treatment on cell apoptosis. 2.1 Experimental Grouping Normal H1299 cells, preserved in our laboratory, were collected. H1299 is a lung cancer cell line. Cells were randomly divided into 6 groups in equal quantities: 1 control group and 5 experimental groups. Each sample was tested in triplicate.

[0087] 2.2 Experimental Methods Day 1: (1) Digest, centrifuge, resuspend and count the cells cultured one day in advance.

[0088] (2) Seed 1000 μL of 50 kJ-100 kJ / well cells into a 6-well plate and culture for 24 hours at 37°C, 5% CO2 and 90% humidity.

[0089] the next day: (1) Observe the cell growth status and density under a microscope to ensure that the cells are in good growth status and that the distribution and density are uniform before conducting the experiment.

[0090] (2) Prepare drug sample solutions with different concentration gradients. Set up three replicates for each concentration, add the drug solution to the 96-well plate, and incubate for an appropriate time, such as 24 or 48 hours, under the conditions of 37°C, 5% CO2 and 90% humidity.

[0091] Day 3 / Day 4: (1) Digest the cells with trypsin without EDTA, then collect the cells by centrifugation at 300 g and 4°C for 5 min. The digestion time with trypsin should not be too long to avoid false positives.

[0092] (2) Wash the cells twice with PBS pre-cooled to 4°C, each time using 300 g, and centrifuge at 4°C for 5 min.

[0093] (3) Discard the PBS and add 100 μl of 1×Binding Buffer (diluted with double distilled water) to resuspend the cells.

[0094] (4) Add 5 μl Annexin V-FITC and 5 μl PI Staining Solution to the cells and shake gently.

[0095] (5) Incubate the cells at room temperature in the dark for 10-15 minutes.

[0096] (6) Add 400 μl of 1×Binding Buffer (diluted with double-distilled water) to the cells, mix well, and place on ice. Detect cell apoptosis using flow cytometry within 1 hour.

[0097] (7) Use Excel and Graphpad Prism to process the data and analyze the results.

[0098] 2.3 Experimental Results and Analysis like Figure 6 China J and Figure 6 As shown in Figure K, the apoptosis rate of H1299 cells treated with 2 μM DL51 was 1.38%, the apoptosis rate of H1299 cells treated with 0.5 μM VE-822 was 13.49%, and the apoptosis rate of H1299 cells treated with a combination of 0.5 μM VE-822 and 2 μM DL51 was 31.76%. This result further indicates that the combined use of DL51 and VE-822 can increase the degree of apoptosis in H1299 cells.

[0099] 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. A quinoline compound, characterized in that, The chemical formula of the quinoline compound is C 23 H 26 ClN3O, the structural formula is shown in Formula I: Formula I.

2. The use of the quinoline compound of claim 1 in the preparation of DKC1 inhibitors.

3. The use of the quinoline compound of claim 1 in the preparation of reagents or drugs that inhibit DKC1 pseudouridine synthase activity.

4. The use of the quinoline compound of claim 1 in the preparation of reagents or drugs that bind to DKC1 protein.

5. The use of the quinoline compound of claim 1 in the preparation of a drug for treating tumors associated with high DKC1 expression.

6. The application according to claim 5, characterized in that, The tumors associated with high DKC1 expression include one or more of the following: liver cancer, gastric adenocarcinoma, lung adenocarcinoma, colon cancer, and breast cancer.

7. A combination drug composition for treating DKC1-overexpressing tumors, characterized in that, Includes the quinoline compounds and ATR inhibitors as described in claim 1.

8. The combination drug composition according to claim 7, characterized in that, The molar ratio of the quinoline compound to the ATR inhibitor is 1~20:1~2.

9. The combination drug composition according to claim 7 or 8, characterized in that, The ATR inhibitors include VE-822.

10. The combination drug composition according to claim 7 or 8, characterized in that, The tumors associated with high DKC1 expression include one or more of the following: liver cancer, gastric adenocarcinoma, lung adenocarcinoma, colon cancer, and breast cancer.

Images