SPOP multivalent inhibitor as well as preparation method and application thereof

Abstract

The invention discloses an SPOP multivalent inhibitor and a preparation method and application thereof.The inhibitor is a linear polypeptide polymer formed by polypeptide polymerization, the SPOP multivalent inhibitor has a large molecular structure and higher specificity, specific protein or protein complexes can be targeted more accurately, binding to non-target spots is reduced, and the SPOP multivalent inhibitor has the advantages that the SPOP multivalent inhibitor can be used for preparing the SPOP multivalent inhibitor; and the multivalent inhibitor can interact with the target through a plurality of binding sites, so that the affinity and the stability with the target can be increased, the effect and the durability of the drug can be improved, a new strategy is expected to be provided for tumor treatment, and particularly, the multivalent inhibitor can be used for treating tumors which are insensitive or difficult to treat in the existing therapy. Therefore, the traditional Chinese medicine composition has a promising application prospect.

Description

Technical Field

[0001] The invention relates to a SPOP multivalent inhibitor and also to a preparation method and application of the inhibitor. Background Art

[0002] SPOP protein, as an adaptor protein of E3 ubiquitin ligase, has specific domains including MATH domains, which are essential for its binding to substrate proteins and the performance of biological functions. Under normal physiological conditions, SPOP is involved in regulating a variety of cellular activities, mainly involving two basic cellular processes: genome modification and cell signal transduction. Specifically, in terms of gene modification, SPOP is involved in regulating alternative splicing, DNA damage repair response, and the persistent inactivation of X chromosomes in female species; in terms of cell signal transduction, the Cul3 / SPOP complex regulates the functions of a variety of proteins through ubiquitination pathways, affecting physiological processes such as cell proliferation, apoptosis, and migration.

[0003] In most tumor types, SPOP plays the role of a tumor suppressor gene. For example, in prostate cancer, it degrades key oncogene proteins including androgen receptor (AR), steroid receptor coactivator 3 (SRC3), TRIM24 and DEK through ubiquitination to maintain the balance of intracellular signaling pathways and inhibit the occurrence and development of tumors. However, in a few tumors such as renal cancer, SPOP exhibits the characteristics of an oncogene, which may be related to its abnormal expression, localization or mutation in tumor cells. For example, in clear cell renal carcinoma, the SPOP protein is mistakenly localized from the nucleus to the cytoplasm, and the activation of its upstream regulatory factors leads to its overexpression, thereby promoting the formation of renal cancer. In prostate cancer, the mutation rate of the SPOP gene is high. About 10%-15% of prostate cancer patients have SPOP gene mutations. These mutations are mainly concentrated in the MATH domain that binds to the substrate, which destroys the interaction between SPOP and the substrate, resulting in its loss of function, and then leads to abnormal accumulation of key proteins, promoting the development of prostate cancer.

[0004] Given the important role of SPOP in tumorigenesis and development, as well as its specific expression and function in different tumors, SPOP has become a promising target for tumor therapy. 50The SPOP inhibitors with a Kd value of 2.1 μM and the SPOP small molecule inhibitor 221C7 with a novel β-lactam structure as the core have poor antiproliferative activity in renal cancer cell lines; SPOP peptide inhibitors, such as peptide-38Ac-EVSIIQGADSTT-NH2, have an affinity Kd value of 5.6±0.08 μM with the SPOP-MATH domain. In summary, the current SPOP small molecule inhibitors and peptide inhibitors are generally low in activity, all at the micromolar level, and new SPOP activity inhibitors are urgently needed to be developed. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a highly active multivalent SPOP inhibitor, and also to provide a method for preparing the above inhibitor and its use in the preparation of anti-tumor drugs.

[0006] Technical solution: The present invention discloses a SPOP multivalent inhibitor, which is a linear polypeptide polymer formed by polypeptide polymerization, and the structure is shown in Formula I:

[0007]

[0008] Wherein, n=10-30; Petide is selected from YEADSTT-NH2, YEADSTTRRRR-NH2, wherein Y is tyrosine, E is glutamic acid, A is alanine, D is aspartic acid, S is serine, T is threonine, R is arginine, T-NH2 means the carboxyl end of threonine is blocked by NH2, and R-NH2 means the carboxyl end of arginine is blocked by NH2.

[0009] Wherein, the structural formula of YEADSTT-NH2 is as follows:

[0010]

[0011] The YEADSTTRRRR-NH2 structural formula is as follows:

[0012]

[0013] Wherein, the inhibitor, n is preferably 10 or 30; preferably has the following structure:

[0014]

[0015]

[0016] The preparation method of the above-mentioned SPOP multivalent inhibitor comprises the following steps: dissolving a norbornene derivative of the polypeptide in water, adding a tetrahydrofuran solution of a catalyst under nitrogen protection, reacting at room temperature, adding vinyl ethyl ether to the reaction solution for reaction, and dialyzing to obtain the SPOP multivalent inhibitor shown in formula I.

[0017] The invention is characterized in that the molar mass ratio of the catalyst to the norbornene derivative of the polypeptide is 1 / n; wherein the structural formula of the norbornene derivative of YEADSTT-NH2 is as follows:

[0018]

[0019] The structural formula of the norbornene derivative of YEADSTTRRRR-NH2 is as follows:

[0020]

[0021] The present invention also discloses a pharmaceutical composition, comprising the compound shown in formula I and a pharmaceutically acceptable carrier.

[0022] The above-mentioned SPOP multivalent inhibitor can also be used in the preparation of anti-tumor drugs.

[0023] The application is that the inhibitor intervenes in the binding of SPOP to substrate protein and reduces the ubiquitination level of substrate protein, inhibits the phosphorylation level of oncogene pathway protein, and thus inhibits the proliferation of renal cancer cells.

[0024] Wherein, the tumor is gastric cancer, breast cancer, ovarian cancer or kidney cancer.

[0025] Principle of the invention: The SPOP multivalent inhibitor of the present invention is a protein-like polymer: a high-density brush-like polymer formed by ring-opening metathesis polymerization of norbornene-peptide monomers. In the structure, the polymer backbone is wrapped in the peptide side chain to form a spherical structure, which can maintain biological activity while preventing protein hydrolysis. Each molecule of the multivalent inhibitor contains N polypeptide side chains, and each polypeptide side chain can interact with the target. This feature can increase the affinity and stability with the target, improve the effect and durability of the drug, and overcome the problem of low activity caused by existing SPOP small molecule and polypeptide inhibitors.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The activity of the SPOP multivalent inhibitor of the present invention can reach the hundred nanomolar level, the activity is greatly improved, and the side effects are low, and the efficacy and durability are significantly improved; (2) The preparation method is relatively simple, the conditions are mild and the reaction is rapid, and the target compound can be obtained by reacting for only two hours at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the cell viability experimental curve of SPOP multivalent inhibitor compound 1 and monovalent inhibitor of the present invention;

[0028] Figure 2 This is a staining diagram of the cell anti-proliferation experiment of SPOP multivalent inhibitor compound 1 and monovalent inhibitor of the present invention;

[0029] Figure 3 It is the non-denaturing electrophoresis diagram of SPOP multivalent inhibitor compound 1 and monovalent inhibitor of the present invention;

[0030] Figure 4 The protein immunoblot images of the SPOP pathway by the SPOP multivalent inhibitor compound 1 (A) and the monovalent inhibitor (B) of the present invention;

[0031] Figure 5 Tumor volume (A) and mouse body weight curve (B) of mice in different groups;

[0032] Figure 6 Figure 2 shows the tumor weights of mice in different groups after being given different doses.

[0033] Figure 7 In vivo tumor imaging (A) and in vitro tumor imaging (B) after different groups of mice were given different doses of the drug.

[0034] Figure 8 These are the ITC experimental curves of compounds 1 to 4 and monovalent inhibitors prepared in the examples; wherein, Figure A is the monovalent inhibitor YEADSTTRRRR-NH2, Figure B is compound 1, Figure C is compound 2, Figure D is compound 3, and Figure E is compound 4. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0036] Example 1

[0037] The SPOP multivalent inhibitor of the present invention, wherein compound 1 is:

[0038]

[0039] Compound 1 was prepared according to the following route:

[0040]

[0041] The norbornene derivative of the peptide YEADSTTRRRR-NH2 (112 mg, 0.067 mmoL, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.8 mL of a tetrahydrofuran solution of the catalyst (7.5 mg / mL, 0.1 eq) was added and reacted at room temperature for 1 h. 0.5 mL of vinyl ethyl ether was added to the reaction solution and the reaction was stopped after being placed in the air for 1 h. The reaction solution was dialyzed in pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD, and the residual liquid was lyophilized to obtain compound 1 (31.5 mg, 28.12%), which was an off-white flocculent solid; compound 1 was recorded as a multivalent inhibitor with N=10.

[0042] Example 2

[0043] The SPOP multivalent inhibitor of the present invention, wherein compound 2 is:

[0044]

[0045] The norbornene derivative (84.9 mg, 0.051 mmol, 1 eq) of the peptide YEADSTTRRRR-NH2 was dissolved in 5 mL of water. Under nitrogen protection, 0.2 mL of a tetrahydrofuran solution of the catalyst (7.5 mg / mL, 0.03 eq) was added and reacted at room temperature for 1 h. 0.5 mL of vinyl ethyl ether was added to the reaction solution and the reaction was stopped after being placed in the air for 1 h. The reaction solution was dialyzed in pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD, and the residual liquid was lyophilized to obtain compound 2 (21.6 mg, 25.44%), which was a white flocculent solid.

[0046] Example 3

[0047] The SPOP multivalent inhibitor of the present invention, wherein compound 3 is:

[0048]

[0049] The norbornene derivative of the peptide YEADSTT-NH2 (49.15 mg, 0.049 mmol, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.5 mL of tetrahydrofuran solution of the catalyst (7.5 mg / mL, 0.004 eq) was added and reacted at room temperature for 1 h. 0.5 mL of vinyl ethyl ether was added to the reaction solution and the reaction was stopped after being placed in the air for 1 h. The reaction solution was dialyzed in pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD, and the residual liquid was lyophilized to obtain compound 2 (15.6 mg, 31.74%), and compound 3 was a white flocculent solid.

[0050] Example 4

[0051] The SPOP multivalent inhibitor of the present invention, wherein compound 4 is:

[0052]

[0053] The norbornene derivative of the peptide YEADSTT-NH2 (49.15 mg, 0.049 mmol, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.16 mL of a tetrahydrofuran solution of the catalyst (7.5 mg / mL, 0.0016 eq) was added and reacted at room temperature for 1 h. 0.5 mL of vinyl ethyl ether was added to the reaction solution, and the reaction was stopped after being placed in the air for 1 h. The reaction solution was dialyzed in pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD, and the residual liquid was lyophilized to obtain compound 2 (14.3 mg, 29.09%), and compound 3 was a white flocculent solid.

[0054] Example 5

[0055] For cell culture:

[0056] (1) Cell recovery: Start a constant temperature water bath and maintain the water temperature at around 37°C; remove the cells from the liquid nitrogen tank and quickly transfer them to a 37°C constant temperature water bath; shake the cryotubes to accelerate thawing; remove the cells quickly after all the ice crystals have melted, wipe with alcohol and transfer to a clean bench; transfer the cells to a complete culture medium containing 10% fetal bovine serum (FBS), centrifuge at room temperature at 1000 rpm for 5 min, discard the supernatant, add 1 mL of fresh culture medium, pipette and mix the cells, then add the cell suspension to a culture medium containing 10% PBS, and place the culture dish in a 37°C constant temperature incubator containing 5% CO2 for static culture.

[0057] (2) Cell passaging: The culture conditions for each cell type are different. Cells should be cultured in the required culture medium as needed. After the cells have grown fully, the supernatant should be discarded. The cells should be rinsed twice with 1× PBS, and then 1 mL of trypsin should be added for digestion. When the cells become round and bright and a small number of cells begin to float under the microscope, the culture medium containing 10% FBS can be added to terminate the digestion. The cells should be collected in a 15 mL centrifuge tube and centrifuged at room temperature at 1000 rpm for 5 min. The supernatant should be discarded, and 1 mL of fresh culture medium should be added for subculture at a subculture ratio of 1:2.

[0058] (3) Cryopreservation of cells: prepare a cryopreservation medium containing 90% FBS and 10% DMSO; digest the cells with trypsin and collect them into a 15 mL centrifuge tube, centrifuge at room temperature at 1000 rpm for 5 min, discard the supernatant and add the cryopreservation medium to the cell pellet. The final density of the cryopreserved cells is approximately 5×106 cells / mL to 1×107 cells / mL; divide the cells into cryopreservation tubes, label the cell name and date, place them in a cryopreservation box, place them in a -80°C refrigerator to slowly cool overnight, and then move them to a liquid nitrogen tank for long-term storage.

[0059] Example 6

[0060] For the SPOP multivalent inhibitor of the present invention, a cell viability experiment was performed:

[0061] (1) Operation steps of Cell Counting Kit-8 (CCK-8):

[0062] Prepare cell suspension and count cells.

[0063] Inoculate the cell suspension in a 96-well plate, about 100 μl per well, and repeat the same sample three times. Place the culture plate in an incubator for a period of time (37°C, 5% CO2), and it takes about 2-4 hours for the cells to adhere to the wall.

[0064] Add 10 μl of CCK-8 solution to each well and gently shake the plate to help mix. Note that bubbles should be avoided during the addition process.

[0065] Place the culture plate in the incubator and incubate for 4 h.

[0066] The absorbance (OD) at 450 nm was measured by an enzyme-labeled instrument.

[0067] (2) The vitality calculation formula is as follows:

[0068] Cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100%

[0069] A (drug added): OD value of the wells with cells, CCK-8 solution and drug solution

[0070] A (0 drug addition): OD value of the well with cells, CCK-8 solution but no drug solution

[0071] A (blank): OD value of the well without cells

[0072] (3) Cell viability test steps:

[0073] Renal cancer cell A498 was cultured and seeded in a 96-well plate at 4000-5000 cells per well. After the cells adhered to the wall, the monovalent inhibitor, i.e., YEADSTTRRRR-NH2, and the multivalent inhibitor compound 1 of the present invention were administered at two-fold dilutions at the highest concentration of 200 μM and 20 μM, respectively. After 96 hours of action, the cell viability was tested using the CCK-8 method. The measured data was calculated using the cell viability calculation method, and the processed data was plotted using GraphPad Prism7. The fitting calculation results were as follows: Figure 1 The calculation data parameters are shown in Table 1.

[0074] Table 1. Cell viability test results

[0075]

[0076] The results showed that compound 1 was significantly more active than the monomer compound, and the activity was increased by more than ten times.

[0077] Example 7

[0078] For the SPOP multivalent inhibitor of the present invention, a cell anti-proliferation experiment was performed:

[0079] The experimental operation steps are as follows: A498 (MEM culture medium supplemented with 10% PBS, 1% double antibody) cells are seeded at a density of 1000 cells / well in a six-well plate, and different concentrations of compounds are added after adhesion. The cells are cultured at 37°C and 5% CO2. The monovalent compound YEADSTTRRRR-NH2 and the multivalent inhibitor compound 1 of the present invention are administered for 12 hours, and the culture medium is replaced every 3 days. After incubation for 8-12 days, the cell culture medium is removed after cell colonies are formed, and the cells are washed 3 times with 1x PBS, and then fixed with 4% paraformaldehyde fixative for more than 30 minutes, the fixative is washed off, and 1% crystal violet is stained for 10 minutes. The staining solution is poured off, and the cells are rinsed with water and dried before taking pictures. The experimental results are as follows Figure 2 .

[0080] from Figure 2 The data showed that compound 1 exhibited a significant inhibitory effect on A498 renal cancer cells at a concentration of 2.5μM, and the reduction in the number of cells was identifiable with the naked eye; when the concentration was increased to 5μM, the number of cells dropped sharply. In contrast, even at a concentration of up to 50μM, the inhibitory effect of the monomer compound on cells was still minimal. Combining these observations, it can be concluded that compound 1 has excellent efficacy in inhibiting the proliferation of A498 renal cancer cells, and its ability to inhibit cell proliferation is significantly enhanced compared to the monomer compound.

[0081] Example 8

[0082] A non-denaturing electrophoresis experiment was performed for the SPOP multivalent inhibitor of the present invention:

[0083] The monovalent inhibitor, YEADSTTRRRR-NH2 and compound 1, were diluted 2X from 200 μM and 20 μM, respectively, and incubated with an equal volume of SPOP full-length protein (protein concentration of 80 μM) on ice. The samples were run on gels and stained with Coomassie blue for 30 min before decolorization. The results are shown in Figure 2. Figure 3 .

[0084] The results showed that compared with monovalent inhibitors, the multimeric inhibitor compound 1 can significantly destroy the dimeric structure of SPOP, causing the full-length SPOP protein to transform into a highly polymerized state. This transformation not only affects the function of SPOP, but may also change its interactions and stability in cells, thereby having a profound impact on related signaling pathways. Through this mechanism, multimeric inhibitors may show stronger effects in the treatment of diseases related to SPOP.

[0085] Example 9

[0086] Western blotting experiments were performed on the SPOP multivalent inhibitors of the present invention:

[0087] The immunoblotting experiment was performed as follows:

[0088] First, A498 cells were mixed and washed with 1*PBS (Gibco, 10010023), aspirated, and weak RIPA lysis buffer (100 μL) (Biyuntian, P0013) was added to lyse the cells. The lysis time was 30 minutes, and centrifuged at 13000rpm and 4°C for 15 minutes. 80 μL of supernatant was taken and 20 μL of loading buffer (Biyuntian, P0015) was added, and heated at 100°C for 10 minutes, cooled on ice, and 10 μL was loaded onto a 10% SDS-PAGE gel. The gel was run at a constant voltage of 60V on a Bio-Rad protein electrophoresis instrument until the marker was separated, and then switched to 120V and ran to the bottom. After electrophoresis, the gel was removed and a PVDF membrane of appropriate size was cut according to the size. The order from white board to black board was filter paper, PVDF membrane, gel, filter paper, and black gauze. The proteins were transferred to PVDF membrane (PerkinElmer, Northwalk, CT, USA) using wet transfer for 2 h.

[0089] After completion, immerse the PVDF membrane in the Ponceau staining solution for 2 to 5 minutes, rinse with distilled water, cut the target band, and wash off the Ponceau with 1*TBST (Biyuntian, ST671). Block with blocking buffer (1*TBST containing 5% w / v skim milk powder) at 37°C for 1 hour. After blocking, wash three times with 15ml 1*TBST, 5 minutes each time. Place the primary antibody in 10mL primary antibody dilution buffer (1*TBST containing 5% skim milk powder, to prepare 20ml, add 1.0g skim milk powder to 20ml 1*TBST, and then mix thoroughly) according to the appropriate dilution recommended in the product manual, incubate overnight at 4°C and shake gently from time to time. After completion, wash three times with 1*TBST, 10 minutes each time. Then incubate the membrane with the color developing solution (SignalFireTMECLReagent#6883) at room temperature for 1 minute, pour off the excess solution (keep the membrane wet) and expose; the results are as follows Figure 4 .

[0090] The results showed that PTEN is a tumor suppressor factor that regulates cell proliferation and apoptosis in ccRCC through the SPOP signaling pathway in the cytoplasm. Previous articles have shown that SPOP inhibitors at the biochemical level can inhibit the activity of SPOP protein in cells to stabilize the content of substrate protein PTEN to achieve therapeutic effects. The increase in PTEN protein content inhibits the AKT-driven cancer signaling pathway.

[0091] Western Bolt experiments were performed to verify this view. The results showed that after A498 cells were treated with compound 1 and the monovalent inhibitor YEADSTTRRRR-NH2 for 12 hours, PTEN protein accumulated stably in a concentration-dependent manner. The accumulation of PTEN in the cytoplasm led to a decrease in p-AKT in A498 cells. The total abundance of AKT protein in A498 cells remained unchanged. The results show that compound 1 can interfere with the binding of SPOP to substrate proteins and reduce the ubiquitination level of substrate proteins, inhibit the phosphorylation level of oncogene pathway proteins, and thus inhibit the proliferation of renal cancer cells.

[0092] Example 10

[0093] A mouse xenograft tumor transplantation experiment was conducted for the SPOP multivalent inhibitor of the present invention:

[0094] Experimental steps:

[0095] (1) Mouse modeling: 6-week-old female BALB / c nude mice were prepared, and 8×106 A498-Luc cells mixed with Matrigel (Corning) were injected into the right armpit of the mice. About 100 μL was injected into each mouse. After subcutaneous injection, the mice were cultured for about 6-8 weeks until tumors formed.

[0096] (2) Grouping: The mice were randomly divided into 4 groups according to their body weight, with 6 mice in each group: blank control group (normal saline), YEADSTTRRRR-NH2 (10 mg / Kg), low-dose group of compound 1 (2 mg / Kg) and high-dose group of compound 1 (10 mg / Kg).

[0097] (3) Administration: Each group had 6 mice. The mice were injected intratumorally every other day. The body weight and tumor size of the mice were measured before each administration. The mice were administered at 10:28 every day. After three weeks, the mice were observed using a non-invasive in vivo imaging system (IVIS), and tumor tissues and organs were dissected and removed.

[0098] (4) Result processing: Plot the previously recorded and collected data to obtain Figure 5 .

[0099] The results showed that during the 3-week dosing period, the body weight of the experimental mice did not fluctuate significantly. This observation indicated that compound 1 had little effect on the overall health and physiological function of the mice and showed good tolerance. By comparing the tumor growth curve, compound 1 showed significant superiority in inhibiting tumor volume growth compared with the monovalent compound YEADSTTRRRR-NH2. This inhibitory effect was particularly prominent in the high-concentration dose group, which may be attributed to the direct inhibitory effect of compound 1 on tumor cell proliferation or its regulatory effect on the tumor microenvironment.

[0100] After the mice were dissected, the tumor volume of the compound 1-treated group was significantly smaller than that of the control group. This result was consistent with the monitoring results of IVIS (in vivo imaging system), further confirming the effectiveness of compound 1 in inhibiting tumor growth. Therefore, the SPOP multivalent inhibitor of the present invention has strong activity and low side effects, and its efficacy and durability are significantly improved; it has shown significant potential in inhibiting tumor growth, and has shown positive results in terms of safety and effectiveness, and has great application prospects.

[0101] Embodiment 11

[0102] For the SPOP multivalent inhibitor of the present invention, an ITC (isothermal titration calorimetry) experiment was performed:

[0103] The ITC experiment uses the MicroCal ITC 200 instrument to measure the binding affinity between proteins and ligands. The ITC experiment can fit and quantitatively calculate the binding thermodynamic parameters, including the number of binding sites (n), binding constant (Ka), reaction enthalpy change (ΔH), entropy change (ΔS), etc., by detecting the heat change (endothermic or exothermic) when the protein solution and the small molecule (ligand) solution interact.

[0104] Experimental steps:

[0105] Through extraction, dialysis and ultrafiltration, high-concentration and high-purity SPOP protein was obtained. During the dialysis process, the buffer consistent with the protein component system was retained as much as possible. First, 400 μL SPOP protein (1 μM) and 100 μL compound (10 μM) were prepared, and the DMSO content in the protein and compound needed to be consistent. Then, an isothermal titration calorimeter (MicroCaliTC200) was used, and the operating parameters were set: temperature 25°C; speed 750 rpm. The sample pool and titration needle were cleaned, and about 280 μL of SPOP protein was slowly added to the sample pool. About 40 μL of compound was inhaled into the titration needle, and 2 μL was dripped into the sample pool at an interval of 2.5 minutes per drop, for a total of 19 drops. After the experiment, MicroCal.Origin version 7.0 software was used for data processing and analysis to obtain the entropy change ΔS, enthalpy change ΔH and free energy ΔG change of the compound binding to the SPOP protein, and then the KA value and KD value (KD = 1 / KA) were obtained. The smaller the KD value, the better the binding strength and selectivity of the compound to the protein.

[0106] The binding activity of compounds 1 to 4 to SPOP protein was determined, as shown in Table 2 and Figure 8 shown.

[0107] Table 2. Binding activity of compounds to SPOP protein

[0108]

[0109] The results showed that compared with the monovalent compound YEADSTTRRRR-NH2, compounds 1, 2 and 3 showed good binding affinity with SPOP protein, their binding activities were greatly improved, and their activities were much higher than those of the currently published peptide inhibitors.

[0110] Therefore, the SPOP multivalent inhibitor of the present invention has a larger molecular structure and higher specificity, and can more accurately target specific proteins or protein complexes, reduce binding to non-target sites, and thus reduce side effects. In addition, the multivalent inhibitor can interact with the target through multiple binding sites, thereby increasing the affinity and stability with the target, improving the effect and durability of the drug, and has great application prospects.

Claims

1. A SPOP multivalent inhibitor, characterized in that The inhibitor is a linear polypeptide polymer formed by polypeptide polymerization, and its structure is shown in Formula I: Wherein, n=10-30; Petide is selected from YEADSTT-NH2 or YEADSTTRRRR-NH2, wherein Y is tyrosine, E is glutamic acid, A is alanine, D is aspartic acid, S is serine, T is threonine, and R is arginine, T-NH2 means that the carboxyl end of threonine is blocked with NH2, and R-NH2 means that the carboxyl end of arginine is blocked with NH2.

2. The inhibitor according to claim 1, characterized in that The YEADSTT-NH2 structural formula is as follows: The YEADSTTRRRR-NH2 structural formula is as follows:

3. The inhibitor according to claim 1, characterized in that For the inhibitor, n is 10 or 30.

4. A method for preparing the SPOP multivalent inhibitor according to claim 1, characterized in that: The following steps are involved: The norbornene derivative of the polypeptide is dissolved in water, and a tetrahydrofuran solution of the catalyst is added under nitrogen protection, reacted at room temperature, vinyl ethyl ether is added to the reaction solution for reaction, and dialyzed to obtain the SPOP multivalent inhibitor shown in formula I.

5. The preparation method according to claim 4, characterized in that: The norbornene derivative of the polypeptide is a norbornene derivative of YEADSTT-NH2 or a norbornene derivative of YEADSTTRRRR-NH2; wherein the norbornene derivative of YEADSTT-NH2 has the following structural formula: The structural formula of the norbornene derivative of YEADSTTRRRR-NH2 is as follows:

6. A pharmaceutical composition, characterized in that Comprising the compound shown in formula I of claim 1 and a pharmaceutically acceptable carrier.

7. Use of the SPOP multivalent inhibitor according to claim 1 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The application is that the inhibitor intervenes in the binding of SPOP and substrate protein and reduces the ubiquitination level of the substrate protein, inhibits the phosphorylation level of oncogene pathway protein, and thus inhibits the proliferation of renal cancer cells.

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