Application of a proteolysis-targeting chimera

Abstract

The present invention discloses the application of a proteolysis-targeting chimera, relating to the technical field of drug preparation. The key points of its technical solution are as follows: The present invention has developed a selective RNF4 degrader, RD6, which can effectively induce the degradation of RNF4 and apply it in liver cancer cell lines, including HEPG2, HCCLM6 and Huh7. In addition, the degrader RD6 exhibits significant anti-proliferation characteristics in liver cancer cell lines and shows promising in vivo efficacy in a mouse model transplanted with HCCLM6 cells. This provides a brand-new chemical tool and new insights for exploring liver cancer and its treatment methods.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and more specifically, it relates to the application of a proteolytic targeting chimera. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor with a high metastasis rate and high mortality. Currently, the drugs used for the clinical treatment of liver cancer still cannot meet the demand for effectively alleviating the disease. Therefore, there is an urgent need to explore new therapeutic targets and mechanisms of action.

[0003] A proteolytic targeting chimera (PROTAC) is a bifunctional small molecule, a technology that has developed rapidly in recent years and has made remarkable progress in the treatment of various diseases. PROTACs have the characteristics of low dose and low toxicity, do not interfere with protein function, but mediate the degradation of target proteins. This technology makes the originally drug-resistant or non-drug-resistant targets druggable. PROTACs consist of three components: a target protein ligand, an E3 ubiquitin ligase ligand, and a linker connecting these two parts. PROTACs promote the formation of a ternary complex between the target protein and the E3 ligase, thereby ubiquitinating the target protein, which is subsequently degraded by the ubiquitin-proteasome system.

[0004] RNF4 belongs to the auxiliary chaperone protein (STUb1) family and is an E3 ligase that targets small ubiquitin-like modifier (SUMO). SUMO is one of the ubiquitin-like proteins discovered in recent years. This protein modifies target proteins by forming covalent bonds with specific lysine residues on the target proteins, a process similar to ubiquitination and usually referred to as SUMOylation. It is an important post-translational modification of proteins in eukaryotes and regulates biological processes such as transcription factor activation and DNA damage repair. RNF4 consists of a RING domain and four tandem SUMO interaction motifs (SIMs). Its C-terminal RING domain exhibits enzymatic activity only in dimer form, while the N-terminal SIMs effectively bind to long poly-sumo chains. These motifs play a crucial role in mediating the ubiquitination of SUMOylated substrates. RNF4 has been shown to be able to mediate the ubiquitination of MDC1 and BRCA1, thus playing a key role in the homologous recombination repair process. The expression level of RNF4 is elevated in liver cancer cells, suggesting its oncogenic role in the development of this malignant tumor. And the silencing of RNF4 has been shown to induce cell cycle arrest and apoptosis, thus exerting a significant anti-tumor effect. Summary of the Invention

[0005] The object of the present invention is to provide an application of proteolysis-targeting chimeras, and a selective RNF4 degrader RD6 has been developed, which can effectively induce the degradation of RNF4, especially in liver cancer cell lines, including HepG2, HCCLM6 and Huh7. In addition, the degrader RD6 exhibits significant anti-proliferative properties in liver cancer cell lines and shows promising in vivo efficacy in a mouse model transplanted with HCCLM6 cells. This provides a novel chemical tool and new insights for exploring liver cancer and its treatment methods.

[0006] The above technical object of the present invention is achieved through the following technical solutions: The present invention provides a pharmaceutical composition synthesized from the proteolysis-targeting chimera.

[0007] The present invention further provides an application of the proteolysis-targeting chimera in the preparation of a drug for treating liver cancer.

[0008] Furthermore, the proteolysis-targeting chimera is applied to the preparation of a selective RNF4 degrader.

[0009] Furthermore, the proteolysis-targeting chimera can induce the degradation of RNF4

[0010] Furthermore, the drug for treating liver cancer prepared from the proteolysis-targeting chimera realizes the treatment of liver cancer by regulating HepG2, HCCLM6 and Huh7.

[0011] Furthermore, the proteolysis-targeting chimera exhibits significant anti-proliferative properties in liver cancer cell lines.

[0012] In summary, the present invention has the following beneficial effects:

[0013] Through the molecular docking study of the RNF4 inhibitor R1 and RNF4, a series of RNF4 degrading agents were synthesized to recruit VHL with R1. By improving the length and chemical properties of the linker in PROTAC, RD1-RD14 were successfully developed, and the most excellent one is RD6. RD6 is a highly efficient and selective RNF4 degrading agent. RD6 shows a strong anti-tumor effect and does not cause any change in the body weight of mice, thus indicating that it has no potential toxicity.

[0014] In conclusion, the invention focuses on RNF4 as a key therapeutic target for liver cancer, and finally realizes the rational design and synthesis of its effective degrader, providing new treatment strategies and insights for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1a: Molecular docking analysis of R1 and RNF4; b: Structure of the RD1-RD4 compounds; c: RD3 and RD4 can effectively induce the degradation of RNF4 in HepG2 cells (concentration 5 μM); d: Neither R1 nor the VHL ligand MDK-7526 can induce the degradation of RNF4;

[0016] Figure 2 a: The time-dependent degradation of RNF4 triggered by RD4 administration reaches a peak after 6 hours of treatment; b: A 6-hour dose-response study of RD4 confirmed that the degradation of RNF4 depends on the concentration of RD4; c: RD4 triggers the phosphorylation of histone H2AX; d: The 72-hour IC50 values of RD4 in three cell lines were accurately measured, 1.788 μM for HepG2, 0.4813 μM for HCCLM6, and 0.6642 μM for Huh7;

[0017] Figure 3 a: The anti-proliferative effect of RD4-RD14; b: The RNF4 degradation efficiency of RD4-RD14; c: The protac formed by replacing the flexible linker with a rigid linker; d: The biological activity of the protac with a rigid linker;

[0018] Figure 4 Shows the inhibitory effect of R1 on the growth of HepG2 cells;

[0019] Figure 5 Is the result graph of flow cytometry detecting apoptosis induced by compound RD4;

[0020] Figure 6 Is the result graph of the IC50 of RD5, RD6, and RD7 obtained by modifying compound RD4 on liver cancer cell lines;

[0021] Figure 7 Is the result graph of WB detecting the activity of compounds RD5, RD6, and RD7 obtained by modifying compound RD4 to degrade RNF4;

[0022] Figure 8 Is the result graph of WB detecting the ability of compound RD6 to induce DNA damage;

[0023] Figure 9 Is the result graph of verifying the in vivo anti-tumor function of compound RD6. Detailed implementation mode

[0024] The following combines the attached Figures 1-9 Further detailed description of the present invention is made.

[0025] Example 1: Design and characterization of RNF4 degrading agents

[0026] Select a covalent RNF4 inhibitor R1 as a targeting ligand for RNF4. Molecular docking analysis of R1 with RNF4 ( Figure 1 showed in a) that the methyl group of the methoxy group in R1 was significantly exposed to the solvent environment, providing an ideal binding pocket for the development of potential PROTACs. Select cereblon (CRBN) and Von hipel-lindau (VHL) as alternative ligands for the E3 ubiquitin ligase, and synthesize RD1-RD4 compounds together with the RNF4 inhibitor R1 ( Figure 1 in b), with the ultimate goal of targeting RNF4 for degradation. The research results showed that RD4 had an anti-proliferative efficacy comparable to that of the RNF4 inhibitor R1. In addition, at a concentration of 5 μM, RD3 and RD4 could effectively induce the degradation of RNF4 in HepG2 cells. And the PROTAC degrader RD4 using VHL as the E3 ligase recruiter showed better effects compared with the PROTAC degrader using CRBN.

[0027] Example 2: Preparation method of proteolysis-targeting chimera

[0028] The proteolysis-targeting chimera includes RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14, and its structure is composed of a general structure (warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker;

[0029] The warhead R1 structure of RNF4 is as follows:

[0030]

[0031] The VHL ligand structure is as follows:

[0032]

[0033] The CRBN ligand structure is as follows:

[0034]

[0035] The synthetic routes of RD1 and RD3 are as follows:

[0036]

[0037] The synthetic route of RD2 is as follows:

[0038]

[0039] The synthetic route of RD4 is as follows:

[0040]

[0041] In the synthesis route of RD4, replacing the linker of RD4 in step a can obtain RD5 - RD14;

[0042] The linker of the said RD5 is

[0043] The linker of the said RD6 is

[0044] The linker of the said RD7 is

[0045] The linker of the said RD8 is

[0046] The linker of the said RD9 is

[0047] The linker of the said RD10 is

[0048] The linker of the said RD11 is

[0049] The linker of the said RD12 is

[0050] The linker of the said RD13 is

[0051] The linker of the said RD14 is

[0052] The reagents and reaction conditions in its preparation method are as follows:

[0053] (a) K2CO3, acetone, reflux, 3d; (b) chloroacetyl chloride, TEA, CH2Cl2, 0 → room temperature, 1 hour; (c) TFA, CH2Cl2, room temperature, 1 hour; (d) succinic anhydride, DMAP, TEA, CH2Cl2, room temperature, 2 hours; (e) HATU, DIPEA, DMF, room temperature, 2 hours.

[0054] RD4 has an anti - proliferative efficacy equivalent to that of the RNF4 inhibitor R1. Subsequently, the IC50 values of R1 and RD4 after treating HepG2 cells for 72 hours were measured. The results showed that the IC50 values of R1 and RD4 were 2.19 μM and 1.788 μM respectively ( Figure 2 in d)).

[0055] At a concentration of 5 μM, RD3 and RD4 can effectively induce the degradation of RNF4 in HepG2 cells ( Figure 1 in c)).

[0056] It was verified by western blotting experiments that neither R1 nor the VHL ligand MDK-7526 induced the degradation of RNF4 Figure 1 in d).

[0057] RD4 administration triggered the time-dependent degradation of RNF4, reaching a peak after 6 hours of treatment Figure 2 in a). Therefore, we conducted a 6-hour dose-response study using RD4, and the results confirmed that the degradation of RNF4 was indeed dependent on the concentration of RD4 Figure 2 in b). To further investigate the efficacy of RD4 in degrading RNF4 in different HCC cell types, we evaluated the degradation potential of RNF4 in HCC cell lines HepG2, HCCLM6, and Huh7 using western blotting. The research results showed an obvious dose-dependent correlation between RD4 and RNF4 degradation, and also showed that RD4 could also trigger the phosphorylation of histone H2AX Figure 2 in c). Compared with HepG2 cells, RD4 showed better degradation efficiency in HCCLM6 and Huh7 cells. Therefore, we accurately measured the 72-hour IC50 values of RD4 in these three cell lines, and the results showed that HepG2 was 1.788 μM, HCCLM6 was 0.4813 μM, and Huh7 was 0.6642 μM Figure 2 in d).

[0058] Example 3: Design of RNF4 degrading agents with multiple linkers

[0059] Alkanes and polyethylene glycol were initially selected as linkers to modify the length of the linker of RD4, thus synthesizing a series of novel RNF4 degrading agents with flexible linkers (Table 1). The anti-proliferative efficacy and protein degradation ability of RD5-RD14 in the HepG2 cell line were evaluated using cell viability assays and western blotting. It was found that RD5-RD7 continuously showed strong anti-proliferative activity and effective degradation of RNF4. And the anti-proliferative effects and RNF4 degradation efficiencies of RD5-RD7 were comparable to each other, but significantly exceeded RD4 Figure 3 in a and b in 3). We hypothesized that when the linkers of these degrading agents contained 2-6 atoms, their biological activities would be optimal

[0060] The flexible linkers in Table 1 were replaced with rigid linkers, including heterocyclic scaffolds (such as aziridine, pyrrolidine, piperazine, or piperidine), benzene ring linkers, and spiro rings, to form a series of PROTACs Figure 3 in c). However, the compounds in this series did not show outstanding biological activities Figure 3in d).

[0061] Table 1: Novel RNF4 Degraders with Flexible Linkers

[0062]

[0063]

[0064] Example 4: Detection of the Function of Compound RD4 in Degrading RNF4 and Inducing DNA Damage by WB Experiment

[0065] After treating hepatocellular carcinoma cells HCCLM6, Huh7, and HepG2 with compound RD4 for 6 h, cell lysates were collected for WB experiments. The experimental results showed that RD4 could degrade RNF4 in a concentration-dependent manner, and by detecting the DNA damage marker γH2AX, it was shown that RD4 could enhance the effect of inducing DNA damage with increasing concentration gradient. The results are as shown Figure 2 in c).

[0066] Specific steps:

[0067] Preparation of WB protein samples:

[0068] 1. Take hepatocellular carcinoma cells HCCLM6, Huh7, and HepG2 in the logarithmic growth phase, and inoculate 5x10^ 5 cells / well in a six-well plate, and add DMEM complete medium to a volume of 2 mL;

[0069] 2. After the cells adhere overnight, aspirate and remove the supernatant medium, and add 2 mL of the compound diluted in a concentration gradient (dissolve the compound powder with DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 2 mL of DMEM complete medium to dilute the concentration to 10 μM, and then sequentially dilute it to 5 / 2.5 / 1.25 μM);

[0070] 3. Place it in a CO2 incubator and incubate at 37 °C for 6 h.

[0071] 4. After the drug incubation time is over, aspirate and remove the supernatant medium, and add 1 mL of PBS to each well to wash the cells twice;

[0072] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells, and place it in a CO2 incubator and incubate at 37 °C for 2 mins;

[0073] 6. After the incubation is over, add 500 μL of DMEM complete medium to each well to neutralize the digestion;

[0074] 7. Take the digested cells into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 mins;

[0075] 8. After adding 1 mL of PBS to each tube to resuspend the cells, centrifuge at 1000 rpm for 5 minutes;

[0076] 9. Take out the RIPA lysis buffer, add protease inhibitor and phosphatase inhibitor and set aside.

[0077] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitor to the digested cell pellet.

[0078] 11. After lysing on ice for 30 minutes, gently pipette several times and transfer the cell lysate to a 1.5 mL EP tube.

[0079] 12. After centrifuging at 12000 rpm at 4 °C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube and then store it in a -20 °C refrigerator for later use.

[0080] Protein quantification:

[0081] The protein quantification method used in the present invention is the BCA method, and the BCA protein quantification kit produced by Shanghai Beyotime Biotechnology Co., Ltd. is used. The specific experimental steps are as follows:

[0082] 1. Add the standard products to the standard product wells of the 96-well plate at 0, 1, 2, 4, 8, 12, 16, 20 μl, and add standard product diluent to make up to 20 μl, corresponding to standard product concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml respectively;

[0083] 2. Add 20 μL of the sample to the sample wells of the 96-well plate;

[0084] 3. Add 200 μl of BCA working solution to each well and incubate at 37 °C for 30 minutes;

[0085] 4. Measure A562 with an enzyme-linked immunosorbent assay (ELISA) reader, or the absorbance at other wavelengths between 540 - 595 nm;

[0086] 5. Calculate the protein concentration of the sample according to the standard curve.

[0087] SDS-PAGE gel preparation

[0088] 1. Preparation of the lower layer gel: Take a 50 mL centrifuge tube, add 4 mL of the lower layer gel solution and 4 mL of the lower layer gel buffer respectively using a pipette and mix well;

[0089] 2. Add 80 μl of accelerator APS;

[0090] 3. Pour it into the 1.50 mm plate to about 70% height;

[0091] 4. Use a pipette to add absolute ethanol to make its upper edge flat and solidify (pour out and discard the absolute ethanol after the remaining solution in the centrifuge tube solidifies);

[0092] 5. Preparation of the upper gel: Take a plastic cup and add 1 mL each of the upper gel solution and the upper gel buffer, and mix well;

[0093] 6. Add 20 μL of APS as a coagulant;

[0094] 7. Use a pipette to fill the upper gel to the glass plate;

[0095] 8. Add a comb (pay attention to the scale of the comb) and set it aside after it solidifies.

[0096] Electrophoresis: Load the sample into the electrophoresis tank; Run at 90 V for 20 minutes until the sample runs out of the stacking gel, then change the voltage to 140 V and run until it reaches the wire (15 μg of each protein sample);

[0097] Transfer: Take out the completed SDS-PAGE gel from the electrophoresis tank and place it in deionized water (recover the electrophoresis solution in the tank), and place the NC membrane in the NC membrane buffer;

[0098] Place in the transfer apparatus in the order of + sponge - NC membrane - SDS-PAGE gel - sponge - for transfer;

[0099] Staining: Stain with Ponceau S (recover the Ponceau S after use), and wash with pure water after staining;

[0100] Membrane cutting: Cut the NC membrane stained with Ponceau S according to the requirements (TUBULIN: 55; RNF4: 35 kd; γH2AX: 15 kd), cut off the required band and place it in PBST to wash the Ponceau S staining solution;

[0101] Blocking: Weigh 1 g of skim milk powder, dissolve it in 20 mL of PBST (5%), block with milk, on a shaker at 40 rpm for 20 min, wash three times with PBST on a shaker at 40 rpm for 7 min;

[0102] Primary antibody incubation: Incubate overnight at 4 °C on a shaker at 40 rpm, recover the primary antibody, wash three times with PBST on a shaker at 40 rpm for 7 min;

[0103] Secondary antibody incubation: Incubate on a shaker at 40 rpm for 45 min, wash three times with PBST on a shaker at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 μL antibody, antibody: milk = 1:10000; 6 mL of mouse anti-GAPDH, 6 mL of rabbit anti-);

[0104] Exposure and development: After evenly incubating the hypersensitive ECL developing solution on the NC membrane, detect the protein bands by exposure with a developer.

[0105] Example 5. IC of compound RD4 on various hepatocellular carcinoma cell lines 50

[0106] By detecting the cell viability of three different hepatocellular carcinoma cell lines HCCLM6, Huh7, and HepG2 after being treated with compound RD4 for 72 hours, the IC of RD4 was calculated 50 to be 481.3 nM, 644.2 nM, and 1.788 μM respectively.

[0107] IC of the compound at the cellular level 50 The test was carried out as follows:

[0108] 1. Take hepatocellular carcinoma cells HCCLM6, Huh7, and HepG2 in the logarithmic growth phase, inoculate 5000 cells per well in a 96-well plate, and add DMEM complete medium to make up a volume of 100 μL;

[0109] 2. After the cells adhere overnight, aspirate and remove the supernatant medium, and add 100 μL of the compound diluted according to the concentration gradient (dissolve the compound powder with DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 1 mL of DMEM complete medium to dilute the concentration to 20 μM, and then sequentially dilute it to 20 / 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 / 0.15625 / 0.078125 μM);

[0110] 3. Place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 72 hours;

[0111] 4. After the incubation, detect the cell viability using the CellTiter-Glo Luminescent Cell Viability Assay Kit produced by Promega Corporation;

[0112] 5. According to the cell survival rate, draw an IC50 curve and calculate the IC50 value.

[0113] Example 6. Detection of apoptosis induced by compound RD4 by flow cytometry

[0114] After HepG2 cells were treated with compound RD4 for 6 hours, flow cytometry staining showed that RD4 could increase the proportion of apoptotic cell population in HepG2. The results proved that compound RD6 had the function of inducing apoptosis in hepatocellular carcinoma cells. The results are as Figure 5 shown.

[0115] The detection of apoptosis induced by the compound by flow cytometry was carried out as follows

[0116] 1. Take hepatocellular carcinoma cells HepG2 in the logarithmic growth phase, take 5x10^ 5Inoculate cells / holes in a six-well plate and add complete DMEM medium to make up a volume of 2 mL;

[0117] 2. After the cells adhere overnight, aspirate and remove the supernatant medium, and add 2 mL of the compound diluted in a concentration gradient (dissolve the compound powder with DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 2 mL of complete DMEM medium to dilute the concentration to 10 μM, and then sequentially dilute it to 10 / 5 μM);

[0118] 3. Place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 6 h.

[0119] 4. After the drug incubation time ends, aspirate and remove the supernatant medium, and add 1 mL of PBS to each well to wash the cells once;

[0120] 5. Digest with trypsin without EDTA, centrifuge at 1000 rpm at 4 °C for 5 min to collect the cells.

[0121] 6. Wash the cells twice with pre-cooled PBS, each time centrifuging at 300 g at 4 °C for 5 min. Collect 1 - 5×10 5 cells.

[0122] 7. Aspirate and discard the PBS, and resuspend the cells by adding 100 μL of 1×Binding Buffer.

[0123] 8. Add 5 μL of AnnexinV-FITC and 10 μL of PI Staining Solution, and gently mix.

[0124] 9. Incubate in the dark at room temperature for 15 mins.

[0125] 10. Add 400 μL of 1×Binding Buffer, mix well and place on ice, and detect using a flow cytometer.

[0126] Example 7. The IC 50

[0127] By detecting the cell viability of the liver cancer cell line HepG2 after treatment with compounds RD5, RD6, and RD7 for 72 h, the IC 50 values of RD5, RD6, and RD7 were calculated to be 608.5 nM, 450.7 nM, and 605.8 nM respectively. Compared with the IC50 value of 1.788 μM detected for RD4 on HepG2 cells, the activities of the modified RD5, RD6, and RD7 were significantly improved. The results are as Figure 6 shown.

[0128] IC of the compound at the cellular level 50 The test was carried out as follows:

[0129] 1. Take hepatocellular carcinoma cells HepG2 in the logarithmic growth phase, inoculate 5000 cells / well into a 96-well plate, and add DMEM complete medium to make up a volume of 100 μL;

[0130] 2. After the cells adhere overnight, aspirate and remove the supernatant medium, and add 100 μL of the compound diluted according to the concentration gradient (dissolve the compound powder with DMSO to prepare a 20 mM mother liquor, take 1 μL of the mother liquor and add it to 1 mL of DMEM complete medium to dilute the concentration to 20 μM, and then sequentially dilute it to 20 / 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 / 0.15625 / 0.078125 μM);

[0131] 3. Place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 72 h;

[0132] 4. After the incubation is completed, detect the cell viability by using the CellTiter-Glo Luminescent cell viability detection kit produced by Promega Corporation;

[0133] 5. According to the cell survival rate, draw an IC50 curve and calculate the IC50 value.

[0134] Example 8. Detect the activity of compounds RD5, RD6, and RD7 degraded from compound RD4 to degrade RNF4 by WB

[0135] After treating hepatocellular carcinoma cells HCCLM6, Huh7, and HepG2 cells with compounds RD4, RD5, RD6, and RD7 for 6 h, collect the cell lysates for WB experiments. The experimental results show that RD4 can degrade RNF4 in a concentration-dependent manner, and compared with compound RD4, the compounds RD5, RD6, and RD7 obtained by modification have stronger RNF4 degradation activity, and compound RD6 has the best RNF4 degradation activity. The results are as Figure 7 shown.

[0136] Select Western Blot (WB) to test the RNF4 degradation function of the compound. Specific steps:

[0137] Preparation of WB protein samples:

[0138] 1. Take hepatocellular carcinoma cells HepG2 in the logarithmic growth phase, inoculate 5x10^ 5 cells / well into a six-well plate, and add DMEM complete medium to make up a volume of 2 mL;

[0139] 2. After the cells adhere overnight, aspirate and remove the supernatant medium, and add 2 mL of the compound diluted in a concentration gradient (dissolve the compound powder with DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 4 mL of complete DMEM medium to dilute the concentration to 5 μM, and then sequentially dilute it to 2.5 / 1.25 μM);

[0140] 3. Place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 6 h.

[0141] 4. After the drug incubation time ends, aspirate and remove the supernatant medium, and add 1 mL of PBS to each well to wash the cells twice;

[0142] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells, and place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 2 mins;

[0143] 6. After the incubation ends, add 500 μL of complete DMEM medium to each well to neutralize the digestion;

[0144] 7. Transfer the digested cells into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 mins;

[0145] 8. After adding 1 mL of PBS to each tube to resuspend the cells, centrifuge at 1000 rpm for 5 mins;

[0146] 9. Take out the RIPA lysis buffer, add protease inhibitors and phosphatase inhibitors for standby.

[0147] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0148] 11. After lysing on ice for 30 minutes, gently pipette several times, and transfer the cell lysate to a 1.5 mL EP tube.

[0149] 12. After centrifuging at 12000 rpm at 4 °C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and then place it in a -20 °C refrigerator for storage and standby.

[0150] Protein quantification:

[0151] The protein quantification method used in the present invention is the BCA method, and the BCA protein quantification kit produced by Shanghai Beyotime Biotechnology Co., Ltd. is used. The specific experimental steps are as follows:

[0152] 1. Add the standard products in volumes of 0, 1, 2, 4, 8, 12, 16, and 20 μl to the standard product wells of a 96-well plate, and make up to 20 μl with the standard product diluent, corresponding to standard product concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0153] 2. Add 20 μL of the sample to the sample wells of the 96-well plate;

[0154] 3. Add 200 μl of the BCA working solution to each well and incubate at 37 °C for 30 minutes;

[0155] 4. Measure A562 with an enzyme-linked immunosorbent assay (ELISA) reader, or measure the absorbance at other wavelengths between 540 - 595 nm;

[0156] 5. Calculate the protein concentration of the sample according to the standard curve.

[0157] SDS-PAGE Gel Preparation

[0158] 1. Preparation of the lower layer gel: Take a 50 mL centrifuge tube, and use a pipette to add 4 mL each of the lower layer gel solution and the lower layer gel buffer, and mix well;

[0159] 2. Add 80 μl of the accelerator APS;

[0160] 3. Pour it into a 1.50 mm plate to a height of about 70%;

[0161] 4. Use a pipette to add absolute ethanol to make the upper edge flat and solidify (pour out and discard the remaining solution in the centrifuge tube after it solidifies);

[0162] 5. Preparation of the upper layer gel: Take a plastic cup and add 1 mL each of the upper layer gel solution and the upper layer gel buffer, and mix well;

[0163] 6. Add 20 μL of the accelerator APS;

[0164] 7. Use a pipette to fill the upper layer gel to the brim of the glass plate;

[0165] 8. Add a comb (pay attention to the scale of the comb) and wait for it to solidify for later use.

[0166] Electrophoresis: Load the samples into the electrophoresis tank; Run at 90 V for 20 minutes until the samples run out of the stacking gel, then change the voltage to 140 V and run until reaching the wire (15 μg of each protein sample);

[0167] Blotting: Take out the completed SDS-PAGE gel from the electrophoresis tank and place it in deionized water (recover the electrophoresis buffer in the tank), and place the NC membrane in the NC membrane buffer;

[0168] Place in the following order in the blotting apparatus for blotting: + sponge - NC membrane - SDS-PAGE gel - sponge—

[0169] Staining: Ponceau S staining (the Ponceau S is recycled after use), and wash with pure water after staining;

[0170] Membrane cutting: Cut the NC membrane stained with Ponceau S according to requirements (TUBULIN: 55; RNF4: 35 kd), take the required band and put it into PBST to wash the Ponceau S staining solution;

[0171] Blocking: Weigh 1 g of skim milk powder, dissolve it in 20 mL of PBST (5%), block with milk, shake at 40 rpm for 20 min, wash three times with PBST at 40 rpm for 7 min;

[0172] Primary antibody incubation: Incubate overnight at 4°C with shaking at 40 rpm, recycle the primary antibody, wash three times with PBST at 40 rpm for 7 min;

[0173] Secondary antibody incubation: Incubate with shaking at 40 rpm for 45 min, wash three times with PBST at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 μL antibody, antibody: milk = 1:10000; 6 mL of mouse anti-GAPDH, 6 mL of rabbit anti-);

[0174] Exposure and development: After evenly incubating the hypersensitive ECL developing solution on the NC membrane, detect the protein band by exposure with a developer.

[0175] Example 9. Detection of the ability of compound RD6 to induce DNA damage by WB

[0176] After treating the liver cancer cell line HepG2 with compound RD6 for 6 h, collect the cell lysate for WB experiment. The experimental results show that RD4 can upregulate the expression level of the DNA damage marker H2AX protein in a concentration-dependent manner, proving that compound RD6 can induce DNA damage ability with the increase of concentration. The results are as Figure 8 shown.

[0177] Select Western Blot (WB) to test the RNF4 degradation and DNA induction function of the compound. Specific steps:

[0178] Preparation of WB protein samples:

[0179] 1. Take HepG2 liver cancer cells in the logarithmic growth phase, inoculate 5 x 10^ 5 cells / well in a six-well plate, and add DMEM complete medium to make up a volume of 2 mL;

[0180] 2. After the cells adhere overnight, aspirate and remove the supernatant medium, and add 2 mL of the compound diluted in a concentration gradient (dissolve the compound powder with DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 4 mL of complete DMEM medium to dilute the concentration to 5 μM, and then sequentially dilute it to 2.5 / 1.25 μM);

[0181] 3. Place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 6 h.

[0182] 4. After the drug incubation time ends, aspirate and remove the supernatant medium, and add 1 mL of PBS to each well to wash the cells twice;

[0183] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells, and place it in a CO2 incubator and incubate at a constant temperature of 37 °C for 2 mins;

[0184] 6. After the incubation ends, add 500 μL of complete DMEM medium to each well to neutralize the digestion;

[0185] 7. Transfer the digested cells into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 mins;

[0186] 8. After adding 1 mL of PBS to each tube to resuspend the cells, centrifuge at 1000 rpm for 5 mins;

[0187] 9. Take out the RIPA lysis buffer, add protease inhibitors and phosphatase inhibitors for later use.

[0188] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0189] 11. After lysing on ice for 30 minutes, gently pipette several times, and transfer the cell lysate to a 1.5 mL EP tube.

[0190] 12. After centrifuging at 12000 rpm at 4 °C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and then put it in a -20 °C refrigerator for storage and later use.

[0191] Protein quantification:

[0192] The protein quantification method used in the present invention is the BCA method, and the BCA protein quantification kit produced by Shanghai Beyotime Biotechnology Co., Ltd. is used. The specific experimental steps are as follows:

[0193] 1. Add the standard products in volumes of 0, 1, 2, 4, 8, 12, 16, and 20 μl to the standard product wells of a 96-well plate, and make up to 20 μl with the standard product diluent, corresponding to standard product concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0194] 2. Add 20 μL of the sample to the sample wells of the 96-well plate;

[0195] 3. Add 200 μl of the BCA working solution to each well and incubate at 37 °C for 30 minutes;

[0196] 4. Measure A562 with an enzyme-linked immunosorbent assay (ELISA) reader, or measure the absorbance at other wavelengths between 540 - 595 nm;

[0197] 5. Calculate the protein concentration of the sample according to the standard curve.

[0198] SDS-PAGE Gel Preparation

[0199] 1. Preparation of the lower layer gel: Take a 50 mL centrifuge tube, and use a pipette to add 4 mL each of the lower layer gel solution and the lower layer gel buffer, and mix well;

[0200] 2. Add 80 μl of the accelerator APS;

[0201] 3. Pour it into the 1.50 mm plate to a height of about 70%;

[0202] 4. Use a pipette to add absolute ethanol to make the upper edge flat and solidify (pour out and discard the remaining solution in the centrifuge tube after it solidifies);

[0203] 5. Preparation of the upper layer gel: Take a plastic cup and add 1 mL each of the upper layer gel solution and the upper layer gel buffer, and mix well;

[0204] 6. Add 20 uL of the accelerator APS;

[0205] 7. Use a pipette to fill the upper layer gel to the brim of the glass plate;

[0206] 8. Add a comb (pay attention to the comb scale), and wait for it to solidify for later use.

[0207] Electrophoresis: Load the samples into the electrophoresis tank; Run at 90 V for 20 minutes until the samples run out of the stacking gel, then change the voltage to 140 V and run until reaching the wire (15 μg of each protein sample);

[0208] Transfer: Take out the completed SDS-PAGE gel from the electrophoresis tank and place it in primary water (recover the electrophoresis solution in the tank), and place the NC membrane in the NC membrane buffer;

[0209] Place in the transfer apparatus in the order of + sponge - NC membrane - SDS-PAGE gel - sponge - for transfer;

[0210] Staining: Ponceau S staining (Ponceau S is recycled after use), and wash with pure water after staining;

[0211] Membrane cutting: Cut the NC membrane stained with Ponceau S according to requirements (TUBULIN: 55; γH2AX: 15 kd), take the required strips and put them into PBST to wash the Ponceau S staining solution;

[0212] Blocking: Weigh 1 g of skim milk powder, dissolve it in 20 mL of PBST (5%), block with milk, on a shaker at 40 rpm for 20 min, wash three times with PBST on a shaker at 40 rpm for 7 min;

[0213] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm, recycle the primary antibody, wash three times with PBST on a shaker at 40 rpm for 7 min;

[0214] Secondary antibody incubation: Incubate on a shaker at 40 rpm for 45 min, wash three times with PBST on a shaker at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 μL antibody, antibody: milk = 1:10000; 6 mL of mouse anti-GAPDH, 6 mL of rabbit anti-);

[0215] Exposure and development: After uniformly incubating the hypersensitive ECL developing solution on the NC membrane, detect the protein bands by exposure with a developer.

[0216] Example 10. Verification of the in vivo anti-tumor function of compound RD6

[0217] A Huh7 tumor xenograft model was established in wild-type nude mice and treated with compound RD6 (20 mg / kg or 5 mg / kg, intraperitoneal injection, once every two days) alone. Three weeks after administration, the tumor size in the RD6 administration group was significantly smaller than that in the control group. At the same time, there was no significant change in the body weight of the mice treated with compound RD6, indicating the safety of this treatment regimen. After the administration ended, the tumor tissues were taken out and weighed, and it was found that the tumor mass in the RD6 administration group was also significantly lower than that in the control group. The results are as Figure 9 shown.

[0218] Verification of the mouse subcutaneous hepatocellular carcinoma allograft tumor model, and the specific experimental steps are as follows:

[0219] Inoculation of nude mice with hepatocellular carcinoma cell Huh7:

[0220] Material and instrument preparation: 1 mL syringe, Huh7 single-cell suspension, cotton ball, alcohol.

[0221] 1. Resuspend the Huh7 cells obtained by digestion treatment with serum-free DMEM medium into a single-cell suspension (5x10^7 Prepare for use after reaching a cell density of

[0222] 2. Aspirate an appropriate amount of Huh7 single-cell suspension with a syringe, insert the needle (with the needle incision facing the scale, and expel the air in the needle before injection).

[0223] 3. Take out the nude mouse from the mouse cage, pinch one ear and turn it over, clamp the front and hind limbs on one side of the mouse with the ring finger and little finger, and disinfect the injection site with an alcohol cotton ball.

[0224] 4. Inject 100 μL of single-cell suspension subcutaneously at the scapular region of the mouse in a parallel manner.

[0225] 5. Rotate out the needle, and immediately push the injected liquid from bottom to top with a dry cotton ball to avoid leakage.

[0226] 6. Then administer drugs according to the experimental requirements, and monitor the body weight and tumor size of the mice every other day during this period.

[0227] After the experiment, sacrifice the mice, take out the tumor tissue with scissors and forceps, and take pictures for record.

[0228] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. Those skilled in the art can make modifications to this embodiment without creative contributions according to needs after reading this specification, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. An application of a proteolysis targeting chimera, characterized in that: The proteolysis targeting chimera is RD4, RD5, RD6 or RD7, and the structure of RD4, RD5, RD6 and RD7 is composed of a common structure (the warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker; The structure of the RNF4 warhead R1 is as follows: ； The structure of VHL ligand is as follows: ； The CRBN ligand structure is as follows: ； The synthetic route of RD4 is as follows: ； In the synthetic route of RD4, replacing the linker of RD4 in step a can produce RD5-RD7; The linker of RD5 is ; The linker of RD6 is ; The linker of RD7 is ; The proteolysis targeting chimera is used for preparing drugs for degradation and anti-proliferation of liver cancer cells.

2. The use of a proteolysis targeting chimera according to claim 1, characterized in that: The proteolysis targeting chimera was able to induce degradation of RNF4 in hepatocellular carcinoma.

3. Use of a proteolysis targeting chimera according to claim 1 in the preparation of a drug for treating liver cancer.

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