DHODH polypeptide degradation agent and application thereof in preparation of medicine for preventing and / or treating DHODH-mediated diseases

Abstract

The invention provides a DHODH polypeptide degradation agent and application of the DHODH polypeptide degradation agent in preparation of drugs for preventing and / or treating DHODH-mediated diseases, and belongs to the technical field of biological medicines. The invention provides a DHODH polypeptide degradation agent. The DHODH polypeptide degradation agent comprises a cell-penetrating peptide, a DHODH binding peptide, a linker and an E3 ubiquitin ligase VHL binding peptide which are sequentially connected from an N terminal to a C terminal. The DHODH polypeptide degradation agent provided by the invention can be efficiently combined with DHODH protein, and has a good anti-tumor effect on the cellular level and the animal level. The compound has a good application prospect in preparation of drugs for preventing and treating DHODH-mediated diseases, overcomes the defects that existing drugs for treating DHODH-related diseases are single in variety and insufficient in curative effect, and has important significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a DHODH polypeptide degrader and its application in the preparation of a drug for preventing and / or treating DHODH-mediated diseases. Background Art

[0002] In mammalian cells, there are usually two pathways for pyrimidine anabolic metabolism. One is the de novo synthesis pathway using aspartic acid and glutamine as raw materials, which generates uridine monophosphate (UMP) under the catalysis of the trifunctional enzyme CAD, dihydroorotate dehydrogenase (DHODH), and uridine monophosphate synthetase UMPS. The other is the salvage synthesis pathway using pyrimidine metabolites in the blood circulation as raw materials. When cells are in a resting state or fully differentiated, the pyrimidine salvage synthesis pathway can meet the growth needs of the cells. However, 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 catalyzed by DHODH in the pyrimidine de novo synthesis pathway is the only reaction in the cell that connects the pyrimidine de novo synthesis pathway with the mitochondrial oxidative respiratory chain, directly affecting cellular oxidative phosphorylation and the metabolic adaptability of cells in the face of oxidative stress. DHODH is a bi-enzyme containing flavin mononucleotide (FMN), which catalyzes the fourth reaction in the pyrimidine de novo synthesis pathway, dehydrogenating dihydroorotate to generate orotate. DHODH has been reported to be closely related to the occurrence and development of tumors in various cancers.

[0004] However, currently, almost all DHODH inhibitors target its CoQ binding active pocket and inhibit the bi-enzyme activity of DHODH by competitively binding to CoQ with DHODH, with a single variety. Summary of the Invention

[0005] The purpose of the present invention is to provide a DHODH polypeptide degrader and its application in the preparation of a drug for preventing and / or treating DHODH-mediated diseases, which can overcome the defects and deficiencies of the limited variety of existing DHODH inhibitors.

[0006] The present invention provides a DHODH polypeptide degrader, which comprises, 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 structural general formula of the DHODH-binding peptide is X1KTGVQX2K or X1KTGVQX2KX3, wherein X1, X2, and X3 are each composed of any one or several 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.

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

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

[0009] Preferably, X1 is G and X2 is GIPHG; alternatively, X1 is SA and X2 is GIPHG or PAH; alternatively, X1 is NSA and X2 is GIPHG or PAH; alternatively, 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 present invention also provides a nucleic acid molecule encoding the DHODH polypeptide degrader described in the above solution.

[0012] The present invention also provides the use of the DHODH polypeptide degrader described in the above solution or the nucleic acid molecule in the preparation of a drug for preventing and / or treating DHODH-mediated diseases.

[0013] Preferably, the DHODH-mediated diseases include one or several of tumors, autoimmune diseases, and viral infections.

[0014] Preferably, the tumors include one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, glioblastoma, and blood cancer.

[0015] The present invention also provides a pharmaceutical composition, which comprises the DHODH polypeptide degrader described in the above solution and a pharmaceutically acceptable carrier.

[0016] The present invention provides a DHODH polypeptide degrader, which comprises a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide sequentially connected from the N-terminus to the C-terminus. The DHODH polypeptide degrader provided by the present invention can efficiently bind to the DHODH protein and has good anti-tumor effects at the cellular level and the animal level. It has good application prospects in the preparation of drugs for preventing and treating DHODH-mediated diseases, making up for the defects of the existing drugs for treating DHODH-related diseases, such as single variety and insufficient efficacy, and has important significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

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

[0019] Figure 2 It is a purification gel diagram of the DHODH protein;

[0020] Figure 3 It is a diagram of the expression of the DHODH protein in cells after intervention with the DHODH polypeptide degrader;

[0021] Figure 4 It is a microscopic image of cells after the DHODH polypeptide degrader intervenes in the 3D cloning of tumor cells;

[0022] Figure 5 It is a diagram of the results of the DHODH polypeptide degrader inhibiting the growth of subcutaneous tumors in mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The present invention provides a DHODH polypeptide degrader, which comprises a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide sequentially connected from the N-terminus to the C-terminus; 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, where X1, X2, and X3 are each composed of any one or several 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).

[0024] The structural formula of the DHODH polypeptide degrader of the present invention is asFigure 1 As shown; the N-terminus of the DHODH-binding peptide is linked to a transmembrane peptide, and the C-terminus is linked to an E3 ubiquitin ligase VHL-binding peptide through a linker.

[0025] Starting from the interaction between DHODH and STAT3, the present invention selects the key site on the STAT3 protein that binds to DHODH and designs a DHODH-binding peptide. According to the property of substance transmembrane transport, a transmembrane peptide is linked to the N-terminus of the DHODH-binding peptide. According to the property 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 through a linker.

[0026] The DHODH polypeptide degrader of the present invention binds to the DHODH protein through the DHODH-binding peptide, and the E3 ubiquitin ligase VHL-binding peptide binds to the E3 ubiquitin ligase VHL, inducing the formation of a ternary complex of DHODH protein - polypeptide degrader - 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 polypeptide degrader of the present invention can efficiently bind to the DHODH protein, produce an effective DHODH degradation effect at the cellular level, and has high anti-tumor activity against colorectal cancer, lung cancer, breast cancer, blood cancer, etc.

[0027] In the specific implementation process of the present invention, the amino acid sequence of the DHODH polypeptide degrader is synthesized by Sangon Biotech Co., Ltd.

[0028] In the present invention, the amino acid sequence of KTGVQ is numbered as SEQ ID NO.4.

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

[0030] In the specific implementation process of the present 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); X3 is A.

[0031] In the specific implementation process of the present 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.

[0032] In the specific implementation process of the present invention, the nucleotide sequence of the coding gene of the cell-penetrating peptide RRRRRRRR is shown in SEQ ID NO.50, specifically: cgccgccgccgccgccgccgccgc. In the specific implementation process of the present invention, the nucleotide sequence of the coding gene of the cell-penetrating peptide YGRKKRRQRRR is shown in SEQ ID NO.51, specifically: Tatggccgcaaaaaacgccgccagcgccgccgc.

[0033] In the specific implementation process of the present invention, the nucleotide sequence of the coding gene of the E3 ubiquitin ligase VHL-binding peptide is shown in SEQ ID NO.52, specifically: gcgctggcgccgtatattccg.

[0034] In the specific implementation process 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 NOs. 8 to 21. Further, the amino acid sequence and the nucleotide sequence of the coding gene of the DHODH-binding peptide are shown in Table 1.

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

[0036]

[0037] In the specific implementation process of the present invention, when the cell-penetrating peptide is RRRRRRRR, the linker is AHX, the E3 ubiquitin ligase VHL-binding peptide is ALAPYIP, and the DHODH-binding peptides are 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 peptides are GKTGVQGIPHGK, GKTGVQGIPHGKA, SAKTGVQGIPHGK, SAKTGVQGIPHGKA, SAKTGVQPAHK, SAKTGVQPAHKA, NSAKTGVQGIPHGK, NSAKTGVQGIPHGKA, NSAKTGVQPAHK, NSAKTGVQPAHKA, DINSAKTGVQGIPHGK, DINSAKTGVQGIPHGKA, DINSAKTGVQNTGNK or DINSAKTGVQNTGNKA.

[0038] In the embodiment of the present invention, the DHODH polypeptide degrader is as shown in P 1～28 and is specifically 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 - GKTGVQGIPHGKA - AHX - ALAPYIP (SEQ ID NO.37);

[0055] P 17 : YGRKKRRQRRR - SAKTGVQGIPHGK - AHX - ALAPYIP (SEQ ID NO.38);

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

[0057] P 19 : YGRKKRRQRRR - SAKTGVQPAHK - AHX - ALAPYIP (SEQ ID NO.40);

[0058] P 20 : YGRKKRRQRRR - SAKTGVQPAHKA - AHX - ALAPYIP (SEQ ID NO.41);

[0059] P 21 : YGRKKRRQRRR - NSAKTGVQGIPHGK - AHX - ALAPYIP (SEQ ID NO.42);

[0060] P 22 : YGRKKRRQRRR - NSAKTGVQGIPHGKA - AHX - ALAPYIP (SEQ ID NO.43);

[0061] P 23 : YGRKKRRQRRR - NSAKTGVQPAHK - AHX - ALAPYIP (SEQ ID NO.44);

[0062] P 24 : YGRKKRRQRRR - NSAKTGVQPAHKA - AHX - ALAPYIP (SEQ ID NO.45);

[0063] P 25 : YGRKKRRQRRR - DINSAKTGVQGIPHGK - AHX - ALAPYIP (SEQ ID NO.46);

[0064] P 26 : YGRKKRRQRRR - DINSAKTGVQGIPHGKA - AHX - ALAPYIP (SEQ ID NO.47);

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

[0066] P 28 : YGRKKRRQRRR - DINSAKTGVQNTGNKA - AHX - ALAPYIP (SEQ ID NO.49).

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

[0068] The present invention also provides a nucleic acid molecule of the DHODH polypeptide degrader described in the above - mentioned solution.

[0069] The present invention also provides the use of the DHODH polypeptide degrader or the nucleic acid molecule described in the above - mentioned solution in the preparation of a drug for preventing and / or treating DHODH - mediated diseases.

[0070] In the specific implementation process of the present invention, the DHODH - mediated diseases include one or more of tumors, autoimmune diseases, and viral infections.

[0071] In the specific implementation process of the present invention, the drug includes anticancer drugs.

[0072] In the specific implementation process of the present invention, the tumors include one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, glioblastoma, and blood cancer.

[0073] The present invention also provides a pharmaceutical composition, which includes the DHODH polypeptide degrader described in the above - mentioned solution and a pharmaceutically acceptable carrier.

[0074] In the specific implementation process of the present invention, the DHODH polypeptide degrader is the only active ingredient of the pharmaceutical composition.

[0075] In the specific implementation process of the present invention, the dosage form of the drug or pharmaceutical composition includes injection, capsule, or tablet.

[0076] To further illustrate the present invention, the following describes in detail a DHODH polypeptide degrader provided by the present invention and its use in the preparation of a drug for preventing and / or treating DHODH - mediated diseases with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

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

[0078] Example 1 Design and Synthesis of DHODH Polypeptide Degraders

[0079] 1. Design of Targeted DHODH Polypeptide Degraders

[0080] Starting from the interaction between DHODH and STAT3, the key sites on the STAT3 protein that bind to DHODH were selected to design the DHODH-binding peptide P (X) . According to the property of substance transmembrane transport, a cell-penetrating peptide was linked to the N-terminus of the DHODH-binding peptide. The amino acid sequence of this cell-penetrating peptide was RRRRRRRR or YGRKKRRQRRR. According to the property of the degrading peptide, a binding peptide for the E3 ubiquitin ligase VHL was designed, and its amino acid sequence was ALAPYIP. The DHODH-binding peptide and the binding peptide for the E3 ubiquitin ligase VHL were linked by a linker: 6-aminohexanoic acid (AHX).

[0081] 2. Synthesis of DHODH Polypeptide Degraders

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

[0083] Table 2 Amino Acid Sequences of DHODH Polypeptide Degraders

[0084]

[0085]

[0086] Example 2 Detection of the Binding Ability of DHODH Polypeptide Degraders to DHODH Protein by Isothermal Titration Calorimetry (ITC)

[0087] I. In Vitro Prokaryotic Expression and Purification of DHODH Protein

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

[0089]

[0090] After the PCR product was correctly identified by sequencing, it was ligated to the pET-19b vector digested with Nde I and BamHI. The product was transformed into DH5α competent cells and spread on LB solid medium (containing 0.1 mg / mL ampicillin), and cultured overnight at 37°C. The grown colonies were picked and plasmid extraction was performed, followed by sequencing. The plasmid with correct sequencing, namely pET-19b-DHODH, was selected for subsequent operations.

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

[0092] (1) Amplification of target fragment

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

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

[0095]

[0096]

[0097] (2) Preparation of digested vector

[0098] This method is mainly used for the double digestion reaction of pET-19b plasmid with Nde I and BamHI restriction endonucleases. The digestion reaction system is shown in Table 4.

[0099] Table 4 Digestion reaction system

[0100]

[0101] The reagents in the above table were added successively into the PCR tube. After blowing evenly, it was left standing in an oven at 37°C for 1 h. Then it was transferred to inactivate at 65°C for 10 min.

[0102] The PCR amplified target fragment and the vector digestion product were subjected to nucleic acid gel electrophoresis, and DNA gel extraction was performed using Sangon DNA gel extraction kit according to the target molecular weight.

[0103] (3) Ligation of target fragment and digested vector

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

[0105] Table 5 Ligation reaction system

[0106] reagent volume linear vector 50 ng target fragment 100 ng 2×Seamless Cloning Mix 10 μL <![CDATA[ddH2O]]> to 20 μL

[0107] Add the reagents into the PCR tube successively according to the above system, mix well, place it in a 50°C metal bath for reaction for 30 min, after the reaction, place it in an ice bath for 5 min, and then carry out transformation.

[0108] (4) Transformation

[0109] This reaction is carried out by treating chemically competent cells with CaCl2 to make plasmid DNA adhere to the cell membrane, and opening the cell membrane by heat shock, so that plasmid DNA enters the cell. The specific operation is as follows:

[0110] Place the competent cells stored in the -80°C refrigerator on ice to melt, add the pre-cooled ligation product, gently flick the bottom of the tube with your finger to mix the whole system evenly, incubate on ice for 30 min, and pay attention not to shake. Place the EP tube in a 42°C metal bath for heat shock for 90 s, then immediately take it out and place it on ice for 2 min. Pay attention not to move the sample during this period. Then add 800 μL of antibiotic-free LB medium in the biosafety cabinet, and shake and resuscitate at 200 rpm on a 37°C shaker for 60 min. Centrifuge the resuscitated cells at 12000 rpm for 1 min, leave 100 μL of the supernatant in the biosafety cabinet to resuspend the cell pellet, and spread it on a culture plate containing antibiotics. Invert the plate and culture it overnight in a 37°C incubator. After the clones grow out, pick single clones for amplification culture, take part of the bacterial liquid and send it to a sequencing company for sequencing, and select the clones with correct test results for subsequent operations.

[0111] 2. Prokaryotic expression of DHODH protein in vitro

[0112] Transfer the successfully constructed pET-19b-DHODH in step 1 above into protein expression BL21 competent cells, culture the bacterial liquid at 37°C and 220 rpm until OD600 = 0.8 - 1.0, add 0.5 mM inducer IPTG, and induce at 16°C and 220 rpm for 16 h.

[0113] 3. Purification of DHODH protein

[0114] Harvest the bacterial cells expressing DHODH protein in step 2 above by centrifugation at 3000 rpm for 30 min, resuspend the bacterial cells in Lysis Buffer, and perform ultrasonic disruption for 30 min. Separate the inclusion bodies, cell debris, and supernatant by high-speed centrifugation at 12000 rpm for 30 min to obtain the supernatant containing a large amount of DHODH protein. Slowly add the supernatant to the NiNTA resin column to allow the target protein to fully bind to the resin. Wash the miscellaneous proteins on the resin with buffers containing low-concentration imidazole (50 mM, 100 mM) in sequence, elute the target protein with a buffer containing 500 mM imidazole, and concentrate the protein and reduce the imidazole concentration in the protein through an ultrafiltration concentrator tube. For the entire process of protein purification, verify the experimental results by using SDS-PAGE. The results show that a large amount of DHODH protein has been successfully purified in vitro, as Figure 2 shown.

[0115] Three buffers are required for 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. Detect the binding ability of the DHODH polypeptide degrader to the DHODH protein

[0120] Experimental materials: DHODH protein prepared in step 1; DHODH polypeptide degrader prepared in Example 1.

[0121] Dialyze the DHODH protein purified in step 1 overnight with dialysis buffer, and concentrate it to 6 mg / mL for standby after dialysis (the final concentration used in the ITC experiment is 60 μmol / L). Dissolve the DHODH polypeptide degrader of Example 1 with an appropriate amount of buffer after overnight dialysis of the DHODH protein to prepare a DHODH polypeptide degrader solution with a concentration of 600 μmol / L, and then perform the ITC experiment.

[0122] The data was processed by MicroCal PEAQ-ITC Analysis Software, and various parameters were automatically calculated and generated by this software. Finally, the data was exported. From the analysis of the data fitting curve, the affinity constant K of the designed and synthesized DHODH polypeptide degrader with the in vitro purified DHODH protein d value is shown in Table 6. The results show that the DHODH polypeptide degrader designed and synthesized in Example 1 of the present invention has a strong binding ability with the in vitro purified DHODH protein.

[0123] Table 6 Binding ability of DHODH polypeptide degrader with in vitro purified DHODH protein

[0124] polypeptide name <![CDATA[DHODH binding activity (K d value)]]> polypeptide name <![CDATA[DHODH binding activity (K d value)]]> <![CDATA[P1]]> 3.70 μM <![CDATA[P 15 > 2.53 μM <![CDATA[P2]]> 2.43 μM <![CDATA[P 16 > 4.16 μM <![CDATA[P3]]> 3.02 μM <![CDATA[P 17 > 4.32 μM <![CDATA[P4]]> 3.11 μM <![CDATA[P 18 > 3.41 μM <![CDATA[P5]]> 4.58 μM <![CDATA[P 19 > 4.16 μM <![CDATA[P6]]> 2.15 μM <![CDATA[P 20 > 3.03 μM <![CDATA[P7]]> 1.37 μM <![CDATA[P 21 > 2.16 μM <![CDATA[P8]]> 1.51 μM <![CDATA[P 22 > 2.00 μM <![CDATA[P9]]> 2.26 μM <![CDATA[P 23 > 1.98 μM <![CDATA[P 10 > 917 nM <![CDATA[P 24 > 1.03 μM <![CDATA[P 11 > 1.08 μM <![CDATA[P 25 > 967 nM <![CDATA[P 12 > 1.02 μM <![CDATA[P 26 > 1.32 μM <![CDATA[P 13 > 523 nM <![CDATA[P 27 > 761 nM <![CDATA[P 14 > 603 nM <![CDATA[P 28 > 790 nM

[0125] Example 3 Inhibitory activity of DHODH polypeptide degrader on tumor cells

[0126] 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:

[0127] 1. Cultivation of mammalian cells

[0128] 1) Cell resuscitation: Take out the cryopreserved cells in liquid nitrogen, place them in a water bath at 42°C to thaw. After the cells are completely melted, centrifuge at 1000 rpm for 5 min, and then transfer them to a biosafety cabinet for aseptic operation. Open the lid of the cryopreservation tube, aspirate the supernatant, and resuspend the cell pellet thoroughly with 1 mL of complete medium (DMEM + 10% FBS + 1% penicillin-streptomycin double antibody). Transfer it to a culture dish or culture flask, supplement the medium, and shake the culture dish at multiple angles to make the cells evenly distributed. Place it in an incubator at 37°C and 5% CO2 for culture.

[0129] 2) Cell passage: When the cell density in the culture dish reaches 80% - 90% confluence, subculture is required. Take out the cells from the incubator and place them in a biosafety cabinet. After aspirating the old medium, slowly add 1 mL of sterile PBS solution along the wall of the culture dish, cover the dish lid, and gently shake the culture dish to make the PBS fully rinse the bottom of the whole dish. Aspirate the PBS with a pipette and repeat once. Take 1 mL of trypsin digestion solution to moisten the bottom of the culture dish, and then remove the trypsin digestion solution. Place the cells in an incubator at 37°C for digestion. The digestion time varies with different cell types. After digestion, take out the culture dish from the incubator and place it under a microscope to observe the morphological changes of the cells to judge whether the cells are completely digested. In the biosafety cabinet, terminate the digestion with complete medium containing 10% FBS and repeatedly pipette the adherent cells with a pipette to obtain a cell suspension. Transfer an appropriate amount of the cell suspension to a new culture dish, supplement the medium and gently shake it to make the cells evenly distributed in the culture dish, and continue cell culture in the cell incubator.

[0130] 2. The DHODH polypeptide degrader of the present invention effectively inhibits the proliferation of tumor cells.

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

[0132] (2) Reagents: Thiazolyl blue (MTT) was purchased from Beijing LanJieKe Technology Co., Ltd., and dimethyl sulfoxide (DMSO) was purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0133] (3) Experimental instruments: microplate reader (Bio Tek, USA);

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

[0135] The specific procedure 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 seed them in a 96-well culture plate at a seeding density of 3000 cells / 100 μL per well. After 24 h, add the DHODH polypeptide degrader solution prepared in Example 1 of the present invention with different concentrations. Set up 9 drug treatment concentration gradients: 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, and set up a blank control group at the same time. Then place them in an incubator at 37 °C for culture. After 72 h, add 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) to each well and continue to culture for 4 h. Remove the culture medium supernatant, add 150 μL of DMSO to each well, and place it on a shaker for low-speed oscillation for 10 min. Measure the absorbance value of each well at OD 490 nm with an enzyme-linked immunosorbent detector. The experimental results show that the DHODH polypeptide degrader prepared in Example 1 of the present invention has a good inhibitory effect on various tumor cells. The cell inhibition IC 50 values are shown in Table 7.

[0137] Table 7 Inhibitory effect of DHODH polypeptide degrader on tumor cells IC 50

[0138]

[0139]

[0140] Example 4 DHODH polypeptide degrader P 13 DHODH degradation activity in HCT116 cells and A549 cells

[0141] This example discusses the DHODH polypeptide degrader P prepared in Example 1 13 The degradation activity of DHODH in HCT116 cells and A549 cells, and 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 degrader of the present invention 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 a 6 cm cell culture plate with a plating density of 20% - 30%. After 24 h, the DHODH polypeptide degrader P prepared in Example 1 of the present invention containing different concentrations 13 solution was added. Three drug treatment concentration gradients were set up: 20 μM, 10 μM, 5 μM, and a blank control group was set up at the same time. Then it was placed in an incubator at 37°C for culture. After 48 h, the cells were collected and lysed for immunoblotting experiments. The results are as Figure 3 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

[0146] Example 5 DHODH polypeptide degrader P 13 Inhibiting tumor cell colony formation

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

[0148] The specific implementation method of the 3D colony formation experiment is as follows: Prepare 1.2% (w / v) and 0.7% (w / v) agarose, autoclave it and place it in a 42°C water bath for later use. Prepare 2X DMEM medium containing 20% FBS and 2% penicillin-streptomycin double antibody. Take 1.2% (w / v) agarose gel and mix it with 2X DMEM medium at a ratio of 1:1, and spread it in a 6-well plate, 2 mL per well, and wait for it to cool and solidify at room temperature. Then take HCT116 cells and human lung cancer cells A549 in the logarithmic growth phase, digest them into single cell suspensions with trypsin, adjust the cell density to 5×10 4cells / mL. Mix 0.7% (w / v) agarose and 2X DMEM medium in a 1:1 ratio. Take 2 mL and place it in an EP tube. Add 20 μL of the cell suspension. After thorough mixing, add it to a 6-well plate with a lower layer of gel. After the upper layer of agar solidifies, incubate it in a 37°C incubator for 2 weeks, and take pictures under a microscope. As 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.

[0149] The above experiments demonstrated that the DHODH degrader of the present invention could efficiently bind to the DHODH protein and effectively degrade the DHODH protein. In addition, the DHODH degrader of the present invention could effectively inhibit the growth and migration of tumor cells and had great potential for pharmaceutical development.

[0150] Example 6 DHODH polypeptide degrader P 13 Inhibiting the growth of subcutaneous tumors in mice

[0151] This example discussed the DHODH polypeptide degrader P prepared in Example 1 13 The inhibitory effect on the growth of subcutaneous tumors in mice constructed using human colon cancer cell line HCT116. The specific implementation method is as follows:

[0152] 1. Experimental animals and grouping: 15 male nude mice at 6 weeks old were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. and randomly divided into 3 groups: control group: 100 μL of normal saline, experimental group 1: polypeptide P 13 (10 mg / kg, 100 μL), experimental group 2: polypeptide P 13 (20 mg / kg, 100 μL), with 5 mice in each group.

[0153] 2. Preparation and inoculation of tumor cells: The adherently cultured HCT116 cells were digested with trypsin, washed three times with PBS, and filtered once through a filter. Suspend 5×10 6 tumor cells in 100 μL of PBS and inoculate them subcutaneously at the groin of nude mice.

[0154] 3. Observation of tumor growth: When the subcutaneous tumor reached 150 mm 3 , randomly group and start treatment. Mice in experimental group 1 were injected with polypeptide P 13 (10 mg / kg) via the tail vein, mice in experimental group 2 were injected with polypeptide P 13 (20 mg / kg) via the tail vein, and mice in the control group were injected with an equal volume of normal saline once a day. The tumor size was measured with a vernier caliper, and the tumor volume was calculated (volume = long diameter × width diameter 2 / 2). The results are as Figure 5 shown. Compared with the control group, the polypeptide degrader P 13 could significantly inhibit the growth of tumor cells in mice.

[0155] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A DHODH polypeptide degradation agent, characterized in that: From the N-terminus to the C-terminus, it includes a cell-penetrating peptide, a DHODH-binding peptide, a linker, and an E3 ubiquitin ligase VHL-binding peptide that are sequentially connected; 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 respectively composed of any one or several amino acids of G, A, I, P, S, T, Q, D, E, K and H; 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-aminohexanoic acid.

3. The DHODH polypeptide degrading agent according to claim 1, characterized in that The X1 is G, SA, NSA or DINSA; the X2 is PAH, GIPHG or NTGNK; and the X3 is A.

4. The DHODH polypeptide degrading agent according to claim 3, characterized in that The X1 is G and X2 is GIPHG; or, the X1 is SA and X2 is GIPHG or PAH; or, the X1 is NSA and X2 is GIPHG or PAH; or, the X1 is DINSA and X2 is NTGNK or GIPHG.

5. The DHODH polypeptide degrading agent according to claim 4, characterized in that The amino acid sequence of the DHODH binding peptide includes one or more of the amino acid sequences shown in SEQ ID NOs. 8 to 21.

6. A nucleic acid molecule encoding the DHODH polypeptide degrading agent according to any one of claims 1 to 5.

7. Use of the DHODH polypeptide degrading agent according to any one of claims 1 to 5 or the nucleic acid molecule according to claim 6 in the preparation of a drug for preventing and / or treating a DHODH-mediated disease.

8. The use according to claim 7, characterized in that: The DHODH-mediated diseases include one or more of tumors, autoimmune diseases and viral infections.

9. The use according to claim 8, characterized in that: The tumor includes one or more of colorectal cancer, lung cancer, bladder cancer, breast cancer, brain glioma and blood cancer.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the DHODH polypeptide degrading agent according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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