A dhodh polypeptide degrading agent and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a DHODH polypeptide degrading agent and application thereof. The application designs a DHODH binding peptide based on the interaction site of DHODH and STAT3, and designs the DHODH polypeptide degrading agent of the application based on the DHODH binding peptide, and combines the mechanism of proteasome degradation and substance crossing cell membrane. The structure of the DHODH polypeptide degrading agent is X1-DHODH binding peptide-Linker-X2, wherein X1 is a membrane penetrating peptide, and X2 is an E3 ubiquitin ligase VHL binding sequence. The DHODH polypeptide degrading agent of the application can efficiently bind to DHODH protein, effectively produce DHODH degradation effect at the cell level, and has high anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a DHODH polypeptide degrading agent and its application. Background Technology

[0002] In mammalian cells, pyrimidine synthesis typically occurs via two pathways: a de novo pathway using aspartate and glutamine as raw materials, catalyzed by the trifunctional enzymes CAD, dihydroorotate dehydrogenase (DHODH), and uridine monophosphate synthase (UMPS) to produce adenosine monophosphate (UMP); and a salvage pathway using pyrimidine metabolites from the bloodstream. When cells are quiescent or fully differentiated, the salvage pathway can meet their growth needs. However, in rapidly proliferating cells such as cancer cells, the de novo pathway is the primary source of pyrimidine products. Numerous studies have shown that cancer cells exhibit high levels of de novo pyrimidine metabolic flux during tumorigenesis and development driven by oncogene mutations. Therefore, targeting the de novo pyrimidine pathway could be a potential strategy for targeted cancer therapy.

[0003] The key rate-limiting step catalyzed by DHODH in the de novo pyrimidine synthesis pathway is the only reaction in the cell that connects the de novo pyrimidine synthesis pathway to the mitochondrial oxidative respiratory chain, directly affecting cellular oxidative phosphorylation and cellular metabolic adaptation to oxidative stress. DHODH is a flavin mononucleotide (FMN)-containing enzyme that catalyzes the fourth step in the de novo pyrimidine synthesis pathway, dehydrogenating dihydroorotic acid to orotic acid. DHODH has been reported to be closely related to the occurrence and development of tumors in various cancers, such as lung cancer, colorectal cancer, breast cancer, leukemia, lymphoma, bone cancer, cervical cancer, and prostate cancer. Studies have found that DHODH inhibitors can effectively inhibit tumor cell proliferation, thereby exerting anti-tumor effects. Several DHODH inhibitors (such as leflunomide, ASLAN003, JNJ-74856665, and IMU-935) have entered clinical research stages. In addition, studies have shown that inhibiting DHODH can reduce the number of activated immune cells, thus achieving an immunosuppressive effect; and DHODH inhibitors have good antiviral activity and can inhibit the synthesis of viral RNA in cells.

[0004] Currently, almost all DHODH inhibitors target its CoQ binding active pocket, inhibiting the bioenzymatic activity of DHODH by competitively binding to CoQ. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the limited types of existing DHODH inhibitors and to provide a DHODH peptide degrader.

[0006] The purpose of this invention is to provide a DHODH-binding peptide.

[0007] Another object of the present invention is to provide the application of the DHODH binding peptide in the preparation of DHODH polypeptide degrading agents.

[0008] Another object of the present invention is to provide the application of the DHODH peptide degrader in the preparation of drugs for the prevention and treatment of DHODH-mediated diseases.

[0009] Another object of the present invention is to provide a pharmaceutical composition.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] A DHODH-binding peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] This invention starts from the interaction between DHODH and STAT3, selects the key sites on the STAT3 protein that bind to DHODH, and designs a DHODH-binding peptide. The amino acid sequence of this peptide is shown in SEQ ID NO.1 (DINSAKTGVQNGTNK).

[0013] This invention also protects the use of the DHODH binding peptide in the preparation of DHODH polypeptide degrading agents.

[0014] A DHODH polypeptide degrading agent, comprising a membrane-penetrating peptide, the DHODH binding peptide of the present invention, a linker, and an E3 ubiquitin ligase VHL binding sequence; wherein the N-terminus of the DHODH binding peptide is linked to the membrane-penetrating peptide, and the C-terminus is linked to the E3 ubiquitin ligase VHL binding sequence via the linker.

[0015] The DHODH peptide degrader of this invention binds to the DHODH protein via a DHODH-binding peptide and to the E3 ubiquitin ligase VHL-binding sequence, inducing the formation of a DHODH protein-peptide degrader-E3 ubiquitin ligase VHL ternary complex. This ternary complex effectively recruits the E3 ligase to polyubiquitinate the DHODH protein, which is then degraded by the 26S proteasome.

[0016] Furthermore, the amino acid sequence of the transmembrane peptide is shown in SEQ ID NO.2 (RRRRRRRR), SEQ ID NO.3 (YGRKKRRQRRR), or SEQ ID NO.4 (RQIKIWFQNRRMKWKK).

[0017] Furthermore, the amino acid sequence of the E3 ubiquitin ligase VHL binding sequence is shown in SEQ ID NO.5 (ALAPYIP) or SEQ ID NO.6 (LAP(OH)YI).

[0018] In the sequence LAP(OH)YI of this invention, P(OH) is a hydroxylated proline.

[0019] Furthermore, the linker is selected from polypeptides or 6-aminocaproic acid with amino acid sequences as shown in SEQ ID NO.7 (GSGS).

[0020] In this invention, the full English name of 6-aminohexanoic acid is Aminohexanoic acid, which can be abbreviated as AHX.

[0021] Preferably, the DNA sequence corresponding to the DHODH binding peptide of the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.8.

[0022] Preferably, the DNA sequence encoding the transmembrane peptide is shown in SEQ ID NO.9 (corresponding to SEQ ID NO.2 amino acid sequence), SEQ ID NO.10 (corresponding to SEQ ID NO.3 amino acid sequence), and SEQ ID NO.11 (corresponding to SEQ ID NO.4 amino acid sequence).

[0023] Preferably, the DNA sequence encoded by the E3 ubiquitin ligase VHL binding sequence is shown in SEQ ID NO.12 (corresponding to SEQ ID NO.5 amino acid sequence) and SEQ ID NO.13 (corresponding to SEQ ID NO.6 amino acid sequence).

[0024] Preferably, the DNA sequence encoded by the linker is as shown in SEQ ID NO.14 (corresponding to the amino acid sequence of SEQ ID NO.7).

[0025] Preferably, the amino acid sequence structure of the DHODH polypeptide degrader is shown in any of the following:

[0026] RRRRRRRR-DINSAKTGVQNGTNK-AHX(6-Aminohexanoic acid)-ALAPYIP,

[0027] RRRRRRRR-DINSAKTGVQNGTNK-AHX(6-aminocaproic acid)-LAP(OH)YI,

[0028] RRRRRRRR-DINSAKTGVQNGTNK-GSGS-ALAPYIP,

[0029] RRRRRRRR-DINSAKTGVQNGTNK-GSGS-LAP(OH)YI、

[0030] YGRKKRRQRRR-DINSAKTGVQNGTNK-AHX(6-aminocaproic acid)-ALAPYIP,

[0031] YGRKKRRQRRR-DINSAKTGVQNGTNK-AHX(6-aminocaproic acid)-LAP(OH)YI,

[0032] YGRKKRRQRRR-DINSAKTGVQNGTNK-GSGS-ALAPYIP,

[0033] YGRKKRRQRRR-DINSAKTGVQNGTNK-GSGS-LAP(OH)YI、

[0034] RQIKIWFQNRRMKWKK-DINSAKTGVQNGTNK-AHX(6-aminocaproic acid)-ALAPYIP,

[0035] RQIKIWFQNRRMKWKK-DINSAKTGVQNGTNK-AHX(6-aminocaproic acid)-LAP(OH)YI,

[0036] RQIKIWFQNRRMKWKK-DINSAKTGVQNGTNK-GSGS-ALAPYIP,

[0037] RQIKIWFQNRRMKWKK-DINSAKTGVQNGTNK-GSGS-LAP(OH)YI.

[0038] Furthermore, this invention also protects the use of the DHODH peptide degrader in the preparation of drugs for the prevention and treatment of DHODH-mediated diseases. Specifically, DHODH-mediated diseases refer to diseases that can be treated by inhibiting / degrading DHODH.

[0039] Furthermore, the DHODH-mediated diseases are selected from tumors, autoimmune diseases, or viral infections.

[0040] Furthermore, the tumor is selected from one or more of lung cancer, colorectal cancer, breast cancer, and leukemia.

[0041] In specific embodiments, the DHODH polypeptide degrader of the present invention can effectively inhibit tumor cell growth, and has good inhibitory effects on human colon cancer cells HCT116, human colorectal adenocarcinoma cells RKO, lung cancer cells A549, breast cancer cells MCF7, and leukemia cells MOLM13. Its IC50 value is [not specified]. 50 The values ​​were 9.8 μM, 10.5 μM, 20.6 μM, 18.5 μM, and 23.3 μM, respectively.

[0042] The present invention provides a pharmaceutical composition comprising any of the DHODH peptide degrading agents described herein and a pharmaceutically acceptable carrier.

[0043] The present invention has the following beneficial effects:

[0044] This invention provides a DHODH peptide degrader that targets and degrades DHODH. The peptide degrader comprises a membrane-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL binding sequence. The peptide degrader provided by this invention exhibits good anti-tumor effects. Experiments show that the DHODH peptide degrader of this invention binds to DHODH protein purified from in vitro prokaryotic expression with a KD value of approximately 523 nM, demonstrating efficient binding to DHODH protein. Furthermore, the DHODH peptide degrader of this invention degrades DHODH protein in cells in a concentration-dependent manner, achieving a degradation efficiency of 90% at a concentration of 20 μM, showing promising application prospects in the preparation of drugs for the prevention and treatment of DHODH-mediated diseases. Simultaneously, the DHODH peptide degrader of this invention exhibits good inhibitory effects on different tumor cell types and can effectively inhibit tumor cell colony formation, thus exerting a good anti-tumor effect. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the structure of the DHODH polypeptide degrader.

[0046] Figure 2 This is a gel image of DHODH protein expression in prokaryotes.

[0047] Figure 3 A gel image showing the purification of DHODH protein.

[0048] Figure 4 This is a statistical graph showing the binding activity of DHODH peptide degraders with in vitro purified DHODH protein.

[0049] Figure 5 This is a diagram showing the expression of DHODH protein in cells after intervention with a DHODH peptide degrader.

[0050] Figure 6Cell micrographs of tumor cells after intervention with DHODH peptide degrading agent in 2D cloning.

[0051] Figure 7 Cell micrographs of tumor cells after intervention with DHODH peptide degrading agent in 3D cloning. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0053] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0054] Example 1: Design and Synthesis of DHODH Peptide Degrading Agent

[0055] 1. Design of DHODH peptide degraders

[0056] Starting from the interaction between DHODH and STAT3, a key binding site on the STAT3 protein for DHODH was selected to design a DHODH-binding peptide, the amino acid sequence of which is shown in SEQ ID NO.1: DINSAKTGVQNGTNK. Based on the transmembrane transport properties of substances, a transmembrane peptide was linked to the N-terminus of the DHODH-binding peptide, the amino acid sequence of which is shown in SEQ ID NO.2: RRRRRRRR. Based on the properties of the degradation peptide, an E3 ubiquitin ligase VHL binding sequence was designed, its amino acid sequence being shown in SEQ ID NO.5: ALAPYIP. The DHODH-binding peptide and the E3 ubiquitin ligase VHL binding sequence are linked via a linker: 6-aminocaproic acid.

[0057] 2. Synthesis of DHODH peptide degrading agent

[0058] The amino acid sequence of the DHODH peptide degrader described in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the structural formula of the peptide degrader is as follows: Figure 1 As shown, the amino acid sequence structure of this DHODH polypeptide degrader is: X1-DINSAKTGVQNGTNK-Linker-X2, where X1 is a membrane-penetrating peptide and X2 is the E3 ubiquitin ligase VHL binding sequence.

[0059] Example 2: Design and Synthesis of DHODH Peptide Degrading Agent

[0060] Compared with Example 1, the amino acid sequence of the E3 ubiquitin ligase VHL binding sequence in this example is shown in SEQ ID NO. 6: LAP(OH)YI, and the rest of the experimental procedures and sequences are the same.

[0061] Example 3: Design and Synthesis of DHODH Peptide Degrading Agent

[0062] Compared with Example 1, the connector in this example is GSGS, and the rest of the experimental operations and sequences are the same.

[0063] Example 4: Design and Synthesis of DHODH Peptide Degrading Agent

[0064] Compared with Example 2, the connector in this example is GSGS, and the rest of the experimental operations and sequences are the same.

[0065] Example 5: Design and Synthesis of DHODH Peptide Degrading Agent

[0066] Compared with Example 1, the amino acid sequence of the transmembrane peptide in this example is shown in SEQ ID NO.3: YGRKKRRQRRR, and the rest of the experimental procedures and sequences are the same.

[0067] Example 6: Design and Synthesis of DHODH Peptide Degrading Agent

[0068] Compared with Example 5, the amino acid sequence of the E3 ubiquitin ligase VHL binding sequence in this example is shown in SEQ ID NO. 6: LAP(OH)YI, and the rest of the experimental procedures and sequences are the same.

[0069] Example 7: Design and Synthesis of DHODH Peptide Degrading Agent

[0070] Compared with Example 5, the connector in this example is GSGS, and the rest of the experimental operations and sequences are the same.

[0071] Example 8: Design and Synthesis of DHODH Peptide Degrading Agent

[0072] Compared with Example 6, the connector in this example is GSGS, and the rest of the experimental operations and sequences are the same.

[0073] Example 9: Design and Synthesis of DHODH Peptide Degrading Agent

[0074] Compared with Example 1, the amino acid sequence of the membrane-penetrating peptide in this example is shown in SEQ ID NO.4: RQIKIWFQNRRMKWKK, and the rest of the experimental procedures and sequences are the same.

[0075] Example 10: Design and Synthesis of DHODH Peptide Degrading Agent

[0076] Compared with Example 9, the amino acid sequence of the E3 ubiquitin ligase VHL binding sequence in this example is shown in SEQ ID NO. 6: LAP(OH)YI, and the rest of the experimental procedures and sequences are the same.

[0077] Example 11: Design and Synthesis of DHODH Peptide Degrading Agent

[0078] Compared with Example 9, the connector in this example is GSGS, and the rest of the experimental operations and sequences are the same.

[0079] Example 12: Design and Synthesis of DHODH Peptide Degrading Agent

[0080] Compared with Example 10, the connector in this example is GSGS, and the rest of the experimental operations and sequences are the same.

[0081] Example 13: In vitro prokaryotic expression and purification of DHODH protein

[0082] 1. Constructing a prokaryotic expression plasmid for the DHODH protein

[0083] The DHODH protein gene sequence (NCBI ID: NM_001361.5) was obtained by PCR amplification. After the PCR product was confirmed to be correct by sequencing, it was ligated into the pET-19b vector digested with Nde I and BamHI. The product was transformed into DH5α competent cells and plated on LB agar (containing 0.1 mg / mL ampicillin) and incubated overnight at 37°C. The grown colonies were extracted, and plasmids were extracted and sequenced. The plasmid with correct sequencing, pET-19b-DHODH, was selected for subsequent operations.

[0084] 2. In vitro prokaryotic expression of DHODH protein

[0085] The pET-19b-DHODH successfully constructed in step 1 above was transformed into BL21 competent cells for protein expression. The bacterial culture was incubated at 37°C and 220 rpm until the OD 600 (optical density at 600 nm) reached 0.8–1.0. Then, 0.5 mM IPTG (isopropyl galactothioglycoside) was added, and the cells were induced at 16°C and 220 rpm for 16 h. The protein expression results are as follows. Figure 2 As shown.

[0086] 3. Purification of DHODH protein

[0087] The bacterial cells expressing DHODH protein were harvested by centrifugation at 12000 rpm for 30 min. The cells were resuspended in Lysis Buffer and sonicated for 30 min. Inclusion bodies, cell debris, and supernatant were separated by high-speed centrifugation at 12000 rpm for 30 min, yielding a supernatant containing a large amount of DHODH protein. The supernatant was slowly added to a NiNTA resin column to ensure the target protein was fully bound to the resin. Impurities on the resin were washed sequentially with low-concentration imidazole buffer (50 mM, 100 mM), and the target protein was eluted with buffer containing 500 mM imidazole. The protein was then concentrated using an ultrafiltration concentrator to reduce the imidazole concentration. The entire protein purification process was validated using SDS-PAGE, which showed successful in vitro purification of a large amount of DHODH protein. Figure 3 As shown.

[0088] This experiment requires three buffer solutions, the formulations of which are as follows:

[0089] Lysis Buffer: 50mM Hepes, 300mM NaCl, 10mM Imidazole, 1% (v / v) Triton X-100, 10% Glycerol pH 7.7.

[0090] Wash Buffer: 50mM Hepes, 300mM NaCl, imidazole (50mM and 100mM), 10% glycerol pH 7.7.

[0091] Dialysis buffer: 30 mM Hepes, 200 mM NaCl, 10% glycerol, 1 mM TCEP, pH 7.7. Example 14 uses isothermal titration calorimetry (ITC) to determine the binding affinity of the DHODH peptide degrader to DHODH protein.

[0092] Experimental materials: DHODH protein prepared in Example 13; DHODH polypeptide degrading agent prepared in Example 1.

[0093] The DHODH protein purified in Example 13 was dialyzed overnight using a dialysis buffer, and then concentrated to 6 mg / mL after dialysis. An appropriate amount of the buffer from the overnight DHODH protein dialysis was used to dissolve the lyophilized DHODH peptide degrading agent powder prepared in Example 1 to a specific concentration (it is essential to prepare a high concentration at the beginning), and then an ITC experiment was performed.

[0094] The data was processed using MicroCal PEAQ-ITC Analysis Software, which automatically calculated and generated various parameters. Finally, the data was exported and used with Prism 8.0 to obtain high-resolution data graphs, as shown below. Figure 4 As shown in the figure. Analysis of the data fitting curves shows that the affinity constant KD value between the designed and synthesized DHODH peptide degrader and the in vitro purified DHODH protein is 523 nM ± 174 nM, indicating that the designed and synthesized DHODH peptide degrader has a strong binding ability with the in vitro purified DHODH protein.

[0095] Example 15: Inhibitory activity of DHODH peptide degrader against tumor cells

[0096] This example discusses the inhibitory activity of the DHODH polypeptide degrader prepared in Example 1 on tumor cells. The specific implementation method is as follows:

[0097] 1. Culture of mammalian cells

[0098] Cell resuscitation: Remove cells frozen in liquid nitrogen and thaw them in a 42°C water bath. After complete thawing, centrifuge at 1000 rpm for 5 min, then transfer to a biosafety cabinet for aseptic handling. Open the cryovial cap, aspirate the supernatant, and thoroughly resuspend the cell pellet with 1 mL of complete culture medium (DMEM + 10% FBS + 1% penicillin-streptomycin). Transfer the pellet to a culture dish or flask, add the culture medium to the required amount, and gently agitate the dish at multiple angles to ensure even cell distribution. Incubate at 37°C in a 5% CO2 incubator.

[0099] Cell passage: When the cell density in the culture dish reaches 80%–90% confluence, passage culture is required. Remove the cells from the incubator and place them in a biosafety cabinet. Discard the old culture medium and slowly add 1 mL of sterile PBS solution along the wall of the culture dish. Cover the dish and gently agitate it to ensure the PBS thoroughly washes the bottom. Remove the PBS with a pipette and repeat once. Soak the bottom of the culture dish with 1 mL of trypsin digestion solution, then remove the trypsin digestion solution. Place the cells in a 37°C incubator for digestion. The digestion time varies depending on the cell type. After digestion, remove the culture dish from the incubator and observe the cell morphology changes under a microscope to determine if digestion is complete. In the biosafety cabinet, terminate digestion with complete culture medium containing 10% FBS and repeatedly pipette the adherent cells to obtain a cell suspension. Transfer an appropriate amount of the cell suspension to a new culture dish, add culture medium, and gently agitate to ensure even distribution of cells. Continue cell culture in a cell culture incubator.

[0100] 2. The DHODH polypeptide degrader described in this invention effectively inhibits tumor cell proliferation.

[0101] Human colon cancer cells HCT116, human colorectal adenocarcinoma cells RKO, lung cancer cells A549, breast cancer cells MCF7, and leukemia cells MOLM13 in logarithmic growth phase were seeded in 96 culture plates at a density of 3000 cells / 100 μL per well. After 24 h, solutions containing different concentrations of the DHODH polypeptide degrading agent prepared in Example 1 of this invention were added. Ten drug treatment concentration gradients were established: 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, and 0.01 μM, with a blank control group included. The plates were then incubated at 37 °C. After 48 h, 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) was added to each well, and the plates were incubated for another 4 h. The supernatant was removed, and 150 μL of dimethyl sulfoxide was added to each well. The plates were then shaken at low speed for 10 min on a shaker. The absorbance of each well was measured at OD490nm using an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results showed that the DHODH polypeptide degrading agent prepared in Example 1 of this invention had a good inhibitory effect on various tumor cells. Cell inhibition IC50 (IC50) 50 The values ​​are shown in Table 1.

[0102] Table 1. Inhibitory effect of DHODH peptide degrader on tumor cells (IC50) 50

[0103]

[0104] Example 16: DHODH peptide degrading activity in cells

[0105] This example discusses the degradation activity of the DHODH polypeptide degrader prepared in Example 1 on DHODH in cells. The specific implementation method is as follows:

[0106] 1. Culture of mammalian cells

[0107] The specific implementation method is the same as in Example 15, 1. Culture of mammalian cells.

[0108] 2. The DHODH polypeptide degrading agent of this invention degrades DHODH protein in cells in a concentration-dependent manner.

[0109] Human colon cancer cells HCT116 and human colorectal adenocarcinoma cells RKO were seeded in 6 cm cell culture plates at a density of 20%–30%. After 24 hours, solutions containing different concentrations of the DHODH polypeptide degrading agent prepared in Example 1 of this invention were added. Three drug treatment concentration gradients were established: 20 μM, 10 μM, and 5 μM, with a blank control group included. Cells were then incubated at 37°C, and after 48 hours, they were collected and lysed for Western blotting experiments. The results are as follows: Figure 5As shown, the DHODH polypeptide degrader of the present invention degrades DHODH protein in cells in a concentration-dependent manner, and the degradation effect reaches 90% at 20 μM.

[0110] Example 17: DHODH peptide degrader inhibits tumor cell clone formation

[0111] Experimental materials: DHODH polypeptide degrading agent prepared in Example 1.

[0112] The specific implementation method of the 2D colony formation experiment is as follows: Logarithmically growing colon cancer cells HCT116 and colon adenocarcinoma cells RKO were seeded into 6-well cell culture plates, with 1000 cells per well. Different concentrations of the DHODH polypeptide degrading agent described in this invention were set for treatment groups: 0 μM, 10 μM, and 20 μM. After two weeks of culture, the culture supernatant was removed, and 1 mL of 4% paraformaldehyde was added for fixation for 15 min. Then, the paraformaldehyde was removed, and 1 mL of crystal violet was added for staining for 10 min. The plates were carefully rinsed with tap water to remove excess crystal violet staining solution, and the 6-well plates were photographed. Figure 6 The results showed that the 20 μM DHODH polypeptide degrader described in this invention effectively inhibited the formation of 2D clones of tumor cells.

[0113] The specific implementation method of the 3D colony formation experiment is as follows: Prepare 1.2% (w / v) and 0.7% (w / v) agarose gels, autoclave them, and place them in a 42℃ water bath for later use. Prepare 2X DMEM medium containing 20% ​​FBS and 2% penicillin-streptomycin antibiotics. Mix 1.2% (w / v) agarose gel with 2X DMEM medium at a 1:1 ratio and spread it into 6-well plates, 2 mL per well, and allow it to cool and solidify at room temperature. Then, take HCT116 cells in the logarithmic growth phase, digest them with trypsin to form a single-cell suspension, count the cells, and adjust the cell density to 5 x 10⁻⁶ cells / well. 4 cells / mL. Mix 0.7% (w / v) agarose and 2X DMEM medium at a 1:1 ratio. Take 2 mL and place it in an EP tube. Add 20 μL of cell suspension, mix thoroughly, and then add to a 6-well plate with the bottom layer of gel already deposited. After the top agar solidifies, incubate at 37°C for 2 weeks and take pictures under a microscope. Figure 7 The results showed that the 20 μM DHODH polypeptide degrader described in this invention effectively inhibited the formation of 3D clones of tumor cells.

[0114] The above experiments demonstrate that the DHODH degrading agent of this invention can efficiently bind to and effectively degrade DHODH protein. Furthermore, the DHODH degrading agent of this invention effectively inhibits tumor cell growth and migration, exhibiting significant potential for pharmaceutical development.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A DHODH polypeptide degrading agent, characterized in that, The DHODH polypeptide degrader consists of a membrane-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding sequence; wherein, the N-terminus of the DHODH-binding peptide is linked to the membrane-penetrating peptide, and the C-terminus is linked to the E3 ubiquitin ligase VHL-binding sequence via the linker. The amino acid sequence structure of the DHODH polypeptide degrader is as follows: RRRRRRRR-DINSAKTGVQNGTNK-AHX (6-Aminohexanoic acid)-ALAPYIP; The amino acid sequence of the DHODH-binding peptide is shown in SEQ ID NO.

1.

2. The use of the DHODH polypeptide degrader of claim 1 in the preparation of drugs for preventing and treating DHODH-mediated diseases, wherein the DHODH-mediated diseases are selected from tumors; and the tumors are selected from one or more of lung cancer, colorectal cancer, breast cancer, and leukemia.

3. A pharmaceutical composition, characterized in that, It includes the DHODH peptide degrader of claim 1 and a pharmaceutically acceptable carrier.

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