A SPOP multivalent inhibitor, its preparation method and application

Abstract

This invention discloses a SPOP multivalent inhibitor, its preparation method, and its application. The inhibitor is a linear polypeptide polymer formed by polypeptide polymerization. The SPOP multivalent inhibitor of this invention has a larger molecular structure and higher specificity, enabling it to more accurately target specific proteins or protein complexes, reduce binding to non-target sites, and thus reduce side effects. Furthermore, the multivalent inhibitor can interact with the target through multiple binding sites, which can increase the affinity and stability with the target, improve the efficacy and durability of the drug, and is expected to provide a new strategy for tumor treatment, especially for tumors that are insensitive to or difficult to treat with existing therapies, such as gastric cancer, breast cancer, ovarian cancer, and kidney cancer, which has great application potential.

Description

Technical Field

[0001] This invention relates to a SPOP multivalent inhibitor, as well as a method for preparing the above-mentioned inhibitor and its application. Background Technology

[0002] SPOP proteins, as adaptor proteins of E3 ubiquitin ligases, possess specific domains, including the MATH domain, which are crucial for their binding to substrate proteins and the performance of their biological functions. Under normal physiological conditions, SPOPs participate in the regulation of various cellular activities, primarily involving two fundamental cellular processes: genome modification and cell signal transduction. Specifically, in terms of gene modification, SPOPs are involved in regulating alternative splicing, DNA damage repair responses, and the persistent inactivation of the X chromosome in female species; while in terms of cell signal transduction, the Cul3 / SPOP complex regulates the function of various proteins through the ubiquitination pathway, influencing 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 maintains the balance of intracellular signaling pathways and inhibits tumor development and progression by ubiquitinizing and degrading key oncogene proteins, including the androgen receptor (AR), steroid receptor coactivator 3 (SRC3), TRIM24, and DEK. However, in a few tumors, such as renal cell carcinoma, SPOP exhibits oncogene characteristics, which may be related to its abnormal expression, localization, or mutation in tumor cells. For instance, in clear cell renal cell carcinoma, SPOP protein is mislocalized from the nucleus to the cytoplasm, and activation of its upstream regulators leads to its overexpression, thereby promoting renal cell carcinoma formation. In prostate cancer, the mutation rate of the SPOP gene is high, with approximately 10%-15% of prostate cancer patients having SPOP gene mutations. These mutations are mainly concentrated in the substrate-binding MATH domain, disrupting the interaction between SPOP and its substrate, leading to loss of function, and consequently, abnormal accumulation of key proteins, promoting the development of prostate cancer.

[0004] Given the important role of SPOP in tumorigenesis and development, and its specific expression and function in different tumors, SPOP has become a highly promising target for tumor therapy, for example, A498 anti-proliferative IC50. 50Compounds with a molecular weight of 2.1 μM, such as the SPOP small molecule inhibitor 221C7 with a novel β-lactam structure as its core, exhibit poor anti-proliferative activity in renal cell carcinoma lines. Regarding SPOP peptide inhibitors, for example, peptide-38Ac-EVSIIQGADSTT-NH2 has an affinity (Kd) of 5.6 ± 0.08 μM for the SPOP-MATH domain. In summary, existing SPOP small molecule and peptide inhibitors generally exhibit low activity, mostly at the micromolar level, necessitating the development of novel SPOP activity inhibitors. Summary of the Invention

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

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

[0007]

[0008] Wherein, n = 10 to 30; Petide is selected from YEADSTT-NH2 and YEADSTTRRRR-NH2, where 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 is threonine with the carboxyl terminus blocked by NH2, and R-NH2 is arginine with the carboxyl terminus blocked by NH2.

[0009] The YEADSTT-NH2 structure is as follows:

[0010]

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

[0012]

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

[0014]

[0015]

[0016] The preparation method of the above-mentioned SPOP multivalent inhibitor includes the following steps: dissolving the norbornene derivative of the peptide in water, adding a tetrahydrofuran solution containing a catalyst under nitrogen protection, reacting at room temperature, adding vinyl ether to the reaction solution to carry out the reaction, and dialysis to obtain the SPOP multivalent inhibitor shown in Formula I.

[0017] The characteristic feature is that the molar mass ratio of the catalyst to the norbornene derivative of the peptide 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 a compound of formula I and a pharmaceutically acceptable carrier.

[0022] The aforementioned SPOP multivalent inhibitors can also be used in the preparation of anti-tumor drugs.

[0023] The application involves inhibiting the binding of SPOP to substrate proteins and reducing the ubiquitination level of substrate proteins, thereby inhibiting the phosphorylation level of oncogene pathway proteins and thus inhibiting the proliferation of renal cancer cells.

[0024] The tumor is either stomach cancer, breast cancer, ovarian cancer, or kidney cancer.

[0025] Invention Principle: The SPOP multivalent inhibitor of this invention is a protein-like polymer: a high-density brush-like polymer formed by the ring-opening metathesis polymerization of norbornene peptide monomers. In this structure, the polymer backbone is encapsulated within peptide side chains, forming a globular structure that maintains 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 increases the affinity and stability with the target, improves the drug's efficacy and persistence, and overcomes the low activity problem of 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 level of hundreds of nanomoles, the activity is greatly improved, and the side effects are low, and the efficacy and persistence 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 at room temperature for only two hours. Attached Figure Description

[0027] Figure 1 Cell viability experimental curves 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 This is a non-denaturing electrophoresis image of SPOP multivalent inhibitor compound 1 and monovalent inhibitor of the present invention;

[0030] Figure 4 This is a Western blot diagram of the SPOP pathway by the multivalent inhibitor compound 1 (A) and the monovalent inhibitor (B) of the present invention.

[0031] Figure 5 Tumor volume (A) and body weight curves (B) for mice in different groups;

[0032] Figure 6 Tumor weight in mice from different groups after administration of different doses of the drug;

[0033] Figure 7 In vivo tumor imaging (A) and in vitro tumor imaging (B) of mice in different groups after administration of different doses;

[0034] Figure 8 The figures show the ITC experimental curves of compounds 1-4 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 Implementation

[0035] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be purchased through conventional means.

[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 polypeptide YEADSTTRRRR-NH2 (112 mg, 0.067 mmol / L, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.8 mL of a tetrahydrofuran solution containing the catalyst (7.5 mg / mL, 0.1 eq) was added, and the reaction was carried out at room temperature for 1 h. 0.5 mL of vinyl ether was added to the reaction solution, and the reaction was stopped after being exposed to air for 1 h. The reaction solution was dialyzed against pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD. The residual liquid was then lyophilized to give compound 1 (31.5 mg, 28.12%), which was a grayish-white flocculent solid. Compound 1 was designated as an N=10 multivalent inhibitor.

[0042] Example 2

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

[0044]

[0045] The norbornene derivative of the polypeptide YEADSTTRRRR-NH2 (84.9 mg, 0.051 mmol / L, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.2 mL of a tetrahydrofuran solution containing the catalyst (7.5 mg / mL, 0.03 eq) was added, and the reaction was carried out at room temperature for 1 h. 0.5 mL of vinyl ether was added to the reaction solution, and the reaction was stopped after being exposed to air for 1 h. The reaction solution was dialyzed against pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD. The residual liquid was then lyophilized to give 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 polypeptide YEADSTT-NH2 (49.15 mg, 0.049 mmol / L, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.5 mL of a tetrahydrofuran solution containing the catalyst (7.5 mg / mL, 0.004 eq) was added, and the reaction was carried out at room temperature for 1 h. 0.5 mL of vinyl ether was added to the reaction solution, and the reaction was stopped after being exposed to air for 1 h. The reaction solution was dialyzed against pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD. The residual liquid was then lyophilized to give 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 polypeptide YEADSTT-NH2 (49.15 mg, 0.049 mmol / L, 1 eq) was dissolved in 5 mL of water. Under nitrogen protection, 0.16 mL of a tetrahydrofuran solution containing the catalyst (7.5 mg / mL, 0.0016 eq) was added, and the reaction was carried out at room temperature for 1 h. 0.5 mL of vinyl ether was added to the reaction solution, and the reaction was stopped after being exposed to air for 1 h. The reaction solution was dialyzed against pure water for 72 h using a dialysis bag with a molecular weight cutoff of 3.5 KD. The residual liquid was then lyophilized to give compound 2 (14.3 mg, 29.09%), and compound 3 was a white flocculent solid.

[0054] Example 5

[0055] Perform cell culture:

[0056] (1) Cell thawing: Start the constant temperature water bath and maintain the water temperature at about 37℃; take the cells out of the liquid nitrogen tank and quickly transfer them to the 37℃ constant temperature water bath. Shake the cryovial to accelerate thawing. After all the ice crystals have melted, take them out quickly, wipe them with alcohol and transfer them to the clean bench at the same time; transfer the cells to the complete culture medium containing 10% fetal bovine serum (FBS), centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 1 mL of fresh culture medium, mix the cells by pipetting, and then add the cell suspension to the culture medium containing 10% PBS. Place the culture dish in a 37℃ constant temperature incubator containing 5% CO2 for static culture.

[0057] (2) Cell passage: The culture conditions for each type of cell are different. The cells are cultured in the required culture medium as needed. After the cells have grown to a confluence, the supernatant is discarded, the cells are washed twice with 1×PBS, and then 1mL of trypsin is added for digestion. Under the microscope, when the cells become round and bright and a small number of cells begin to float, culture medium containing 10% FBS is added to stop the digestion. The cells are collected into a 15mL centrifuge tube and centrifuged at 1000rpm for 5min at room temperature. The supernatant is discarded, and then 1mL of fresh culture medium is added for passage culture at a ratio of 1:2.

[0058] (3) Cell cryopreservation: Prepare cryopreservation culture medium containing 90% FBS and 10% DMSO; after digesting the cells with trypsin, collect them into 15mL centrifuge tubes, centrifuge at 1000rpm for 5min at room temperature, discard the supernatant and add cryopreservation solution to the cell pellet. The final density of cryopreserved cells is approximately 5×106 cells / mL to 1×107 cells / mL; aliquot the cells into cryopreservation tubes, label them with cell name and date, place them in a cryopreservation box, and slowly cool them overnight in a -80℃ freezer, and then transfer them to a liquid nitrogen tank for long-term storage.

[0059] Example 6

[0060] Cell viability experiments were conducted using the SPOP multivalent inhibitor of this invention:

[0061] (1) Operating steps of Cell Counting Kit-8 (CCK-8 for short):

[0062] Prepare a cell suspension and count the cells.

[0063] Seed the cell suspension into 96-well plates, approximately 100 μl per well, with three replicates of the same sample. Pre-incubate the plates in an incubator for a period of time (37°C, 5% CO2), as cell adhesion takes approximately 2-4 hours.

[0064] Add 10 μl of CCK-8 solution to each well. After adding the reagents, gently shake the culture plate to help mix. Note that you should try to avoid generating air bubbles during the sample addition process.

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

[0066] The absorbance (OD) at 450 nm was measured using an ELISA reader.

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

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

[0069] A (Drug Addition): OD values ​​of wells containing cells, CCK-8 solution, and drug solution.

[0070] A (0 drug added): OD value of wells containing cells and CCK-8 solution but no drug solution.

[0071] A (Blank): OD value of pores without cells.

[0072] (3) Cell viability experiment procedure:

[0073] Renal cell carcinoma cells A498 were cultured and seeded into 96-well plates at a density of 4000-5000 cells per well. After cell attachment, the monovalent inhibitor YEADSTTRRRR-NH2 and the multivalent inhibitor compound 1 of this invention were administered at two-fold dilutions of 200 μM and 20 μM, respectively. After 96 hours of treatment, cell viability was tested using the CCK-8 assay. The measured data were calculated using cell viability calculation methods, and the processed data were plotted using GraphPad Prism7. The fitting calculation results are shown below. Figure 1 As shown in Table 1, the calculation data parameters are as follows.

[0074] Table 1. Results of cell viability experiment

[0075]

[0076] The results showed that compound 1 had significantly higher activity than the monomeric compound, with an activity increase of more than tenfold.

[0077] Example 7

[0078] Cell proliferation experiments were conducted against the SPOP multivalent inhibitor of this invention:

[0079] The experimental procedure was as follows: A498 cells (MEM medium supplemented with 10% PBS and 1% antibiotics) were seeded at a density of 1000 cells / well in six-well plates. After adhesion, different concentrations of the compound were added, and the plates were cultured at 37°C and 5% CO2. The monovalent compound YEADSTTRRRR-NH2 and the multivalent inhibitor compound 1 of this invention were administered for 12 hours, and the culture medium was changed every 3 days. After incubation for 8-12 days, once cell colonies formed, the cell culture medium was removed, and the cells were washed three times with 1x PBS. The cells were then fixed with 4% paraformaldehyde fixative for at least 30 minutes, the fixative was washed off, and the cells were stained with 1% crystal violet for 10 minutes. The staining solution was discarded, the cells were rinsed with water, air-dried, and photographed. The experimental results are as follows: Figure 2 .

[0080] from Figure 2 Data showed that compound 1 exhibited a significant inhibitory effect on A498 renal cell carcinoma cells at a concentration of 2.5 μM, with a visible reduction in cell number; when the concentration was increased to 5 μM, the cell number decreased sharply. In contrast, even at concentrations as high as 50 μM, the inhibitory effect of the monomeric compound on cells remained negligible. These observations lead to the conclusion that compound 1 possesses superior efficacy in inhibiting the proliferation of A498 renal cell carcinoma cells, and its ability to inhibit cell proliferation is significantly enhanced compared to the monomeric compound.

[0081] Example 8

[0082] Non-denaturing electrophoresis experiments were performed on the SPOP multivalent inhibitor of this invention:

[0083] The monovalent inhibitor YEADSTTRRRR-NH2 and compound 1 were diluted 2X from 200 μM and 20 μM, respectively, and incubated on ice with an equal volume of full-length SPOP protein (protein concentration of 80 μM). Samples were prepared, run on a gel, stained with Coomassie blue for 30 min, and then destained. The results are as follows: Figure 3 .

[0084] The results showed that, compared to monovalent inhibitors, the polymer inhibitor compound 1 significantly disrupted the dimer structure of SPOP, leading to a high-polymerization transition of the full-length SPOP protein. This transition not only affects SPOP function but may also alter its intracellular interactions and stability, thereby profoundly impacting related signaling pathways. Through this mechanism, polymer inhibitors may exhibit stronger efficacy in treating SPOP-related diseases.

[0085] Example 9

[0086] Western blot experiments were performed on the SPOP multivalent inhibitor of this invention:

[0087] The procedure for immunoblotting is as follows:

[0088] First, wash A498 cells with 1*PBS (Gibco, 10010023), aspirate, and add 100 μL of weak RIPA lysis buffer (Beyotime, P0013) to lyse the cells for 30 minutes. Centrifuge at 13000 rpm and 4°C for 15 minutes, collect 80 μL of supernatant, add 20 μL of loading buffer (Beyotime, P0015), and heat at 100°C for 10 minutes. Cool on ice, and load 10 μL onto a 10% SDS-PAGE gel. Run on a Bio-Rad protein electrophoresis apparatus at a constant voltage of 60V until the markers are separated, then switch to 120V and run to the bottom edge. After electrophoresis, remove the gel, cut PVDF membranes to the appropriate size, and arrange them in the following order from white to black: filter paper, PVDF membrane, gel, filter paper, and black gauze. Wet transfer was used to transfer the protein to a PVDF membrane (PerkinElmer, Northwalk, CT, USA) over a time of 2 hours.

[0089] After completion, immerse the PVDF membrane in Ponceau S staining solution for 2-5 minutes, rinse with distilled water, cut out the target band, and wash away Ponceau S with 1*TBST (Beyotime, 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 15 ml of 1*TBST, 5 minutes each time. Place the primary antibody at the appropriate dilution recommended in the product instructions in 10 mL of primary antibody dilution buffer (1*TBST containing 5% skim milk powder; to prepare 20 mL, add 1.0 g of skim milk powder to 20 mL of 1*TBST and mix thoroughly), incubate overnight at 4°C with gentle shaking occasionally, and wash three times with 1*TBST, 10 minutes each time. Then, incubate the membrane with the developing solution (SignalFire™ ECLeagent #6883) at room temperature for 1 minute, discard excess solution (keeping the membrane moist), and expose to sunlight; the results are shown below. 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 demonstrated that at the biochemical level, SPOP inhibitors can inhibit the activity of SPOP protein in cells to stabilize the content of the substrate protein PTEN and achieve a therapeutic effect. The increase in PTEN protein content inhibits the AKT-driven cancer signaling pathway.

[0091] Western blotting experiments were conducted to verify this viewpoint. The results showed that after treating A498 cells 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. However, the total abundance of AKT protein in A498 cells remained unchanged. These results indicate that compound 1 can interfere with the binding of SPOP to its substrate protein and reduce the ubiquitination level of the substrate protein, inhibiting the phosphorylation level of oncogene pathway proteins, thereby inhibiting the proliferation of renal cancer cells.

[0092] Example 10

[0093] Mouse xenograft experiments were conducted using the SPOP multivalent inhibitor of this invention:

[0094] Experimental steps:

[0095] (1) Mouse modeling: Prepare 6-week-old female BALB / c nude mice, mix 8×106 A498-Luc cells with Corning gel and inject into the right axilla of the mice, about 100 μL per mouse. After subcutaneous injection, the mice are cultured for about 6-8 weeks to allow tumor formation.

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

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

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

[0099] The results showed that the body weight of the experimental mice did not fluctuate significantly during the 3-week dosing period, indicating that compound 1 had little impact on the overall health and physiological function of the mice and demonstrated good tolerability. By comparing tumor growth curves, compound 1 showed significant superiority over the monovalent compound YEADSTTRRRR-NH2 in inhibiting tumor volume growth. This inhibitory effect was particularly pronounced 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 dissection of the mice, the tumor volume in the compound 1 treatment group was significantly smaller than that in 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, with significantly improved efficacy and durability; it shows significant potential in inhibiting tumor growth and exhibits positive results in terms of both safety and efficacy, making it highly promising for application.

[0101] Example 11

[0102] For the SPOP multivalent inhibitor of the present invention, ITC (isothermal titration calorimetry) experiments were conducted:

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

[0104] Experimental steps:

[0105] High-concentration, high-purity SPOP protein was obtained through extraction, dialysis, and ultrafiltration. During dialysis, a buffer solution consistent with the protein composition was maintained as much as possible. First, 400 μL of SPOP protein (1 μM) and 100 μL of compound (10 μM) were prepared, ensuring the DMSO content in both the protein and compound was identical. Then, an isothermal titration calorimeter (MicroCali TC200) was used, with the operating parameters set as follows: temperature 25℃; rotation speed 750 rpm. The sample cell and titration needle were cleaned. Approximately 280 μL of SPOP protein was slowly added to the sample cell, and approximately 40 μL of compound was aspirated into the titration needle. The solution was added in 2 μL increments, with 2.5-minute intervals between each 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 changes of the compound binding to the SPOP protein, thereby obtaining the KA value and KD value (KD = 1 / KA). The smaller the KD value, the better the binding strength and selectivity of the compound to the protein.

[0106] The binding activities of compounds 1–4 to SPOP protein were determined, as shown in Table 2 and [other tables]. Figure 8 As 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 exhibited good binding affinity to SPOP protein, with significantly enhanced binding and activity, and their activity was much higher than that of currently published peptide inhibitors.

[0110] Therefore, the SPOP multivalent inhibitor of the present invention has a larger molecular structure and higher specificity, enabling it to more accurately target specific proteins or protein complexes, reduce binding to non-target sites, thereby reducing side effects. Furthermore, the multivalent inhibitor can interact with the target through multiple binding sites, thereby increasing the affinity and stability with the target, improving the efficacy 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, with a structure as shown in Formula I: Wherein, n = 10 to 30; Petide is selected from YEADSTT-NH2 or YEADSTTRRRR-NH2, where 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 is threonine with the carboxyl terminus blocked by NH2, and R-NH2 is arginine with the carboxyl terminus blocked by NH2.

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

3. A method for preparing the SPOP multivalent inhibitor according to claim 1, characterized in that, Includes the following steps: The norbornene derivative of the peptide was dissolved in water, and a tetrahydrofuran solution containing a catalyst was added under nitrogen protection. The reaction was carried out at room temperature, and vinyl ether was added to the reaction solution. Dialysis yielded the SPOP multivalent inhibitor shown in Formula I. The norbornene derivative of the peptide was either a norbornene derivative of YEADSTT-NH2 or a norbornene derivative of YEADSTTRRRR-NH2. The structural formula of the norbornene derivative of YEADSTT-NH2 is as follows: The structural formula of the norbornene derivative of YEADSTTRRRR-NH2 is as follows:

4. A pharmaceutical composition, characterized in that, It comprises the compound of formula I as claimed in claim 1 and a pharmaceutically acceptable carrier.

5. The use of the SPOP multivalent inhibitor of claim 1 in the preparation of a medicament for treating renal cell carcinoma.

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