A DHODH peptide degrader and its application in the preparation of drugs for the prevention and / or treatment of DHODH-mediated diseases.

Abstract

This invention provides a DHODH peptide degrader and its application in the preparation of drugs for the prevention and / or treatment of DHODH-mediated diseases, belonging to the field of biomedical technology. The invention provides a DHODH peptide degrader comprising, from the N-terminus to the C-terminus, a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide linked sequentially. The DHODH peptide degrader provided by this invention can efficiently bind to DHODH protein, exhibiting good anti-tumor effects at both cellular and animal levels. It shows promising application prospects in the preparation of drugs for the prevention and treatment of DHODH-mediated diseases, overcoming the shortcomings of existing drugs for treating DHODH-related diseases, such as limited variety and insufficient efficacy, and is of significant importance.

Description

[0001] This application is a divisional application of the invention entitled "A DHODH polypeptide degrader and its application in the preparation of drugs for the prevention and / or treatment of DHODH-mediated diseases" filed on March 21, 2025, with application number CN202510346581.2 and invention titled "A DHODH polypeptide degrader and its application in the preparation of drugs for the prevention and / or treatment of DHODH-mediated diseases". The applicants are East China University of Science and Technology and Guangdong Provincial Institute for Drug Control (Guangdong Provincial Institute of Drug Quality and Guangdong Provincial Port Drug Inspection Institute). Technical Field

[0002] This invention belongs to the field of biomedical technology, and in particular relates to a DHODH polypeptide degrader and its application in the preparation of drugs for the prevention and / or treatment of DHODH-mediated diseases. Background Technology

[0003] In mammalian cells, pyrimidine synthesis typically occurs via two pathways. One is the de novo synthesis pathway, using aspartate and glutamine as raw materials, which generates adenosine monophosphate (UMP) under the catalysis of the trifunctional enzyme CAD, dihydroorotate dehydrogenase (DHODH), and uridine monophosphate synthase UMPS. The other is the salvage synthesis pathway, using pyrimidine metabolites from the bloodstream as raw materials. When cells are in a resting state or fully differentiated, the pyrimidine salvage pathway can meet the cell's growth needs. However, in rapidly proliferating cells such as cancer cells, the de novo pyrimidine synthesis pathway is the primary source of pyrimidine products.

[0004] 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 tumorigenesis and development in various cancers.

[0005] However, currently, almost all DHODH inhibitors target its CoQ binding active pocket, inhibiting the bioenzymatic activity of DHODH by competitively binding to CoQ, resulting in a limited range of inhibitors. Summary of the Invention

[0006] The purpose of this invention is to provide a DHODH peptide degrader and its application in the preparation of drugs for the prevention and / or treatment of DHODH-mediated diseases, which can overcome the defects and shortcomings of the limited types of existing DHODH inhibitors.

[0007] This invention provides a DHODH polypeptide degrader, comprising, from the N-terminus to the C-terminus, a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide connected in sequence; the cell-penetrating peptide is RRRRRRRR or YGRKKRRQRRR; the general structural formula of the DHODH-binding peptide is X1KTGVQX2K or X1KTGVQX2KX3, wherein X1, X2, and X3 are composed of any one or more amino acids selected from G, A, I, P, S, T, Q, D, E, K, and H; and the E3 ubiquitin ligase VHL-binding peptide is ALAPYIP.

[0008] Preferably, the linker is 6-aminohexanoic acid.

[0009] Preferably, X1 is G, SA, NSA or DINSA; X2 is PAH, GIPHG or NTGNK; and X3 is A.

[0010] Preferably, X1 is G and X2 is GIPHG; or, X1 is SA and X2 is GIPHG or PAH; or, X1 is NSA and X2 is GIPHG or PAH; or, X1 is DINSA and X2 is NTGNK or GIPHG.

[0011] Preferably, the amino acid sequence of the DHODH binding peptide includes one or more of the amino acid sequences shown in SEQ ID NO. 8~21.

[0012] The present invention also provides a nucleic acid molecule encoding the DHODH polypeptide degrader described in the above scheme.

[0013] The present invention also provides the use of the DHODH polypeptide degrader or the nucleic acid molecule described above in the preparation of medicaments for the prevention and / or treatment of DHODH-mediated diseases.

[0014] Preferably, the DHODH-mediated diseases include one or more of tumors, autoimmune diseases, and viral infections.

[0015] Preferably, the tumor includes one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, glioma, and leukemia.

[0016] The present invention also provides a pharmaceutical composition comprising the DHODH peptide degrader described above and a pharmaceutically acceptable carrier.

[0017] This invention provides a DHODH peptide degrader, comprising, from the N-terminus to the C-terminus, a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide linked sequentially. The DHODH peptide degrader provided by this invention can efficiently bind to DHODH protein, exhibiting good anti-tumor effects at both cellular and animal levels. It shows promising application prospects in the preparation of drugs for the prevention and treatment of DHODH-mediated diseases, overcoming the shortcomings of existing drugs for treating DHODH-related diseases, such as limited variety and insufficient efficacy, and is of significant importance. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the structure of the DHODH peptide degrader; Figure 2 A gel image showing the purification of DHODH protein; Figure 3 Diagram showing DHODH protein expression in cells after intervention with DHODH peptide degrading agent; Figure 4 Cell micrographs of tumor cells after intervention with DHODH peptide degrading agent in 3D cloning; Figure 5 The figure shows the results of DHODH peptide degradation agent inhibiting the growth of subcutaneous tumors in mice. Detailed Implementation

[0020] This invention provides a DHODH polypeptide degrader comprising, from the N-terminus to the C-terminus, a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide connected in sequence; the cell-penetrating peptide is RRRRRRRR (SEQ ID NO. 1) or YGRKKRRQRRR (SEQ ID NO. 2); the general structural formula of the DHODH-binding peptide is X1KTGVQX2K or X1KTGVQX2KX3, wherein X1, X2, and X3 are composed of any one or more amino acids selected from G, A, I, P, S, T, Q, D, E, K, and H; the E3 ubiquitin ligase VHL-binding peptide is ALAPYIP (SEQ ID NO. 3).

[0021] The structural formula of the DHODH polypeptide degrader of the present invention is as follows: Figure 1As shown; the N-terminus of the DHODH-binding peptide is linked to a membrane-penetrating peptide, and the C-terminus is linked to the E3 ubiquitin ligase VHL-binding peptide via a linker.

[0022] This invention starts with the interaction between DHODH and STAT3, selecting key binding sites on the STAT3 protein for DHODH, and designing a DHODH-binding peptide. Based on the transmembrane transport properties of substances, a membrane-penetrating peptide is linked to the N-terminus of the DHODH-binding peptide. Based on the properties of the degrading peptide, an E3 ubiquitin ligase VHL-binding peptide is designed. The DHODH-binding peptide and the E3 ubiquitin ligase VHL-binding peptide are linked via a linker.

[0023] The DHODH peptide degrader of this invention induces the formation of a ternary complex of DHODH protein-peptide degrader-E3 ubiquitin ligase VHL by binding the DHODH-binding peptide to the DHODH protein and the E3 ubiquitin ligase VHL-binding peptide to the E3 ubiquitin ligase VHL. This ternary complex effectively recruits the E3 ligase to polyubiquitinate the DHODH protein, which is then degraded by the 26S proteasome. The DHODH peptide degrader of this invention can efficiently bind to DHODH protein, producing an effective DHODH degradation effect at the cellular level, and exhibits highly efficient antitumor activity against colorectal cancer, lung cancer, breast cancer, and leukemia.

[0024] In the specific implementation of this invention, the amino acid sequence of the DHODH polypeptide degrader was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0025] In this invention, the amino acid sequence of KTGVQ is numbered SEQ ID NO.4.

[0026] In the specific implementation of this invention, the linker is 6-aminohexanoic acid, abbreviated as AHX.

[0027] In the specific implementation of this invention, X1 is G, SA, NSA or DINSA (SEQ ID NO.5); X2 is PAH, GIPHG (SEQ ID NO.6) or NTGNK (SEQ ID NO.7); and X3 is A.

[0028] In the specific implementation of this invention, X1 is G and X2 is GIPHG; or, X1 is SA and X2 is GIPHG or PAH; or, X1 is NSA and X2 is GIPHG or PAH; or, X1 is DINSA and X2 is NTGNK or GIPHG.

[0029] In a specific embodiment of the present invention, the nucleotide sequence of the gene encoding the cell-penetrating peptide RRRRRRR is shown in SEQ ID NO.50, specifically: cgccgccgccgccgccgccgccgc. In a specific embodiment of the present invention, the nucleotide sequence of the gene encoding the cell-penetrating peptide YGRKKRRQRRR is shown in SEQ ID NO.51, specifically: tatggccgcaaaaaacgccgccagcgccgccgc.

[0030] In the specific implementation of this invention, the nucleotide sequence of the gene encoding the E3 ubiquitin ligase VHL binding peptide is shown in SEQ ID NO.52, specifically: gcgctggcgccgtatattccg.

[0031] In the specific implementation of the present invention, the amino acid sequence of the DHODH binding peptide includes one or more of the amino acid sequences shown in SEQ ID NO. 8~21. Furthermore, the amino acid sequence of the DHODH binding peptide and the nucleotide sequence of the encoding gene are shown in Table 1.

[0032] Table 1. Amino acid sequence of DHODH-binding peptide and nucleotide sequence of encoding gene

[0033] In the specific implementation of this invention, when the cell transmembrane peptide is RRRRRRR, the linker is AHX, the E3 ubiquitin ligase VHL-binding peptide is ALAPYIP, and the DHODH-binding peptide is GKTGVQGIPHGK, GKTGVQGIPHGKA, SAKTGVQGIPHGK, SAKTGVQGIPHGKA, SAKTGVQPAHK, SAKTGVQPAHKA, NSAKTGVQGIPHGK, NSAKTGVQGIPHGKA, NSAKTGVQPAHK, NSAKTGVQPAHKA, DINSAKTGVQGIPHGK, DINSAKTGVQGIPHGKA, DINSAKTGVQNTGNK, or DINSAKTGVQNTGNKA; or, when the cell transmembrane peptide is YGRKKRRQRRR When the linker is AHX, the E3 ubiquitin ligase VHL-binding peptide is ALAPYIP, and the DHODH-binding peptide is GKTGVQGIPHGK, GKTGVQGIPHGKA, SAKTGVQGIPHGK, SAKTGVQGIPHGKA, SAKTGVQPAHK, SAKTGVQPAHKA, NSAKTGVQGIPHGK, NSAKTGVQGIPHGKA, NSAKTGVQPAHK, NSAKTGVQPAHKA, DINSAKTGVQGIPHGK, DINSAKTGVQGIPHGKA, DINSAKTGVQNTGNK, or DINSAKTGVQNTGNKA.

[0034] In embodiments of the present invention, the DHODH polypeptide degrading agent as follows: P 1~28 As shown, the details are as follows: P 1 ： RRRRRRRR-GKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 22); P 2 ： RRRRRRRR-GKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 23); P 3 ： RRRRRRRR-SAKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 24); P 4 ：RRRRRRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP(SEQ ID NO.25; P 5 ： RRRRRRRR-SAKTGVQPAHK-AHX-ALAPYIP(SEQ ID NO.26); P 6 ： RRRRRRRR-SAKTGVQPAHKA-AHX-ALAPYIP(SEQ ID NO.27); P 7 ： RRRRRRRR-NSAKTGVQGIPHGK-AHX-ALAPYIP(SEQ ID NO.28); P 8 ： RRRRRRRR-NSAKTGVQGIPHGKA-AHX-ALAPYIP(SEQ ID NO.29); P 9 ： RRRRRRRR-NSAKTGVQPAHK-AHX-ALAPYIP(SEQ ID NO.30; P 10 ： RRRRRRRR-NSAKTGVQPAHKA-AHX-ALAPYIP(SEQ ID NO.31; P 11 ： RRRRRRRR-DINSAKTGVQGIPHGK-AHX-ALAPYIP(SEQ ID NO.32); P 12 ： RRRRRRRR-DINSAKTGVQGIPHGKA-AHX-ALAPYIP(SEQ ID NO.33; P 13 ： RRRRRRRR-DINSAKTGVQNTGNK-AHX-ALAPYIP(SEQ ID NO.34); P14 ： RRRRRRRR-DINSAKTGVQNTGNKA-AHX-ALAPYIP(SEQ ID NO.35); P 15 ： YGRKKRRQRRR-GKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO.36) ; P 16 ： YGRKKRRQRRR-GKTGVQGIPHGKA-AHX-ALAPYIP(SEQ ID NO.37)); P 17 ： YGRKKRRQRRR-SAKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO.38) ; P 18 ： YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP(SEQ ID NO.39)); P 19 ： YGRKKRRQRRR-SAKTGVQPAHK-AHX-ALAPYIP (SEQ ID NO.40) P 20 ： YGRKKRRQRRR-SAKTGVQPAHKA-AHX-ALAPYIP (SEQ ID NO.41) ; P 21 ： YGRKKRRQRRR-NSAKTGVQGIPHGK-AHX-ALAPYIP(SEQ ID NO.42)); P 22 ： YGRKKRRQRRR-NSAKTGVQGIPHGKA-AHX-ALAPYIP(SEQ ID NO.43)); P 23 ：YGRKKRRQRRR-NSAKTGVQPAHK-AHX-ALAPYIP (SEQ ID NO. 44); P 24 ： YGRKKRRQRRR-NSAKTGVQPAHKA-AHX-ALAPYIP (SEQ ID NO. 45); P 25 ： YGRKKRRQRRR-DINSAKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 46); P 26 ： YGRKKRRQRRR-DINSAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 47); P 27 ： YGRKKRRQRRR-DINSAKTGVQNTGNK-AHX-ALAPYIP (SEQ ID NO. 48); P 28 ： YGRKKRRQRRR-DINSAKTGVQNTGNKA-AHX-ALAPYIP (SEQ ID NO. 49).

[0035] In the amino acid sequences shown in SEQ ID NO.22~SEQ ID NO.49 of this invention, "-" only indicates a link, for example... P 1 ： RRRRRRRRR-GKTGVQGIPHGK-AHX-ALAPYIP is RRRRRRRRGKTGVQGIPHGKAHXALAPYIP.

[0036] The present invention also provides a nucleic acid molecule of the DHODH polypeptide degrader described in the above scheme.

[0037] The present invention also provides the use of the DHODH polypeptide degrader or the nucleic acid molecule described above in the preparation of medicaments for the prevention and / or treatment of DHODH-mediated diseases.

[0038] In the specific implementation of this invention, the DHODH-mediated diseases include one or more of tumors, autoimmune diseases, and viral infections.

[0039] In the specific implementation of this invention, the drug includes an anticancer drug.

[0040] In the specific implementation of this invention, the tumor includes one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, glioma, and leukemia.

[0041] The present invention also provides a pharmaceutical composition comprising the DHODH peptide degrader described above and a pharmaceutically acceptable carrier.

[0042] In the specific implementation of this invention, the DHODH polypeptide degrader is the only active ingredient in the pharmaceutical composition.

[0043] In the specific implementation of this invention, the dosage form of the drug or drug composition includes injections, capsules, or tablets.

[0044] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a DHODH polypeptide degrading agent provided by the present invention and its application in the preparation of medicaments for the prevention and / or treatment of DHODH-mediated diseases, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] Example 1: Design and Synthesis of DHODH Peptide Degrading Agent 1. Design of DHODH peptide degraders Starting from the interaction between DHODH and STAT3, key sites on the STAT3 protein where DHODH binds were selected to design DHODH-binding peptides. P （X） Based on the transmembrane transport properties of substances, a transmembrane peptide is linked to the N-terminus of the DHODH-binding peptide. The amino acid sequence of this transmembrane peptide is, for example, RRRRRRR or YGRKKRRQRRR. Based on the properties of the degrading peptide, an E3 ubiquitin ligase VHL-binding peptide is designed, with the amino acid sequence ALAPYIP. The DHODH-binding peptide and the E3 ubiquitin ligase VHL-binding peptide are linked via a linker: 6-aminohexanoic acid (AHX).

[0047] 2. Synthesis of DHODH peptide degrading agent 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 DHODH peptide degrader is as follows: Figure 1 As shown, the amino acid sequence structure of this DHODH polypeptide degrader is: RRRRRRR-P （X） -AHX-ALAPYIP or YGRKKRRQRRR- P （X） -AHX-ALAPYIP, where P （x） It is a DHODH-binding peptide. The amino acid sequence of the DHODH peptide degrader is shown in Table 2.

[0048] Table 2. Amino acid sequences of DHODH peptide degraders

[0049] Example 2 uses isothermal titration calorimetry (ITC) to detect the binding ability of DHODH peptide degraders to DHODH proteins. I. In vitro prokaryotic expression and purification of DHODH protein 1. Constructing a prokaryotic expression plasmid for the DHODH protein

[0050] After the PCR product was confirmed to be correct by sequencing, it was combined with... Nde I and BamH The pET-19b vector, digested with enzyme I, was ligated, and the product was transformed into DH5α competent cells and plated onto LB solid medium (containing ampicillin 0.1 mg / mL) and incubated overnight at 37°C. The grown colonies were extracted, and plasmids were extracted and sequenced. The correctly sequenced plasmid, pET-19b-DHODH, was selected for subsequent operations.

[0051] The specific plasmid construction method is as follows: (1) Amplification of the target fragment This method is mainly used for amplification of the gene encoding the DHODH protein and for colony PCR identification of positive clones. The amplification reaction system is shown in Table 3.

[0052] Table 3 PCR amplification reaction system for the target fragment

[0053] (2) Preparation of enzyme digestion vector This method is mainly used for... Nde I and BamH The pET-19b plasmid was subjected to double digestion with restriction endonucleases. The digestion reaction system is shown in Table 4.

[0054] Table 4 Enzyme digestion reaction system

[0055] Add the reagents listed in the table above to the PCR tubes in sequence, mix well, and incubate at 37°C for 1 hour. Then transfer to 65°C for inactivation for 10 minutes.

[0056] The target fragment amplified by PCR and the vector enzyme digestion products were subjected to nucleic acid gel electrophoresis, and the DNA gel was recovered using a Sangon DNA gel recovery kit according to the target molecular weight.

[0057] (3) Ligation of the target fragment with the enzyme digestion vector This method mainly involves ligating the target fragment of the DHODH protein gene and the pET-19b vector fragment to form the target plasmid under the action of ligase. The enzyme ligation reaction system is shown in Table 5.

[0058] Table 5 Enzyme ligation reaction system

[0059] According to the above system, add the reagents to the PCR tube in sequence, mix well, place in a 50℃ metal bath for 30 min, and then place in an ice bath for 5 min after the reaction is completed, followed by transformation.

[0060] (4) Transformation This reaction involves treating chemocompetent cells with CaCl2 to cause plasmid DNA to adhere to the cell membrane. A heat shock then opens the cell membrane, allowing the plasmid DNA to enter the cell. The specific steps are as follows: Thaw competent cells stored at -80°C on ice, add pre-chilled ELISA product, gently tap the bottom of the tube to mix thoroughly, and incubate on ice for 30 min, being careful not to shake. Heat the EP tube in a 42°C metal bath for 90 s, then immediately remove and place on ice for 2 min, being careful not to move the sample during this time. Then, add 800 μL of antibiotic-free LB medium to a biosafety cabinet and incubate at 200 rpm for 60 min at 37°C. Centrifuge the incubated cells at 12000 rpm for 1 min, reserving 100 μL of supernatant in a biosafety cabinet to rehydrate the cell pellet, and spread it onto antibiotic-containing culture plates. Incubate the plates upside down at 37°C overnight. After clones have grown, pick single clones for amplification culture, send a portion of the bacterial culture to a sequencing company for sequencing, and select clones with correct sequencing results for further processing.

[0061] 2. In vitro prokaryotic expression of DHODH protein The pET-19b-DHODH successfully constructed in step 1 above was transformed into BL21 competent cells expressing protein. The bacterial culture was cultured at 37℃ and 220 rpm until OD600 = 0.8~1.0. 0.5 mM inducer IPTG was added and induced at 16℃ and 220 rpm for 16 h.

[0062] 3. Purification of DHODH protein The bacterial cells expressing DHODH protein were harvested by centrifugation at 3000 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 2 As shown.

[0063] This experiment requires three buffer solutions, the formulations of which are as follows: Lysis Buffer: 50 mM Hepes, 300 mM NaCl, 10 mM imidazole, 1% (v / v) Triton X-100 and 10% glycerol, pH 7.7.

[0064] Wash Buffer: 50 mM Hepes, 300 mM NaCl, imidazole (50 mM and 100 mM) and 10% glycerol, pH 7.7.

[0065] Dialysis buffer: 30 mM Hepes, 200 mM NaCl, 10% glycerol and 1 mM TCEP, pH 7.7.

[0066] II. Detection of the binding ability of DHODH peptide degrader to DHODH protein Experimental materials: DHODH protein prepared in step one; DHODH polypeptide degrading agent prepared in Example 1.

[0067] The DHODH protein purified in step one was dialyzed overnight using a dialysis buffer. After dialysis, the concentration was concentrated to 6 mg / mL for later use (the final concentration used in the ITC experiment was 60 μmol / L). An appropriate amount of the buffer from the overnight DHODH protein dialysis was used to dissolve the DHODH peptide degrading agent from Example 1 to prepare a 600 μmol / L DHODH peptide degrading agent solution, which was then used in the ITC experiment.

[0068] Data was processed using MicroCal PEAQ-ITC Analysis Software, which automatically calculated and generated various parameters before exporting the data. Analysis of the fitted curves revealed the affinity constant K between the designed and synthesized DHODH peptide degrader and the in vitro purified DHODH protein. d The values ​​are shown in Table 6. The results show that the DHODH polypeptide degrader designed and synthesized in Example 1 of this invention has a strong binding ability with the DHODH protein purified in vitro.

[0069] Table 6. Binding ability of DHODH peptide degraders to in vitro purified DHODH protein

[0070] Example 3: Inhibitory activity of DHODH peptide degrader against tumor cells 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: 1. Culture of mammalian cells 1) Cell resuscitation: Remove the frozen cells from liquid nitrogen and thaw them in a 42°C water bath. After the cells are completely thawed, centrifuge at 1000 rpm for 5 min, then transfer them 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 shake the dish at multiple angles to ensure even cell distribution. Incubate at 37°C in a 5% CO2 incubator.

[0071] 2) Cell Passaging: When the cell density in the culture dish reaches 80%–90% confluence, passage culture is necessary. 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.

[0072] 2. The DHODH polypeptide degrading agent described in this invention effectively inhibits tumor cell proliferation. (1) Cells: human lung cancer cells A549; human colon cancer cells HCT116; human breast cancer cells MCF7; leukemia cells MOLM13; (2) Reagents: Thiazole blue (MTT) was purchased from Beijing Lanjieke Technology Co., Ltd., and dimethyl sulfoxide (DMSO) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (3) Experimental instruments: ELISA reader (Bio Tek, USA); (4) Experimental method: The cell proliferation experiment used the thiazolyl blue (MTT) assay. The specific principle is that in living cells, succinate dehydrogenase in mitochondria can reduce MTT to blue-purple crystal formazan, which is insoluble in water but soluble in DMSO and has specific absorption at 490 nm.

[0073] The specific process is as follows: Human colon cancer cells HCT116, lung cancer cells A549, breast cancer cells MCF7, and leukemia cells MOLM13 in logarithmic growth phase were seeded in 96-cell culture plates at a density of 3000 cells / 100 μL per well. After 24 h, solutions containing different concentrations of the DHODH peptide degrading agent prepared in Example 1 of this invention were added. Nine 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 for 72 h. After 72 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 DMSO was added to each well. The plates were then shaken at low speed for 10 min. The absorbance of each well was measured at OD490 nm using an ELISA reader. Experimental results show that the DHODH polypeptide degrader prepared in Example 1 of this invention has a good inhibitory effect on various tumor cells. Cell inhibition IC50 50 The values ​​are shown in Table 7.

[0074] Table 7. Inhibitory effect of DHODH peptide degrader on tumor cells (IC50) 50

[0075] Example 4: DHODH Peptide Degrading Agent P 13 DHODH degradation activity in HCT116 and A549 cells This example discusses the DHODH polypeptide degrader prepared in Example 1. P 13 The degradation activity of DHODH in HCT116 and A549 cells was investigated using the following methods: 1. Culture of mammalian cells The specific implementation method is the same as in Example 3, 1. Culture of mammalian cells.

[0076] 2. The DHODH polypeptide degrading agent of this invention degrades DHODH protein in cells in a concentration-dependent manner. Human colon cancer cells HCT116 and human lung cancer cells A549 were seeded in 6 cm cell culture plates at a density of 20%–30%. After 24 h, DHODH polypeptide degrading agents prepared in Example 1 of this invention at different concentrations were added. P 13Solution. Three drug concentration gradients were established: 20 μM, 10 μM, and 5 μM, with a blank control group included. Cells were then incubated at 37°C for 48 h, after which they were collected and lysed for Western blotting. Results are as follows: Figure 3 As 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.

[0077] Example 5: DHODH Peptide Degrading Agent P 13 Inhibit tumor cell clone formation Experimental materials: DHODH peptide degrading agent prepared in Example 1 P 13 .

[0078] 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 and digest them into single-cell suspensions with trypsin. After cell counting, adjust the cell density to 5×10⁻⁶. 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 4 The results showed that the DHODH polypeptide degrader of the present invention at a concentration of 20 μM effectively inhibited the formation of 3D clones of tumor cells.

[0079] 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 can effectively inhibit tumor cell growth and migration, and has great potential for pharmaceutical development.

[0080] Example 6: DHODH Peptide Degrading Agent P 13 Inhibits the growth of subcutaneous tumors in mice This example discusses the DHODH polypeptide degrader prepared in Example 1. P 13The inhibitory effect on the growth of mouse subcutaneous tumors constructed using human colon cancer cells HCT116 was investigated using the following specific methods: 1. Experimental animals and grouping: Fifteen 6-week-old male nude mice were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd., and randomly divided into 3 groups: control group: 100 μL physiological saline; experimental group 1: polypeptide. P 13 (10 mg / kg, 100 μL), Experimental Group 2: Peptide P 13 (20 mg / kg, 100 μL), 5 animals per group.

[0081] 2. Tumor cell preparation and inoculation: Adherent HCT116 cells were digested with trypsin, washed three times with PBS, and filtered once. 5 × 10⁶ cells were then inoculated. 6 One tumor cell was suspended in 100 μL PBS and subcutaneously injected into the groin area of ​​nude mice.

[0082] 3. Tumor growth observation: The subcutaneous tumor reached 150 mm. 3 Mice were randomly divided into groups and began treatment. In experimental group 1, mice were injected with a polypeptide via the tail vein. P 13 (10 mg / kg), mice in experimental group 2 were injected with the polypeptide via the tail vein. P 13 (20 mg / kg) Control group mice received an equal volume of physiological saline via tail vein injection once daily. Tumor size was measured with calipers, and tumor volume was calculated (volume = length × width). 2 / 2). The result is as follows Figure 5 As shown, compared with the control group, the peptide degrading agent P 13 It can significantly inhibit the growth of tumor cells in mice.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A DHODH polypeptide degrading agent, characterized in that, It consists of a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide that are sequentially connected from the N-terminus to the C-terminus; The cell-penetrating peptide is RRRRRRRR or YGRKKRRQRRR; The structural general formula of the DHODH-binding peptide is SAKTGVQPAHX1 or NSAKTGVQPAHX1, where X1 is KA or K; The E3 ubiquitin ligase VHL-binding peptide is ALAPYIP.

2. The DHODH polypeptide degrading agent according to claim 1, characterized in that, The linker is 6-aminocaproic acid.

3. A nucleic acid molecule encoding the DHODH polypeptide degrader according to claim 1 or 2.

4. Use of the DHODH polypeptide degrader according to claim 1 or 2 or the nucleic acid molecule according to claim 3 in the preparation of a drug for treating DHODH-mediated diseases; The DHODH-mediated disease is cancer; The cancer is one or more of colorectal cancer, lung cancer, breast cancer, and blood cancer.

5. Use of the DHODH polypeptide degrader according to claim 1 or 2 or the nucleic acid molecule according to claim 3 in the preparation of a drug for inhibiting cancer cells; The cancer cells are one or more of human lung cancer cells, human colon cancer cells, human breast cancer cells, and blood cancer cells.

6. The application according to claim 5, characterized in that, The human lung cancer cells are human lung cancer cells A549; the human colon cancer cells are human colon cancer cells HCT116; the human breast cancer cells are human breast cancer cells MCF7; the blood cancer cells are blood cancer cells MOLM13.

7. The application according to claim 5, characterized in that, The inhibition of cancer cells is one or more of inhibiting cancer cell proliferation, growth, and migration.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the DHODH polypeptide degrader according to claim

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