A protein hydrolysis-targeted chimera and its preparation method

Abstract

The present invention discloses a proteolysis-targeted chimera and a preparation method thereof, relating to the technical field of drug preparation. The technical scheme thereof mainly comprises: the proteolysis-targeted chimera comprises RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14; the structure of the proteolysis-targeted chimera comprises a universal 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 the VHL ligand is as follows: the structure of the CRBN ligand is as follows:

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and more particularly to a protein hydrolysis targeting chimera and a preparation method thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor with a high metastasis rate and mortality rate. Current drugs used in clinical treatment of HCC still fall short of meeting the demand for effective disease alleviation. Therefore, the exploration of new therapeutic targets and mechanisms of action is urgently needed.

[0003] Proteolytic Targeting Chimera (PROTAC) is a bifunctional small molecule that has developed rapidly in recent years and has made significant progress in the treatment of various diseases. PROTACs are characterized by low dose and low toxicity. They do not interfere with protein function, but mediate the degradation of target proteins. This technology makes targets that were originally resistant or non-resistant drug-resistant drug-resistant. PROTACs are composed of three components: a target protein ligand, an E3 ubiquitin ligase ligand, and a linker connecting the two parts. PROTACs promote the formation of a ternary complex between the target protein and the E3 ligase, thereby ubiquitin-tagging the target protein, which is then degraded by the ubiquitin proteasome system.

[0004] RNF4 belongs to the stubiquitin-chaperone protein (STUb1) family and is an E3 ligase that targets the small ubiquitin-like modifier (SUMO). SUMO is one of the recently discovered ubiquitin-like proteins. This protein modifies target proteins by forming covalent bonds with specific lysine residues on them, a process similar to ubiquitination and commonly referred to as sumoylation. It is a crucial post-translational modification of proteins in eukaryotes, regulating biological processes such as transcription factor activation and DNA damage repair. RNF4 consists of a ring (RING) domain and four tandem SUMO-interacting motifs (SIMs). The C-terminal ring domain exhibits enzymatic activity only in a dimerized form, while the N-terminal SIMs efficiently bind to long SUMO chains. These motifs play a crucial role in mediating the ubiquitination of sumoylated substrates. RNF4 has been shown to mediate the ubiquitination of MDC1 and BRCA1, thereby playing a key role in homologous recombination repair. RNF4 expression is elevated in liver cancer cells, suggesting an oncogenic role in the development of this malignancy. Furthermore, silencing of RNF4 has been shown to induce cell cycle arrest and apoptosis, thereby exerting significant anti-tumor effects. Summary of the Invention

[0005] The present invention provides a proteolysis-targeted chimera and its preparation method. The selective RNF4 degrader RD6 was developed, which effectively induces RNF4 degradation, particularly in liver cancer cell lines, including HEPG2, HCCLM6, and Huh7. Furthermore, the degrader RD6 exhibits significant antiproliferative properties in liver cancer cell lines and demonstrates promising in vivo efficacy in a mouse model transplanted with HCCLM6 cells. This provides a novel chemical tool and new insights into liver cancer and its treatment.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: a proteolysis targeting chimera, wherein the proteolysis targeting chimera includes RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14,

[0007] The structure of the proteolysis targeting chimera consists of a common structure (the warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker;

[0008] The structure of the RNF4 warhead R1 is as follows:

[0009]

[0010] The VHL ligand structure is as follows:

[0011]

[0012] The CRBN ligand structure is as follows:

[0013]

[0014] The present invention further provides a method for preparing a proteolysis-targeted chimera: the synthetic routes of RD1 and RD3 are as follows:

[0015]

[0016] The present invention is further configured as follows: the synthetic route of RD2 is as follows:

[0017]

[0018] The present invention is further configured as follows: the synthetic route of RD4 is as follows:

[0019]

[0020] The present invention is further configured as follows: in the synthetic route of RD4, RD5-RD14 can be obtained by replacing the linker of RD4 in step a;

[0021] The linker of RD5 is

[0022] The linker of RD6 is

[0023] The linker of RD7 is

[0024] The linker of RD8 is

[0025] The linker of RD9 is

[0026] The linker of RD10 is

[0027] The linker of RD11 is

[0028] The linker of RD12 is

[0029] The linker of RD13 is

[0030] The linker of RD14 is

[0031] The present invention is further configured as follows: the reagents and reaction conditions in the preparation method are as follows:

[0032] (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.

[0033] The present invention further provides a pharmaceutical composition synthesized from the proteolysis targeting chimera.

[0034] The present invention further provides the use of the protein hydrolysis targeting chimera in preparing drugs for treating liver cancer.

[0035] In summary, the present invention has the following beneficial effects:

[0036] Through molecular docking studies of the RNF4 inhibitor R1 with RNF4, a series of RNF4 degraders were synthesized, using R1 to recruit VHL. By modifying the linker length and chemical properties of the PROTACs, RD1-RD14 were successfully developed. The most effective of these was RD6, a highly potent and selective RNF4 degrader. RD6 demonstrated potent antitumor efficacy without causing any changes in mouse body weight, indicating a lack of potential toxicity.

[0037] In summary, this invention focuses on RNF4 as a key therapeutic target for liver cancer, and ultimately achieves the rational design and synthesis of its effective degraders, providing new therapeutic strategies and insights for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 a: Molecular docking analysis of R1 and RNF4; b: Structures of 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 induces the degradation of RNF4;

[0039] Figure 2 a: RD4 administration triggers time-dependent degradation of RNF4, reaching a peak after 6 hours of treatment; b: A 6-hour dose-response study of RD4 confirmed that RNF4 degradation is dependent on RD4 concentration; c: RD4 triggers phosphorylation of histone H2AX; d: The 72-hour IC50 values ​​of RD4 in three cell lines were precisely measured: 1.788 μM for HepG2, 0.4813 μM for HCCLM6, and 0.6642 μM for Huh7;

[0040] Figure 3 a: Anti-proliferative effect of RD4-RD14; b: RNF4 degradation efficiency of RD4-RD14; c: Protac formed by replacing the flexible linker with a rigid linker; d: Biological activity of Protac with a rigid linker;

[0041] Figure 4 is the inhibitory effect of R1 on HepG2 cell growth;

[0042] Figure 5 The figure shows the results of flow cytometry detection of cell apoptosis induced by compound RD4;

[0043] Figure 6 The IC50 results of RD5, RD6, and RD7 modified from compound RD4 on liver cancer cell lines are shown in the graph;

[0044] Figure 7This is the result of WB detection of RNF4 degradation activity of compounds RD5, RD6, and RD7 modified from compound RD4;

[0045] Figure 8 This is the result of WB detection of the DNA damage-inducing ability of compound RD6;

[0046] Figure 9 This is the result diagram of the in vivo anti-tumor function verification of compound RD6. DETAILED DESCRIPTION

[0047] The following is combined with Figure 1-9 The present invention is described in further detail.

[0048] Example 1: Design and characterization of RNF4 degraders

[0049] A covalent RNF4 inhibitor R1 was selected as a targeting ligand for RNF4. Molecular docking analysis of R1 and RNF4 ( Figure 1 Figure (a) shows that the methyl group of the methoxy group in R1 is clearly exposed to the solvent environment, providing an ideal binding pocket for the development of potential PROTACs. Cereblon (CRBN) and Von hipel-lindau (VHL) were selected as alternative ligands for E3 ubiquitin ligases and synthesized together with the RNF4 inhibitor R1 to form RD1-RD4 compounds ( Figure 1 In (b), the ultimate goal is to target RNF4 for degradation. The results showed that RD4 had comparable antiproliferative efficacy to the RNF4 inhibitor R1. Furthermore, at a concentration of 5 μM, RD3 and RD4 effectively induced RNF4 degradation in HepG2 cells. Furthermore, the PROTAC degrader RD4, which uses VHL as an E3 ligase recruiter, exhibited superior efficacy compared to PROTAC degraders targeting CRBN.

[0050] Example 2: Preparation of proteolysis-targeted chimeras

[0051] The proteolysis targeting chimeras include RD1, RD2, RD3, RD4, RD5, RD6, RD7, RD8, RD9, RD10, RD11, RD12, RD13 and RD14, and their structures are composed of a common structure (the warhead R1 of RNF4), a VHL ligand or a CRBN ligand, and a linker;

[0052] The structure of the RNF4 warhead R1 is as follows:

[0053]

[0054] The structure of the VHL ligand is as follows:

[0055]

[0056] The CRBN ligand structure is as follows:

[0057]

[0058] The synthetic routes of RD1 and RD3 are as follows:

[0059]

[0060] The synthetic route of RD2 is as follows:

[0061]

[0062] The synthetic route of RD4 is as follows:

[0063]

[0064] In the synthetic route of RD4, replacing the linker of RD4 in step a can produce RD5-RD14;

[0065] The linker of RD5 is

[0066] The linker of RD6 is

[0067] The linker of RD7 is

[0068] The linker of RD8 is

[0069] The linker of RD9 is

[0070] The linker of RD10 is

[0071] The linker of RD11 is

[0072] The linker of RD12 is

[0073] The linker of RD13 is

[0074] The linker of RD14 is

[0075] The reagents and reaction conditions in its preparation method are as follows:

[0076] (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.

[0077] RD4 has an antiproliferative effect comparable to that of the RNF4 inhibitor R1. The IC50 values ​​of R1 and RD4 were then determined in HepG2 cells after 72 hours of treatment. The results showed that the IC50 values ​​of R1 and RD4 were 2.19 μM and 1.788 μM, respectively. Figure 2 (d)

[0078] At a concentration of 5 μM, RD3 and RD4 could effectively induce the degradation of RNF4 in HepG2 cells ( Figure 1 (c)

[0079] Western blotting experiments confirmed that neither R1 nor the VHL ligand MDK-7526 induced the degradation of RNF4 ( Figure 1 (d)

[0080] RD4 administration triggered a time-dependent degradation of RNF4, reaching a peak after 6 h of treatment ( Figure 2 Therefore, we performed a 6-hour dose-response study using RD4, and the results confirmed that RNF4 degradation was indeed dependent on the concentration of RD4 ( Figure 2 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 results showed a clear dose-dependent association between RD4 and RNF4 degradation and also indicated that RD4 also triggered the phosphorylation of histone H2AX ( Figure 2 Compared with HepG2 cells, RD4 showed better degradation efficiency in HCCLM6 and Huh7 cells. Therefore, we precisely measured the 72-hour IC50 values ​​of RD4 in these three cell lines, and the results showed that HepG2 was 1.788μM, HCCLM6 was 0.4813μM, and Huh7 was 0.6642μM ( Figure 2 (d)

[0081] Example 3: Design of RNF4 degraders with multiple linkers

[0082] Initially, alkanes and polyethylene glycol were selected as linkers to modify the length of the linker of RD4, thereby synthesizing a series of novel RNF4 degraders with flexible linkers (Table 1). The antiproliferative efficacy and protein degradation ability of RD5-RD14 in HepG2 cell lines were evaluated using cell viability assays and western blots. It was found that RD5-RD7 continued to exhibit strong antiproliferative activity and effective degradation of RNF4. The antiproliferative effect and RNF4 degradation efficiency of RD5-RD7 were comparable to each other, but significantly exceeded those of RD4 ( Figure 3 We hypothesized that the bioactivity of these degraders would be optimal when their linkers contained 2–6 atoms.

[0083] The flexible linkers in Table 1 were replaced with rigid linkers, including heterocyclic scaffolds (such as nitrogen pyrrole, tetrahydropyrrole, piperazine or piperidine), benzene ring linkers and spiro rings, to form a series of PROTACs ( Figure 3 c). However, the compounds in this series did not show outstanding biological activity ( Figure 3 (d)

[0084] Table 1: Novel RNF4 degraders with flexible linkers

[0085]

[0086]

[0087] Example 4: Western blot assay to detect the degradation of RNF4 and the induction of DNA damage by compound RD4

[0088] After treating liver cancer cells HCCLM6, Huh7 and HepG2 with compound RD4 for 6 hours, cell lysates were collected for WB experiments. The experimental results showed that RD4 could degrade RNF4 in a concentration-dependent manner, and the detection of DNA damage marker γH2AX showed that RD4 could enhance the induction of DNA damage with the concentration gradient. Figure 2 As shown in c.

[0089] Specific steps:

[0090] Western blotting protein sample preparation:

[0091] 1. Take 5x 10^ 5 Each well was inoculated into a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0092] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in 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 dilute it in a gradient to 5 / 2.5 / 1.25 μM).

[0093] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0094] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells twice;

[0095] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells and incubate at 37°C in a CO2 incubator for 2 minutes;

[0096] 6. After incubation, add 500 μL DMEM complete medium to each well for neutralization and digestion;

[0097] 7. Place the incubated cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0098] 8. Add 1 mL of PBS to each tube to resuspend the cells and centrifuge at 1000 rpm for 5 minutes;

[0099] 9. Remove the RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors for later use.

[0100] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0101] 11. After lysis on ice for 30 minutes, gently pipette up and down several times and transfer the cell lysate to a 1.5 mL EP tube.

[0102] 12. Centrifuge at 12000 rpm and 4°C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and store it in a -20°C refrigerator until use.

[0103] Protein quantification:

[0104] The protein quantification method used in the present invention is the BCA method, using the BCA protein quantification kit produced by Shanghai Biyuntian Biotechnology Co., Ltd. The specific experimental steps are as follows:

[0105] 1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0106] 2. Add 20 μL of sample to the sample wells of a 96-well plate;

[0107] 3. Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes.

[0108] 4. Measure the absorbance at A562 or other wavelengths between 540-595 nm using a microplate reader;

[0109] 5. Calculate the protein concentration of the sample based on the standard curve.

[0110] SDS-PAGE gel preparation

[0111] 1. Preparation of lower gel: Take a 50mL centrifuge tube and use a pipette to add 4mL each of lower gel solution and lower gel buffer and mix well;

[0112] 2. Add 80ul of coagulant APS;

[0113] 3. Pour into the 1.50mm board to about 70% height;

[0114] 4. Use a pipetting gun to add anhydrous ethanol to make the upper edge flat and solidify (pour out the anhydrous ethanol and discard the remaining solution in the centrifuge tube after it solidifies);

[0115] 5. Preparation of upper gel: Take a plastic cup and add 1 mL each of upper gel solution and upper gel buffer, mix well;

[0116] 6. Add 20uL of coagulant APS;

[0117] 7. Use a dosing gun to fill the glass plate with the upper layer of glue;

[0118] 8. Add the comb (pay attention to the scale on the comb) and wait for it to solidify before use.

[0119] Electrophoresis: Add samples to the electrophoresis tank; run at 90V for 20 minutes until the samples run out of the stacking gel, then change the voltage to 140V and run until the wires are reached (15 μg of protein sample each);

[0120] Transfer: Take out the SDS-PAGE gel after electrophoresis from the electrophoresis tank and place it in primary water (the electrophoresis solution in the tank is recovered), take out the NC membrane and place it in NC membrane buffer;

[0121] Place the membrane in the transfer apparatus in the order of + sponge-NC membrane-SDS-PAGE gel-sponge for transfer;

[0122] Dyeing: Dye with Ponceau red (Ponceau red is recycled after use), and then rinse in pure water;

[0123] Membrane cutting: Cut the NC membrane stained with Ponceau red according to the requirements (TUBULIN: 55; RNF4: 35kd; γH2AX: 15kd), remove the required bands and place them in PBST to wash out the Ponceau red staining solution;

[0124] Blocking: Weigh 1 g of skim milk powder and dissolve it in 20 mL of PBST (5%). Block with milk and shake on a shaker at 40 rpm for 20 min. Wash three times with PBST and shake on a shaker at 40 rpm for 7 min.

[0125] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm. Recover the primary antibody and wash three times with PBST on a shaker at 40 rpm for 7 min.

[0126] Secondary antibody incubation: Incubate on a shaker at 40 rpm for 45 min, then wash three times with PBST at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 uL antibody: milk = 1:10,000; GAPDH mouse antibody 6 mL, rabbit antibody 6 mL)

[0127] Exposure and development: After evenly incubating the NC membrane with the ultrasensitive ECL developer, the protein bands are exposed and detected using a developer.

[0128] Example 5. IC of compound RD4 on various liver cancer cell lines 50

[0129] The RD4 IC was calculated by detecting the cell viability of three different liver cancer cell lines HCCLM6, Huh7, and HepG2 after treatment with compound RD4 for 72 hours. 50 They are 481.3nM, 644.2nM and 1.788μM respectively.

[0130] Compound cellular IC 50 The specific experimental steps are as follows:

[0131] 1. Take 5000 hepatocellular carcinoma cells HCCLM6, Huh7, and HepG2 in the logarithmic growth phase and inoculate them into a 96-well plate. Add DMEM complete medium to make up the volume to 100 μL.

[0132] 2. After the cells have attached overnight, remove the supernatant medium and add 100 μL of the compound diluted in a concentration gradient (dissolve the compound powder in 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, then dilute it in a gradient to 20 / 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 / 0.15625 / 0.078125 μM);

[0133] 3. Place in a CO2 incubator and incubate at 37°C for 72 hours;

[0134] 4. After incubation, cell viability was detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit produced by Promega;

[0135] 5. Draw the IC50 curve based on the cell viability and calculate the IC50 value.

[0136] Example 6. Flow cytometry detection of cell apoptosis induced by compound RD4

[0137] After HepG2 cells were treated with compound RD4 for 6 hours, flow cytometry staining showed that RD4 could increase the proportion of HepG2 apoptotic cells. The results showed that compound RD6 has the function of inducing apoptosis of liver cancer cells. Figure 5 shown.

[0138] Flow cytometry was used to detect compound-induced cell apoptosis. The specific experimental steps are as follows:

[0139] 1. Take 5x 10^ 5 Each well was inoculated into a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0140] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in 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 dilute it in a gradient to 10 / 5 μM).

[0141] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0142] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells once;

[0143] 5. After digestion with EDTA-free trypsin, collect the cells by centrifugation at 1000 rpm and 4°C for 5 minutes.

[0144] 6. Wash the cells twice with pre-cooled PBS, centrifuging at 300g for 5 min each time at 4°C. Collect 1-5×10 5 cell.

[0145] 7. Aspirate and discard PBS, then add 100 μL 1× Binding Buffer to resuspend the cells.

[0146] 8. Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution and mix gently.

[0147] 9. Protect from light and incubate at room temperature for 15 minutes.

[0148] 10. Add 400 μL 1× Binding Buffer, mix well, place on ice, and detect using flow cytometer.

[0149] Example 7. IC of RD5, RD6, and RD7 obtained by modifying compound RD4 on liver cancer cell lines 50

[0150] The cell viability of the liver cancer cell line HepG2 treated with compounds RD5, RD6, and RD7 for 72 hours was detected and the IC values ​​of RD5, RD6, and RD7 were calculated. 50 The activity of modified RD5, RD6 and RD7 were significantly improved compared with the IC50 value of 1.788μM obtained by RD4 in HepG2 cells. Figure 6 shown.

[0151] Compound cellular IC 50 The specific experimental steps are as follows:

[0152] 1. Take 5000 HepG2 liver cancer cells in the logarithmic growth phase and inoculate them into a 96-well plate. Add DMEM complete medium to make up to 100 μL.

[0153] 2. After the cells have attached overnight, remove the supernatant medium and add 100 μL of the compound diluted in a concentration gradient (dissolve the compound powder in 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, then dilute it in a gradient to 20 / 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 / 0.15625 / 0.078125 μM);

[0154] 3. Place in a CO2 incubator and incubate at 37°C for 72 hours;

[0155] 4. After incubation, cell viability was detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit produced by Promega;

[0156] 5. Draw the IC50 curve based on the cell viability and calculate the IC50 value.

[0157] Example 8. Western blotting to detect RNF4 degradation activity of compounds RD5, RD6, and RD7 modified from compound RD4

[0158] After treating liver cancer cells HCCLM6, Huh7 and HepG2 with compounds RD4, RD5, RD6 and RD7 for 6 hours, cell lysates were collected for WB experiments. The experimental results showed that RD4 could degrade RNF4 in a concentration-dependent manner, and compared with compound RD4, the modified compounds RD5, RD6 and RD7 had stronger RNF4 degradation activity, among which compound RD6 had the best RNF4 degradation activity. Figure 7 shown.

[0159] Western Blot (WB) was used to test the RNF4 degradation function of the compound. Specific steps:

[0160] Western blotting protein sample preparation:

[0161] 1. Take 5x 10^ 5 Each well was inoculated into a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0162] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 4 mL of DMEM complete medium to dilute the concentration to 5 μM, and then dilute it in a gradient to 2.5 / 1.25 μM).

[0163] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0164] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells twice;

[0165] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells and incubate at 37°C in a CO2 incubator for 2 minutes;

[0166] 6. After incubation, add 500 μL DMEM complete medium to each well for neutralization and digestion;

[0167] 7. Place the incubated cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0168] 8. Add 1 mL of PBS to each tube to resuspend the cells and centrifuge at 1000 rpm for 5 minutes;

[0169] 9. Remove the RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors for later use.

[0170] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0171] 11. After lysis on ice for 30 minutes, gently pipette up and down several times and transfer the cell lysate to a 1.5 mL EP tube.

[0172] 12. Centrifuge at 12000 rpm and 4°C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and store it in a -20°C refrigerator until use.

[0173] Protein quantification:

[0174] The protein quantification method used in the present invention is the BCA method, using the BCA protein quantification kit produced by Shanghai Biyuntian Biotechnology Co., Ltd. The specific experimental steps are as follows:

[0175] 1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0176] 2. Add 20 μL of sample to the sample wells of a 96-well plate;

[0177] 3. Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes.

[0178] 4. Measure the absorbance at A562 or other wavelengths between 540-595 nm using a microplate reader;

[0179] 5. Calculate the protein concentration of the sample based on the standard curve.

[0180] SDS-PAGE gel preparation

[0181] 1. Preparation of lower gel: Take a 50mL centrifuge tube and use a pipette to add 4mL each of lower gel solution and lower gel buffer and mix well;

[0182] 2. Add 80ul of coagulant APS;

[0183] 3. Pour into the 1.50mm board to about 70% height;

[0184] 4. Use a pipetting gun to add anhydrous ethanol to make the upper edge flat and solidify (pour out the anhydrous ethanol and discard the remaining solution in the centrifuge tube after it solidifies);

[0185] 5. Preparation of upper gel: Take a plastic cup and add 1 mL each of upper gel solution and upper gel buffer, mix well;

[0186] 6. Add 20uL of coagulant APS;

[0187] 7. Use a dosing gun to fill the glass plate with the upper layer of glue;

[0188] 8. Add the comb (pay attention to the scale on the comb) and wait for it to solidify before use.

[0189] Electrophoresis: Add samples to the electrophoresis tank; run at 90V for 20 minutes until the samples run out of the stacking gel, then change the voltage to 140V and run until the wires are reached (15 μg of protein sample each);

[0190] Transfer: Take out the SDS-PAGE gel after electrophoresis from the electrophoresis tank and place it in primary water (the electrophoresis solution in the tank is recovered), take out the NC membrane and place it in NC membrane buffer;

[0191] Place the membrane in the transfer apparatus in the order of + sponge-NC membrane-SDS-PAGE gel-sponge for transfer;

[0192] Dyeing: Dye with Ponceau red (Ponceau red is recycled after use), and then rinse in pure water;

[0193] Membrane cutting: Cut the NC membrane stained with Ponceau red according to the requirements (TUBULIN: 55; RNF4: 35kd), remove the required bands and place them in PBST to wash out the Ponceau red staining solution;

[0194] Blocking: Weigh 1 g of skim milk powder and dissolve it in 20 mL of PBST (5%). Block with milk and shake on a shaker at 40 rpm for 20 min. Wash three times with PBST and shake on a shaker at 40 rpm for 7 min.

[0195] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm. Recover the primary antibody and wash three times with PBST on a shaker at 40 rpm for 7 min.

[0196] Secondary antibody incubation: Incubate on a shaker at 40 rpm for 45 min, then wash three times with PBST at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 uL antibody: milk = 1:10,000; GAPDH mouse antibody 6 mL, rabbit antibody 6 mL)

[0197] Exposure and development: After evenly incubating the NC membrane with the ultrasensitive ECL developer, the protein bands are exposed and detected using a developer.

[0198] Example 9. Detection of DNA damage induced by compound RD6 by WB

[0199] After treating HepG2 cells with compound RD6 for 6 hours, cell lysates were collected for WB experiments. The experimental results showed that RD6 could increase the expression of DNA damage maker H2AX protein in a concentration-dependent manner, proving that compound RD6 could enhance its ability to induce DNA damage with increasing concentration. Figure 8 shown.

[0200] Western Blot (WB) was used to test the RNF4 degradation and DNA induction functions of the compounds. Specific steps:

[0201] Western blotting protein sample preparation:

[0202] 1. Take 5x 10^ 5 Each well was inoculated into a six-well plate, and DMEM complete medium was added to make up the volume to 2 mL;

[0203] 2. After the cells have attached overnight, remove the supernatant medium and add 2 mL of compound diluted in a concentration gradient (dissolve the compound powder in DMSO to prepare a 20 mM stock solution, take 1 μL of the stock solution and add it to 4 mL of DMEM complete medium to dilute the concentration to 5 μM, and then dilute it in a gradient to 2.5 / 1.25 μM).

[0204] 3. Place in a CO2 incubator and incubate at 37°C for 6 hours.

[0205] 4. After the drug incubation time is over, remove the supernatant medium and add 1 mL of PBS to each well to wash the cells twice;

[0206] 5. Add 500 μL of 0.25% trypsin to each well to digest the cells and incubate at 37°C in a CO2 incubator for 2 minutes;

[0207] 6. After incubation, add 500 μL DMEM complete medium to each well for neutralization and digestion;

[0208] 7. Place the incubated cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0209] 8. Add 1 mL of PBS to each tube to resuspend the cells and centrifuge at 1000 rpm for 5 minutes;

[0210] 9. Remove the RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors for later use.

[0211] 10. Add about 200 μL of RIPA lysis buffer containing protease inhibitors to the digested cell pellet.

[0212] 11. After lysis on ice for 30 minutes, gently pipette up and down several times and transfer the cell lysate to a 1.5 mL EP tube.

[0213] 12. Centrifuge at 12000 rpm and 4°C for 15 minutes, transfer the supernatant to a new 1.5 mL EP tube, and store it in a -20°C refrigerator until use.

[0214] Protein quantification:

[0215] The protein quantification method used in the present invention is the BCA method, using the BCA protein quantification kit produced by Shanghai Biyuntian Biotechnology Co., Ltd. The specific experimental steps are as follows:

[0216] 1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μl, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively;

[0217] 2. Add 20 μL of sample to the sample wells of a 96-well plate;

[0218] 3. Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes.

[0219] 4. Measure the absorbance at A562 or other wavelengths between 540-595 nm using a microplate reader;

[0220] 5. Calculate the protein concentration of the sample based on the standard curve.

[0221] SDS-PAGE gel preparation

[0222] 1. Preparation of lower gel: Take a 50mL centrifuge tube and use a pipette to add 4mL each of lower gel solution and lower gel buffer and mix well;

[0223] 2. Add 80ul of coagulant APS;

[0224] 3. Pour into the 1.50mm board to about 70% height;

[0225] 4. Use a pipetting gun to add anhydrous ethanol to make the upper edge flat and solidify (pour out the anhydrous ethanol and discard the remaining solution in the centrifuge tube after it solidifies);

[0226] 5. Preparation of upper gel: Take a plastic cup and add 1 mL each of upper gel solution and upper gel buffer, mix well;

[0227] 6. Add 20uL of coagulant APS;

[0228] 7. Use a dosing gun to fill the glass plate with the upper layer of glue;

[0229] 8. Add the comb (pay attention to the scale on the comb) and wait for it to solidify before use.

[0230] Electrophoresis: Add samples to the electrophoresis tank; run at 90V for 20 minutes until the samples run out of the stacking gel, then change the voltage to 140V and run until the wires are reached (15 μg of protein sample each);

[0231] Transfer: Take out the SDS-PAGE gel after electrophoresis from the electrophoresis tank and place it in primary water (the electrophoresis solution in the tank is recovered), take out the NC membrane and place it in NC membrane buffer;

[0232] Place the membrane in the transfer apparatus in the order of + sponge-NC membrane-SDS-PAGE gel-sponge for transfer;

[0233] Dyeing: Dye with Ponceau red (Ponceau red is recycled after use), and then rinse in pure water;

[0234] Membrane cutting: Cut the NC membrane stained with Ponceau red according to the requirements (TUBULIN: 55; γH2AX: 15kd), remove the required bands and place them in PBST to wash out the Ponceau red staining solution;

[0235] Blocking: Weigh 1 g of skim milk powder and dissolve it in 20 mL of PBST (5%). Block with milk and shake on a shaker at 40 rpm for 20 min. Wash three times with PBST and shake on a shaker at 40 rpm for 7 min.

[0236] Primary antibody incubation: Incubate overnight at 4°C on a shaker at 40 rpm. Recover the primary antibody and wash three times with PBST on a shaker at 40 rpm for 7 min.

[0237] Secondary antibody incubation: Incubate on a shaker at 40 rpm for 45 min, then wash three times with PBST at 40 rpm for 7 min (secondary antibody = 6 mL milk: 0.6 uL antibody: milk = 1:10,000; GAPDH mouse antibody 6 mL, rabbit antibody 6 mL)

[0238] Exposure and development: After evenly incubating the NC membrane with the ultrasensitive ECL developer, the protein bands are exposed and detected using a developer.

[0239] Example 10. Verification of the anti-tumor function of compound RD6 in vivo

[0240] A Huh7 tumor xenograft model was constructed in wild-type nude mice, and compound RD6 (20 mg / kg or 5 mg / kg, intraperitoneal injection, once every two days) was used for single-drug treatment. After 3 weeks of administration, the tumor size of the RD6-treated group was significantly reduced compared to the control group. At the same time, there was no significant change in body weight in mice treated with compound RD6, indicating the safety of this treatment regimen. After the administration, we removed the tumor tissue and weighed it, and found that the tumor mass of the RD6-treated group was also significantly reduced compared to the control group. The results are as follows Figure 9 shown.

[0241] Validation of the mouse subcutaneous liver cancer xenograft model. The specific experimental steps are as follows:

[0242] Nude mice were inoculated with Huh7 liver cancer cells:

[0243] Materials and equipment preparation: 1 mL syringe, Huh7 single cell suspension, cotton balls, and alcohol.

[0244] 1. Use serum-free DMEM medium to resuspend the Huh7 cells obtained by digestion into a single cell suspension (5x10^ 7 / mL) for later use;

[0245] 2. Draw an appropriate amount of Huh7 single cell suspension into the syringe and insert the needle (the needle cut is facing the scale and the air in the needle is expelled before injection);

[0246] 3. Take the nude mouse out of the cage, pinch one ear and turn it over, pinch the front and back limbs of the mouse with the ring finger and pinky finger, and disinfect the injection site with an alcohol cotton ball;

[0247] 4. Inject 100 μL of single cell suspension into the subcutaneous area of ​​the mouse shoulder blade through the needle.

[0248] 5. Remove the needle and use a dry cotton ball to push the injection liquid from bottom to top in time to avoid leakage;

[0249] 6. Then administer the drug according to the experimental requirements, and monitor the weight and tumor size of the mice every other day.

[0250] After the experiment, the mice were killed, and the tumor tissues were removed with scissors and forceps, and photographed and recorded.

[0251] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A proteolysis-targeting chimera, characterized by: The proteolysis targeting chimera is RD4, RD5, RD6 or RD7, The structure of the proteolysis targeting chimera consists of the RNF4 warhead R1, the VHL ligand, and a linker; The structure of the RNF4 warhead R1 is as follows: The VHL ligand structure is as follows: The synthetic route of described RD4 is as follows: In the synthetic route of RD4, RD5-RD7 can be obtained by replacing the linker of RD4 in the first step; The linker of RD5 is The linker of RD6 is The linker of RD7 is

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