A dhodh polypeptide degrading agent and use thereof in the preparation of a medicament for preventing and / or treating dhodh-mediated diseases

Abstract

The application provides a DHODH polypeptide degrading agent and application thereof in preparation of a medicine for preventing and / or treating a DHODH-mediated disease, and belongs to the technical field of biological medicine.The application provides a DHODH polypeptide degrading agent, which comprises, from N-terminal to C-terminal, a cell-penetrating peptide, a DHODH binding peptide, a linker and an E3 ubiquitin ligase VHL binding peptide connected in sequence.The DHODH polypeptide degrading agent provided by the application can efficiently bind to DHODH protein, and has good antitumor effect at the cellular level and the animal level.The DHODH polypeptide degrading agent has a good application prospect in preparation of a medicine for preventing and treating a DHODH-mediated disease, and can make up for the defects of single type and insufficient efficacy of existing medicines for treating DHODH-related diseases, and has important significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a DHODH polypeptide degrading agent and application thereof in preparation of a medicine for preventing and / or treating a DHODH-mediated disease. BACKGROUND

[0002] In mammalian cells, there are generally two pathways for pyrimidine synthesis metabolism, one is a de novo synthesis pathway taking aspartate and glutamine as raw materials to generate uridine monophosphate (UMP) under the catalysis of a trifunctional enzyme CAD, dihydroorotate dehydrogenase (DHODH) and uridine monophosphate synthetase UMPS, and the other is a salvage synthesis pathway taking pyrimidine metabolites in the blood circulation as raw materials. When the cell is in a resting state or completely differentiated, the pyrimidine salvage synthesis pathway can meet the growth needs of the cell, and in rapidly proliferating cells such as cancer cells, the pyrimidine de novo synthesis pathway is the main source of pyrimidine products.

[0003] The key rate-limiting step in the pyrimidine de novo synthesis pathway catalyzed by DHODH is the only reaction connecting the pyrimidine de novo synthesis pathway with the mitochondrial oxidative respiratory chain in the cell, which directly affects the oxidative phosphorylation of the cell and the metabolic adaptability of the cell when facing oxidative stress. DHODH is a biological enzyme containing flavin mononucleotide (FMN), which catalyzes the fourth reaction in the pyrimidine de novo synthesis pathway to dehydrogenate dihydroorotate to orotate. DHODH has been reported to be closely related to the occurrence and development of tumors in various cancers.

[0004] However, at present, all DHODH inhibitors are almost directed to the CoQ binding active pocket thereof, and inhibit the biological enzyme activity of DHODH by competitively binding DHODH with CoQ, and the types are single. SUMMARY

[0005] The purpose of the present application is to provide a DHODH polypeptide degrading agent and application thereof in preparation of a medicine for preventing and / or treating a DHODH-mediated disease, which can overcome the defects and deficiencies of the limited types of existing DHODH inhibitors.

[0006] The application provides a DHODH polypeptide degradation agent, which comprises, from N terminus to 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 DHODH binding peptide has a general structure of X1KTGVQX2K or X1KTGVQX2KX3, wherein X1, X2 and X3 are respectively composed of any one or several 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.

[0007] Preferably, the linker is 6-aminohexanoic acid.

[0008] Preferably, X1 is G, SA, NSA or DINSA; X2 is PAH, GIPHG or NTGNK; and X3 is A.

[0009] 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.

[0010] Preferably, the amino acid sequence of the DHODH binding peptide comprises one or several of the amino acid sequences shown in SEQ ID NO. 8-21.

[0011] The application also provides a nucleic acid molecule encoding the DHODH polypeptide degradation agent described in the above scheme.

[0012] The application also provides use of the DHODH polypeptide degradation agent described in the above scheme or the nucleic acid molecule in the preparation of a medicament for preventing and / or treating a DHODH-mediated disease.

[0013] Preferably, the DHODH-mediated disease comprises one or several of a tumor, an autoimmune disease and a viral infection.

[0014] Preferably, the tumor comprises one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, brain glioma and blood cancer.

[0015] The application also provides a pharmaceutical composition comprising the DHODH polypeptide degradation agent described in the above scheme and a pharmaceutically acceptable carrier.

[0016] The application provides a DHODH polypeptide degradation agent, which comprises, from N terminus to C terminus, a cell-penetrating peptide, a DHODH binding peptide, a linker and an E3 ubiquitin ligase VHL binding peptide connected in sequence. The DHODH polypeptide degradation agent provided by the application can efficiently bind to DHODH protein, and has good antitumor effect at the cellular level and the animal level. The DHODH polypeptide degradation agent has good application prospect in the preparation of drugs for preventing and treating DHODH-mediated diseases, and can make up for the defects of single drug type and insufficient efficacy in the existing treatment of DHODH-related diseases, and has important significance. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 It is a structural schematic diagram of the DHODH polypeptide degradation agent;

[0019] Figure 2 It is a DHODH protein purification gel map;

[0020] Figure 3 It is a DHODH protein expression map in cells after intervention of the DHODH polypeptide degradation agent;

[0021] Figure 4 It is a cell micrograph after 3D cloning of tumor cells intervened by the DHODH polypeptide degradation agent;

[0022] Figure 5 It is a result map of inhibition of subcutaneous tumor growth in mice by the DHODH polypeptide degradation agent. DETAILED DESCRIPTION

[0023] The application provides a DHODH polypeptide degradation agent, which comprises, from N terminus to 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 structural general formula of the DHODH binding peptide is X1KTGVQX2K or X1KTGVQX2KX3, wherein X1, X2 and X3 are respectively composed of any one or several 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 (SEQ ID NO. 3).

[0024] The structural formula of the DHODH polypeptide degradation agent of the application is as follows:Figure 1 The N-terminal of the DHODH binding peptide is connected with a transmembrane peptide, and the C-terminal is connected with an E3 ubiquitin ligase VHL binding peptide through a linker.

[0025] Based on the interaction between DHODH and STAT3, the key site on the STAT3 protein combined with DHODH is selected to design a DHODH binding peptide. According to the transport properties of substances across the cell membrane, the N-terminal of the DHODH binding peptide is connected with a transmembrane peptide. According to the degradation peptide properties, an E3 ubiquitin ligase VHL binding peptide is designed. The DHODH binding peptide and the E3 ubiquitin ligase VHL binding peptide are connected through a linker.

[0026] The DHODH polypeptide degrading agent described in the application binds to the DHODH protein through the DHODH binding peptide, binds to the E3 ubiquitin ligase VHL through the E3 ubiquitin ligase VHL binding peptide, and induces the formation of a DHODH protein-polypeptide degrading agent-E3 ubiquitin ligase VHL ternary complex. The ternary complex effectively recruits the E3 ligase to polyubiquitinate the DHODH protein, which is then degraded by the 26S proteasome. The DHODH polypeptide degrading agent of the application can efficiently bind to the DHODH protein, effectively degrade the DHODH at the cellular level, and has high antitumor activity against colorectal cancer, lung cancer, breast cancer, blood cancer and the like.

[0027] In the specific implementation process of the application, the amino acid sequence of the DHODH polypeptide degrading agent is synthesized by Sangon Biotech.

[0028] In the application, the amino acid sequence of KTGVQ is SEQ ID NO. 4.

[0029] In the specific implementation process of the application, the linker is 6-aminohexanoic acid, abbreviated as AHX.

[0030] In the specific implementation process of the application, 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.

[0031] In the specific implementation process of the application, 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.

[0032] In the embodiment of the present application, the nucleotide sequence of the gene encoding the cell penetrating peptide RRRRRRRR is shown as SEQ ID NO. 50, specifically: cgccgccgccgccgccgccgccgc. In the embodiment of the present application, the nucleotide sequence of the gene encoding the cell penetrating peptide YGRKKRRQRRR is shown as SEQ ID NO. 51, specifically: Tatggccgcaaaaaacgccgccagcgccgccgc.

[0033] In the embodiment of the present application, the nucleotide sequence of the gene encoding the E3 ubiquitin ligase VHL binding peptide is shown as SEQ ID NO. 52, specifically: gcgctggcgccgtatattccg.

[0034] In the embodiment of the present application, the amino acid sequence of the DHODH binding peptide comprises one or more of the amino acid sequences shown as SEQ ID NO. 8-21, and further, the amino acid sequence and the nucleotide sequence of the gene encoding the DHODH binding peptide are shown as Table 1.

[0035] Table 1 Amino acid sequence and nucleotide sequence of the gene encoding the DHODH binding peptide

[0036]

[0037] In the implementation of the present application, when the cell penetrating peptide is RRRRRRRR, 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 penetrating peptide is YGRKKRRQRRR, 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.

[0038] In the embodiment of the present application, the DHODH polypeptide degrading agent as P 1～28 is shown as follows:

[0039] P1: RRRRRRRR-GKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 22);

[0040] P2: RRRRRRRR-GKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 23);

[0041] P3: RRRRRRRR-SAKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 24);

[0042] P4: RRRRRRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 25);

[0043] P5: RRRRRRRR-SAKTGVQPAHK-AHX-ALAPYIP (SEQ ID NO. 26);

[0044] P6: RRRRRRRR-SAKTGVQPAHKA-AHX-ALAPYIP (SEQ ID NO. 27);

[0045] P7: RRRRRRRR-NSAKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 28);

[0046] P8: RRRRRRRR-NSAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 29);

[0047] P9: RRRRRRRR-NSAKTGVQPAHK-AHX-ALAPYIP (SEQ ID NO. 30);

[0048] P 10 : RRRRRRRR-NSAKTGVQPAHKA-AHX-ALAPYIP (SEQ ID NO. 31);

[0049] P 11 : RRRRRRRR-DINSAKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 32);

[0050] P 12 : RRRRRRRR-DINSAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 33);

[0051] P 13 : RRRRRRRR-DINSAKTGVQNTGNK-AHX-ALAPYIP (SEQ ID NO. 34);

[0052] P 14 : RRRRRRRR-DINSAKTGVQNTGNKA-AHX-ALAPYIP (SEQ ID NO. 35);

[0053] P 15 : YGRKKRRQRRR-GKTGVQGIPHGK-AHX-ALAPYIP (SEQ ID NO. 36);

[0054] P 16: YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0055] P 17 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0056] P 18 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0057] P 19 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0058] P 20 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0059] P 21 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0060] P 22 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0061] P 23 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0062] P 24 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0063] P 25 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0064] P 26 : YGRKKRRQRRR-SAKTGVQGIPHGKA-AHX-ALAPYIP (SEQ ID NO. 39);

[0065] P 27 : YGRKKRRQRRR-DINSAKTGVQNTGNK-AHX-ALAPYIP (SEQ ID NO. 48);

[0066] P 28 : YGRKKRRQRRR-DINSAKTGVQNTGNK-AHX-ALAPYIP (SEQ ID NO. 48);

[0067] In the amino acid sequences of SEQ ID NO. 22-SEQ ID NO. 49 of the present application, "-" only represents a connection, for example, P1: RRRRRRRR-GKTGVQGIPHGK-AHX-ALAPYIP is RRRRRRRRGKTGVQGIPHGKAHXALAPYIP.

[0068] The present application also provides a nucleic acid molecule of the DHODH polypeptide degrading agent described in the above scheme.

[0069] The present application also provides the use of the DHODH polypeptide degrading agent described in the above scheme or the nucleic acid molecule in the preparation of a medicament for preventing and / or treating a DHODH-mediated disease.

[0070] In the specific implementation of the present application, the DHODH-mediated disease includes one or more of tumors, autoimmune diseases, and viral infections.

[0071] In the specific implementation of the present application, the medicament includes an anticancer drug.

[0072] In the specific implementation of the present application, the tumor includes one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, brain glioma, and blood cancer.

[0073] The present application also provides a pharmaceutical composition comprising the DHODH polypeptide degrading agent described in the above scheme and a pharmaceutically acceptable carrier.

[0074] In the specific implementation of the present application, the DHODH polypeptide degrading agent is the only active ingredient of the pharmaceutical composition.

[0075] In the specific implementation of the present application, the dosage form of the medicament or pharmaceutical composition includes an injection, a capsule, or a tablet.

[0076] In order to further illustrate the present application, a DHODH polypeptide degrading agent and its use in the preparation of a medicament for preventing and / or treating a DHODH-mediated disease are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0077] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0078] Example 1 Design and synthesis of DHODH polypeptide degrader

[0079] 1. Design of DHODH polypeptide degrader targeting DHODH

[0080] Based on the interaction between DHODH and STAT3, the key site on STAT3 protein that binds to DHODH was selected, and DHODH binding peptide P was designed (X) . According to the transport properties of substances across the cell membrane, a transmembrane peptide was connected to the N terminus of the DHODH binding peptide, and the amino acid sequence of the transmembrane peptide was RRRRRRRR or YGRKKRRQRRR. According to the properties of the degradation peptide, the E3 ubiquitin ligase VHL binding peptide was designed, and the amino acid sequence was ALAPYIP. The DHODH binding peptide and the E3 ubiquitin ligase VHL binding peptide were connected by a linker: 6-aminohexanoic acid (AHX).

[0081] 2. Synthesis of DHODH polypeptide degrader

[0082] The amino acid sequence of the DHODH polypeptide degrader described in the present application is synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Sangon Biotech), and the structure of the DHODH polypeptide degrader is as shown in Figure 1 , the amino acid sequence structure of the DHODH polypeptide degrader is: RRRRRRRR-P (X) -AHX-ALAPYIP or YGRKKRRQRRR-P (X) -AHX-ALAPYIP, wherein P (x) is a DHODH binding peptide. The amino acid sequence of the DHODH polypeptide degrader is shown in Table 2.

[0083] Table 2 Amino acid sequence of DHODH polypeptide degrader

[0084]

[0085]

[0086] Example 2 Detection of the binding ability of DHODH polypeptide degrader and DHODH protein by Isothermal Titration Calorimetry (ITC)

[0087] I. In vitro prokaryotic expression and purification of DHODH protein

[0088] 1. Construction of DHODH protein prokaryotic expression plasmid

[0089]

[0090] PCR product is sequenced to identify the correct one, then it is connected with Nde I and BamHI enzyme cut pET-19b vector, the product is transformed into DH5a competent and coated to LB solid medium (containing ampicillin 0.1 mg / mL), placed at 37℃ overnight. The growing colonies are extracted and plasmid extraction is performed, then sequencing is performed, and the correct plasmid pET-19b-DHODH is selected for subsequent operation.

[0091] The specific plasmid construction method is as follows:

[0092] (1) Target fragment amplification

[0093] This method is mainly used for amplification of DHODH protein coding gene and colony PCR identification of positive clones. The amplification reaction system is shown in Table 3.

[0094] Table 3 PCR amplification reaction system of target fragment

[0095]

[0096]

[0097] (2) Preparation of enzyme cut vector

[0098] This method is mainly used for double enzyme cutting reaction of pET-19b plasmid using Nde I and BamHI restriction endonuclease. The enzyme cutting reaction system is shown in Table 4.

[0099] Table 4 Enzyme cutting reaction system

[0100]

[0101] Add the reagents in the above table into the PCR tube in turn, blow evenly, and then place in a 37℃ oven for 1h. Then transfer to 65℃ for 10min.

[0102] Perform nucleic acid gel electrophoresis on the PCR amplified target fragment and the enzyme cut product, and use the Genomics DNA gel recovery kit to recover the DNA gel according to the desired molecular weight.

[0103] (3) Connection of target fragment and enzyme cut vector

[0104] This method is mainly to connect the DHODH protein gene target fragment and the pET-19b vector fragment under the action of ligase to form the target plasmid. The enzyme ligation reaction system is shown in Table 5.

[0105] Table 5 Enzyme ligation reaction system

[0106] Reagent Volume Linear carrier 50 ng Fragment of interest 100 ng 2x Seamless Cloning Mix 10 μL ddH2O to 20 μL

[0107] According to the above system, reagents are sequentially added into the PCR tube, mixed uniformly, and placed in a 50°C metal bath for reaction for 30 min. After reaction, ice bath for 5 min, and then transformation is performed.

[0108] (4) Transformation

[0109] The reaction is performed by treating the chemically competent cells with CaCl2, adhering the plasmid DNA to the cell membrane, opening the cell membrane by heat shock, and allowing the plasmid DNA to enter the cell. The specific operation is as follows:

[0110] The competent cells stored in a -80°C refrigerator are thawed on ice, and the pre-cooled enzyme product is added. The tube bottom is gently stirred with fingers to mix the whole system. Incubate on ice for 30 min, and do not shake. Place the EP tube in a 42°C metal bath for heat shock for 90 s, and immediately take it out and place it on ice for 2 min. Do not move the sample during this period. Then add 800 μL of antibiotic-free LB medium in a biological safety cabinet, and shake in a 37°C shaker at a speed of 200 rpm for 60 min. Centrifuge the recovered cells at 12000 rpm for 1 min. Blow the cell pellet with 100 μL of supernatant in a biological safety cabinet, and spread it on a culture plate containing an antibiotic. The plate is inverted in a 37°C incubator and incubated overnight. After the clones grow, a single clone is picked for amplification culture. Part of the bacterial solution is sent to a sequencing company for sequencing, and the clone with correct test results is selected for subsequent operation.

[0111] 2. In vitro prokaryotic expression of DHODH protein

[0112] The pET-19b-DHODH successfully constructed in the above step 1 is transferred into the protein expression BL21 competent cells. The bacterial solution is cultured at 37°C and 220 rpm until OD600 = 0.8-1.0. 0.5 mM inducer IPTG is added, and the induction is performed at 16°C and a speed of 220 rpm for 16 h.

[0113] 3. Purification of DHODH protein

[0114] The DHODH protein-expressing bacteria in Step 2 above were harvested by centrifugation at 3000 rpm for 30 min, resuspended in Lysis Buffer, and subjected to ultrasonic disruption for 30 min. The inclusion bodies and cell debris were separated from the supernatant by high-speed centrifugation at 12000 rpm for 30 min, and the supernatant containing a large amount of DHODH protein was obtained. The supernatant was slowly added to a NiNTA resin column, and the target protein was fully bound to the resin. The impurities on the resin were washed with a low-concentration imidazole (50 mM, 100 mM) buffer, and the target protein was eluted with a buffer containing 500 mM imidazole. The protein was concentrated and the imidazole concentration in the protein was reduced by using an ultrafiltration concentration tube. For the entire process of protein purification, the experimental results were verified by using SDS-PAGE, and the results showed that a large amount of DHODH protein was successfully purified in vitro, as shown in Figure 2

[0115] Three buffers were used in this experimental operation, and the formulations are as follows:

[0116] Lysis Buffer: 50 mM Hepes, 300 mM NaCl, 10 mM imidazole, 1% (v / v) Triton X-100, and 10% glycerol, pH 7.7.

[0117] Wash Buffer: 50 mM Hepes, 300 mM NaCl, imidazole (50 mM and 100 mM), and 10% glycerol, pH 7.7.

[0118] Dialysis buffer: 30 mM Hepes, 200 mM NaCl, 10% glycerol, and 1 mM TCEP, pH 7.7.

[0119] II. Detection of the binding ability of DHODH polypeptide degrading agent to DHODH protein

[0120] Experimental materials: DHODH protein prepared in Step 1; DHODH polypeptide degrading agent prepared in Example 1.

[0121] The DHODH protein purified in Step 1 was dialyzed overnight using the dialysis buffer, and after dialysis, it was concentrated to 6 mg / mL for standby use (the final concentration for ITC experiment was 60 μmol / L). An appropriate amount of DHODH polypeptide degrading agent after overnight dialysis was dissolved in the buffer to prepare a DHODH polypeptide degrading agent solution with a concentration of 600 μmol / L, and then the ITC experiment was performed.

[0122] ​The data was processed by MicroCal PEAQ-ITC Analysis Software, various parameters were automatically calculated by the software, and finally the data was exported. From the data fitting curve analysis, the affinity constant K d of the DHODH polypeptide degrading agent designed and synthesized in Example 1 to the in vitro purified DHODH protein was calculated, and the value is shown in Table 6. The results show that the DHODH polypeptide degrading agent designed and synthesized in Example 1 has strong binding capacity to the in vitro purified DHODH protein.

[0123] Table 6 Binding capacity of DHODH polypeptide degrading agent to in vitro purified DHODH protein

[0124] Polypeptide name DHODH binding activity (K d values) Polypeptide name DHODH binding activity (K d values) [P2] 3.70 μM P 15 ]]> 2.53 μM [P2] 2.43 μM P 16 ]]> 4.16 μM [P3] 3.02 μM P 17 ]]> 4.32 μM [P4] 3.11 μM P 18 ]]> 3.41 μM [P5] 4.58 μM P 19 ]]> 4.16 μM [P6] 2.15 μM P 20 ]] 3.03 μM [P7] 1.37 μM P 21 ]] 2.16 μM [P8] 1.51 μM P 22 ]]> 2.00 μM [P9] 2.26 μM P 23 ]]> 1.98 μM P 10 ]]> 917 nM P 24 ]]> 1.03 μM P 11 ]]> 1.08 μM P 25 ]]> 967 nM P 12 ]]> 1.02 μM P 26 ]]> 1.32 μM P 13 ]]> 523 nM P 27 ]]> 761 nM P 14 ]]> 603 nM P 28 ]]> 790 nM

[0125] Example 3 Inhibition activity of DHODH polypeptide degrading agent on tumor cells

[0126] This example discusses the inhibition activity of the DHODH polypeptide degrading agent prepared in Example 1 on tumor cells, and the specific implementation method is as follows:

[0127] 1. Culture of mammalian cells

[0128] 1) Cell recovery: Take out the frozen cells in liquid nitrogen and place them in a 42°C water bath to thaw. After the cells are completely melted, centrifuge at 1000 rpm for 5 min, then transfer to a biological safety cabinet for sterile operation. Open the frozen tube cap, aspirate the supernatant, and suspend the cell pellet with 1 mL of complete medium (DMEM + 10% FBS + 1% penicillin-streptomycin double antibiotic), transfer to a culture dish or culture bottle, supplement the medium, and shake the culture dish at multiple angles to make the cells evenly distributed, and place it in a 37°C, 5% CO2 incubator for culture.

[0129] 2) Cell passage: When the cell density in the culture dish reaches 80%-90% confluence, it needs to be passaged. Take the cells out of the incubator and place them in a biological safety cabinet. After aspirating the old medium, slowly add 1 mL of sterile PBS solution along the wall of the culture dish, cover the dish, and gently shake the culture dish to fully rinse the bottom of the dish with PBS. Remove the PBS with a gun, and repeat 1 time. Immerse the culture dish bottom with 1 mL of trypsin digestion solution, then remove the trypsin digestion solution. Digest the cells in a 37°C incubator, and the digestion time varies depending on the type of cells. After digestion, take the culture dish out of the incubator and observe the cell morphology under a microscope to determine whether the cells are completely digested. In the biological safety cabinet, terminate the digestion with complete medium containing 10% FBS and repeatedly blow the adherent cells with a gun to obtain a cell suspension. Transfer an appropriate amount of cell suspension to a new culture dish, supplement the medium, and gently shake to evenly distribute the cells in the culture dish. Continue cell culture in the cell culture incubator.

[0130] 2、The DHODH polypeptide degradation agent effectively inhibits tumor cell proliferation

[0131] (1) Cells: human lung cancer cells A549, human colon cancer cells HCT116, human breast cancer cells MCF7, and blood cancer cells MOLM13;

[0132] (2) Reagents: thiazolyl blue (MTT) purchased from Beijing Lanjiekeli Technology Co., Ltd., and dimethyl sulfoxide (DMSO) purchased from Shanghai Maklin Biochemical Technology Co., Ltd.;

[0133] (3) Experimental instruments: enzyme label instrument (Bio Tek, USA);

[0134] (4) Experimental method: the cell proliferation experiment in this case uses the thiazolyl blue (MTT) test method. The specific principle is that in living cells, succinate dehydrogenase in mitochondria can reduce MTT to blue-purple crystalline formazan, and formazan is insoluble in water but can be dissolved in DMSO, and has specific absorption at 490 nm.

[0135] The specific process is as follows:

[0136] Take human colon cancer cells HCT116, lung cancer cells A549, breast cancer cells MCF7, and blood cancer cells MOLM13 in the logarithmic growth phase and place them in a 96 culture plate, with a plating density of 3000 cells per 100 μL per well. After 24 h, add a solution containing different concentrations of the DHODH polypeptide degradation agent prepared in Example 1 of the present application. Nine drug treatment concentration gradients are set: 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. Then place in a 37°C incubator for culture, and after 72 h, add 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) per well, and continue to culture for 4 h. Remove the culture medium supernatant, add 150 μL of DMSO per well, and place on a shaking bed for low-speed shaking for 10 min. Measure the absorbance value of each well at OD 490 nm on an enzyme-linked immunodetection instrument. The experimental results show that the DHODH polypeptide degradation agent prepared in Example 1 of the present application has good inhibitory effect on each tumor cell. The cell inhibition IC 50 value is shown in Table 7.

[0137] Table 7: Inhibitory effect of DHODH polypeptide degradation agent on tumor cells IC 50

[0138]

[0139]

[0140] Example 4 DHODH polypeptide degrading agent P 13 DHODH degrading activity in HCT116 cells and A549 cells

[0141] This example discusses the DHODH polypeptide degrading agent P prepared in Example 1 13 DHODH degrading activity in HCT116 cells and A549 cells, the specific implementation method is as follows:

[0142] 1. Cultivation of mammalian cells

[0143] The specific implementation method is the same as that of 1. Cultivation of mammalian cells in Example 3.

[0144] 2. The DHODH polypeptide degrading agent of the present application degrades DHODH protein in cells in a concentration-dependent manner

[0145] Human colon cancer cells HCT116 and human lung cancer cells A549 were plated in 6 cm cell culture plates at a plating density of 20% to 30%. After 24 h, different concentrations of the DHODH polypeptide degrading agent P prepared in Example 1 of the present application were added to the solution. Three drug treatment concentration gradients were set: 20 μM, 10 μM, 5 μM, and a blank control group was set. Then they were placed in a 37 °C incubator for culture, and after 48 h, the cells were collected and lysed for immunoblotting experiments. The results are shown in 13 Figure 3 The DHODH polypeptide degrading agent of the present application degrades DHODH protein in cells in a concentration-dependent manner, and the degradation effect is 90% at 20 μM.

[0146] Example 5 DHODH polypeptide degrading agent P 13 Inhibition of tumor cell colony formation

[0147] Experimental materials: DHODH polypeptide degrading agent P prepared in Example 1 13 .

[0148] 3D colony formation experiment The specific implementation method is as follows: 1.2% (w / v) and 0.7% (w / v) agarose were prepared, sterilized by autoclaving, and then placed in a 42 °C water bath for standby. 2X DMEM medium containing 20% FBS and 2% penicillin-streptomycin double antibody was prepared. Take 1.2% (w / v) agarose gel and 2X DMEM medium at a ratio of 1:1, and pour 2 mL per well in a 6-well plate, and cool it at room temperature until it solidifies. Then take logarithmic growth phase HCT116 cells and human lung cancer cells A549, and digest them into single cell suspension with trypsin. After cell counting, the cell density was adjusted to 5 × 10 4 ​cells / mL. Mix 0.7% (w / v) agarose and 2X DMEM medium at a ratio of 1:1, take 2 mL into an EP tube, add 20 μL of cell suspension, mix thoroughly, and then add to the 6-well plate with the underlayer glue. After the upper layer agar solidifies, place it in a 37°C incubator for 2 weeks. Take pictures under a microscope. Figure 4 The results show that the DHODH polypeptide degrading agent of the present application at a concentration of 20 μM effectively inhibits 3D colony formation of tumor cells.

[0149] The above experiments prove that the DHODH degrading agent of the present application can efficiently bind to DHODH protein and effectively degrade DHODH protein. In addition, the DHODH degrading agent of the present application can effectively inhibit tumor cell growth and migration, and has great potential for medical development.

[0150] Example 6 DHODH polypeptide degrading agent P 13 Inhibition of subcutaneous tumor growth in mice

[0151] This example discusses the DHODH polypeptide degrading agent P 13 prepared in Example 1 for inhibiting the growth of subcutaneous tumors in mice constructed using human colon cancer cells HCT116, and the specific implementation method is as follows:

[0152] 1. Experimental animals and grouping: 15 six-week-old male nude mice were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. and randomly divided into three groups: control group: 100 μL of normal saline, experimental group 1: polypeptide P 13 (10 mg / kg, 100 μL), and experimental group 2: polypeptide P 13 (20 mg / kg, 100 μL), with 5 mice in each group.

[0153] 2. Tumor cell preparation and inoculation: Adherent cultured HCT116 cells were trypsinized, washed with PBS three times, and filtered once through a mesh. 5 x 10 6 cells were suspended in 100 μL of PBS and inoculated subcutaneously into the groin of the nude mice.

[0154] 3. Tumor growth observation: When the subcutaneous tumor reached 150 mm 3 , the mice were randomly grouped and treatment began. Experimental group 1 mice were injected with polypeptide P 13 (10 mg / kg) via the tail vein, experimental group 2 mice were injected with polypeptide P 13 (20 mg / kg) via the tail vein, and the control group mice were injected with the same volume of normal saline via the tail vein once a day. The tumor size was measured using a vernier caliper, and the tumor volume was calculated (volume = long diameter x wide diameter2 / 2). The results are shown in Figure 5 Compared with the control group, polypeptide degrading agent P 13 can significantly inhibit the growth of tumor cells in mice.

[0155] Although the above embodiments have been described in detail, they are only some embodiments of the present application, not all embodiments. Other embodiments can be obtained on the basis of the above embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A DHODH polypeptide degrading agent, characterized in that, consists of, from N-terminus to 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 DHODH binding peptide is DINSAKTGVQNTGNK or DINSAKTGVQNTGNKA; the E3 ubiquitin ligase VHL binding peptide is ALAPYIP.

2. The DHODH polypeptide degrading agent of claim 1, wherein, the linker is 6-aminohexanoic acid.

3. Use of the DHODH polypeptide degradation agent of claim 1 or 2 in the preparation of a medicament for preventing and / or treating a DHODH-mediated disease; the DHODH-mediated disease is a tumor; the tumor is one or more of colorectal cancer, lung cancer, breast cancer and blood cancer.

4. Use of the DHODH polypeptide degradation agent of claim 1 or 2 in the preparation of a medicament for inhibiting tumor cells; the tumor cells are one or more of human lung cancer cells A549, human colon cancer cells HCT116, human breast cancer cells MCF7 and blood cancer cells MOLM13; the inhibition of tumor cells is one or more of inhibition of tumor cell proliferation, growth and migration.

5. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises the DHODH polypeptide degradation agent of claim 1 or 2 and a pharmaceutically acceptable carrier.

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