Isolated polypeptide targeting MPP8 protein and application thereof

Abstract

The invention relates to isolated polypeptide targeting MPP8 protein and application of the isolated polypeptide, and belongs to the technical field of biological medicine. According to the scheme, a bifunctional molecule with high affinity is formed by utilizing a small molecule chip synthesis technology and a solid phase polypeptide synthesis technology and combining an azide-alkyne cycloaddition reaction, and the affinity and functions of the bifunctional molecule are comprehensively verified through methods such as SPR, Western blot and cell proliferation. Compared with an existing technical scheme, the method has the advantages that the synthesis cost is reduced, the reaction speed is increased, the reliability of quality control and function verification is enhanced, the stability and consistency of the compound are ensured, and the method is suitable for large-scale production and application and has a wide market application scene.

Description

Technical Field

[0001] The present application relates to an isolated polypeptide targeting MPP8 protein and its application, belonging to the field of biomedical technology. Background Art

[0002] UNC5246 is a peptidomimetic ligand against the chromatin domain of mitotic phosphoprotein 8 (MPP8). MPP8 is a member of the human silencing hub (HUSH) complex and also a reader of histone 3 lysine 9 trimethylation (H3K9me3), which is crucial for heterochromatin formation and has a specific role in cancer metastasis.

[0003] However, as a peptidomimetic ligand, UNC5246 has a relatively complex structure, with multiple synthesis steps and difficult purification. This not only increases the synthesis cost but also may lead to quality differences between batches, affecting the stability and consistency of the compound.

[0004] The development technology of UNC5246 compound shows significant advantages in screening and identifying high-affinity ligands, but it also has disadvantages such as high cost, slow reaction speed, and high error rate. These disadvantages limit its large-scale application and rapid development to a certain extent.

[0005] Small molecule microarray synthesis technology (SMM) integrates different small molecule drugs (or compounds to be screened) on a solid-phase carrier to form a microarray, also known as a small molecule drug chip. This technology can efficiently synthesize and screen a large number of compounds.

[0006] Therefore, it is necessary to provide a compound product with a relatively simple structure, a more convenient synthesis and purification process, easier quality control, and a lower error rate. Summary of the Invention

[0007] To solve the above problems, an isolated polypeptide targeting MPP8 protein and its application are provided. It is synthesized by small molecule microarray synthesis technology, and polypeptides with good affinity are screened using fluorescently labeled MPP8 as the target protein. A bifunctional molecule is formed by coupling an alkyne fragment with a pomalidomide azide derivative, and the screening results and its function are further confirmed by methods such as surface plasmon resonance (SPR), western blot, and cell proliferation, which have important significance for the drug research and development targeting MPP8 protein.

[0008] The present application provides an isolated polypeptide targeting MPP8 protein, and the polypeptide has any one of the following structures: 。

[0010] In this application, an isolated polypeptide with high-efficiency targeting of the MPP8 protein is screened through chip combined with fluorescence quantitative detection. It has a strong ability to target the MPP8 protein and can be used for the preparation of drug compounds targeting the MPP8 protein.

[0011] Optionally, at least one amino acid in the isolated polypeptide is modified; The modification includes at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification, C-amidation modification, N-palmitoylation modification, N-myristoylation modification, and lipidation modification.

[0012] This application provides the use of the above-mentioned isolated polypeptide targeting the MPP8 protein in the preparation of drugs targeting the MPP8 protein, molecular diagnosis, and protein interaction research.

[0013] This application provides a polypeptide compound targeting the MPP8 protein, which includes the above-mentioned isolated polypeptide targeting the MPP8 protein, Lys, an alkynyl-containing linking unit, and an azide-modified E3 ligand connected in sequence.

[0014] Optionally, a linking peptide is further provided between the isolated polypeptide targeting the MPP8 protein and the Lys. The linking peptide is selected from divalent structural fragments composed of one or more of the following groups: C1 alkylene, 6-10 membered arylene, 5-6 membered heteroarylene, -(R-)-, carboxyl, -O-, residues of amino acids or their analogs; the amino acids are natural amino acids or non-natural amino acids; Optionally, the amino acids are selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met.

[0015] The linking peptide is used to connect the target protein ligand molecules, which are the polypeptide targeting the MPP8 protein and the E3 ubiquitin ligase in this application. It mainly plays a connecting function. By adjusting the length and chemical structure of the linking peptide, the degradation efficiency and in vivo half-life of the bifunctional degradation molecule can be optimized. The selection of the linking peptide is a conventional technique for those skilled in the art and can be selected according to needs.

[0016] Optionally, the amino acid is bAla. It can be selected according to empirical rules based on its advantages in flexibility and length. In addition, compared with other amino acids, bAla can effectively increase the length of the linking peptide and has no obvious side reaction sites.

[0017] Optionally, the alkynyl-containing linking unit is selected from 4-pentynoic acid, 6-heptynoic acid, 3-butynoic acid, 8-nonynoic acid, 3-(2-propyn-1-yloxy)propanoic acid, Hex-4-ynoic acid, Hexadec-15-ynoic acid, Dec-9-ynoic acid, Dodec-11-ynoic acid, Tridec-12-ynoic acid, Undec-10-ynoic acid, 2,2-dimethylbut-3-ynoic acid, 2,2-dimethylpent-4-ynoic acid, 3,6,9,12-tetraoxapentadec-14-ynoic acid, 3,6,9,12,15-pentaoxaoctadec-17-ynoic acid acid, Propargyl-peg6-acid, Propargyl-peg7-acid, Octadec-17-ynoic acid, 13-Tetradecynoic acid, (E)-Pent-2-en-4-ynoic acid, 4-Oxooct-7-ynoic acid, 4,4-Dimethylnon-8-ynoic acid.

[0018] The above-mentioned alkynyl-containing linking units all contain alkynyl and carboxyl groups, and are C54 (4-Pentynoic acid) or its analogs, which can be connected to the azidated E3 ligand and react with the amino group in Lys, thereby realizing the covalent connection between the azidated E3 ligand and the polypeptide, forming a bifunctional compound that targets the MPP8 protein and promotes the degradation of MPP8.

[0019] Optionally, the E3 ligand is selected from pomalidomide and its derivatives or (S, R, S)-AHPC and its derivatives.

[0020] Pomalidomide and (S,R,S)-AHPC are both common E3 ligands that can be azidated and covalently linked to polypeptides via alkyne-containing linking units. It should be noted that other E3 ligands or their derivatives other than pomalidomide and (S,R,S)-AHPC can also achieve the same function, exert their effects through similar mechanisms, and achieve the same technical effects.

[0021] It should be noted that the selection of the alkyne-linked unit-containing and azidated E3 ligand is one way to link the drug to the polypeptide. In addition, the following alternative strategies can also be used for linking: 1) Esterification reaction; 2) Amidation reaction; 3) Thioether bond reaction; 4) Disulfide exchange reaction; 5) Ether bond reaction; 6) Olefin metathesis reaction. Those skilled in the art can select other drugs other than the E3 ligand according to needs and make selections based on the commonly used drug linking methods in the art.

[0022] This application provides a method for preparing the above-mentioned polypeptide compound targeting the MPP8 protein, and the preparation method includes the following steps: 1) Sequentially perform polypeptide ligation synthesis on each component of the polypeptide compound targeting the MPP8 protein except for the azidated E3 ligand; 2) Prepare the azidated E3 ligand; 3) Perform an addition reaction on the product of step 1) and the product of step 2) to obtain the polypeptide compound targeting the MPP8 protein.

[0023] Optionally, when the E3 ligand in the azidated E3 ligand is selected from pomalidomide, the preparation of the azidated E3 ligand in step 2) includes the following reactions: .

[0024] It should be noted that the azidated E3 ligand in this application is synthesized by itself. Of course, the purchased azidated E3 ligand can also be directly used. The azidation method of the E3 ligand is not specifically limited, and the commonly used azidation methods in the art can be used. The above method is only one of the many methods to achieve the azidation of the E3 ligand.

[0025] This application provides the use of the above-mentioned polypeptide compound targeting the MPP8 protein in the preparation of a drug for degrading the MPP8 protein.

[0026] The beneficial effects of this application include but are not limited to: 1. According to the isolated polypeptide targeting the MPP8 protein and its application in this application, although the UNC5246 small molecule chip synthesis technology in the prior art is efficient but costly, the solid-phase polypeptide synthesis technology is adopted in this application, which can reduce the synthesis cost by about 84%.

[0027] 2. According to the isolated polypeptide targeting the MPP8 protein and its application in this application, the synthesis and purification processes of UNC5246 in the prior art are complex and the quality control is difficult. In this application, by simplifying the synthesis and purification steps, the reliability of quality control is improved, and the stability and consistency of the compound are ensured. The purity verified by HPLC > 98%.

[0028] 3. The isolated polypeptide targeting MPP8 protein according to the present application and its applications. In the prior art, although UNC5246 verified the affinity of the compound by methods such as surface plasmon resonance (SPR), the functional verification methods were relatively single. However, the present invention comprehensively verified the functions of the compound through various experimental methods, such as Western blot and cell proliferation experiments, and verified the targeting and drug efficacy from multiple dimensions.

[0029] 4. The isolated polypeptide targeting MPP8 protein according to the present application and its applications. Compared with UNC5246, the solution of the present application has significant advantages in terms of synthesis cost, reaction speed, and quality control. The development technology of the present application not only reduces the production cost, but also improves the reaction speed and the reliability of quality control, making it more suitable for large-scale production and application.

[0030] 5. The isolated polypeptide targeting MPP8 protein according to the present application and its applications. The alkynyl fragment is coupled with the azide derivative of pomalidomide to form a bifunctional molecule, providing a synergistic targeting degradation effect and having various potential biological activities and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings described herein are used to provide a further understanding of the present application, and constitute a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation to the present application. In the drawings: Figure 1 is the synthesis flow chart of the polypeptide compound targeting MPP8 protein involved in Example 2 of the present application; Figure 2 is the LC-MC identification result chart of the polypeptide compound targeting MPP8 protein involved in Example 2 of the present application; Figure 3 is the result chart of the chip affinity performance test (fluorescence reading value) of the polypeptide compound targeting MPP8 protein and MPP8 protein involved in Test Example 2 of the present application; Figure 4 is the result chart of the degradation of MPP8 protein by the compounds of Examples 2 to 12 involved in Test Example 2 of the present application in molm13 leukemia cells; Figure 5 is the result chart of the degradation of MPP8 protein by the compound of Example 2 involved in Test Example 2 of the present application in molm13, THP-1, and MV4-11 leukemia cells; Figure 6 is the result chart of the degradation of MPP8 protein by the compound of Example 2 involved in Test Example 2 of the present application in MC38 solid tumor cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The present application will be described in detail below in conjunction with embodiments. However, the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0033] The present application relates to small molecule chip synthesis technology. Its technical principle: Small molecule chip synthesis technology (Small Molecule Microarray, SMM) is to integrate different small molecule drugs (or compounds to be screened) on a solid-phase carrier to form a microarray, also known as a small molecule drug chip. This technology can efficiently synthesize and screen a large number of compounds. Specific steps: 1) Chip preparation: Select a suitable solid-phase carrier, such as polystyrene resin or gold surface, and fix small molecule compounds on the carrier. 2) Synthesis: Synthesize the target compound on the chip through chemical synthesis methods, such as solid-phase synthesis or solution-phase synthesis. 3) Screening: Incubate the fluorescently labeled MPP8 protein with the compounds on the chip, and screen out the compounds with high affinity for MPP8 through the fluorescence signal.

[0034] It also relates to solid-phase peptide synthesis technology. Its technical principle: Solid-phase peptide synthesis (Solid-Phase Peptide Synthesis, SPPS) is to construct a polypeptide chain by gradually adding amino acid units on a solid-phase support. This technology has simple operation, mild reaction conditions, and easy purification, and is suitable for large-scale production.

[0035] Specific steps: 1) Solid-phase carrier preparation: Select a suitable solid-phase carrier, such as polystyrene resin, and fix the first amino acid on the carrier through a covalent bond. 2) Amino acid linkage: Sequentially add other amino acids, and gradually construct a polypeptide chain through deprotection and coupling reactions. 4) Purification: Purify the synthesized polypeptide through methods such as high performance liquid chromatography (HPLC) to ensure that the purity meets the requirements.

[0036] The present application relates to azide-alkyne cycloaddition reaction. Technical principle: Azide-alkyne cycloaddition is an efficient click chemical reaction used to couple a polypeptide containing an alkyne fragment with a pomalidomide azide derivative to form a bifunctional molecule. This reaction has mild conditions, high yield, and is easy to control. Specific steps: 1) Synthesize alkyne polypeptide: Synthesize a polypeptide containing an alkyne fragment through solid-phase peptide synthesis technology. 2) Synthesize azide derivative: Synthesize the azide derivative of pomalidomide. 3) Coupling reaction: In a suitable solvent, mix the alkyne polypeptide with the azide derivative and carry out the azide-alkyne cycloaddition reaction to form a bifunctional molecule. 4) Purification: Purify the coupling product through methods such as HPLC to ensure that the purity meets the requirements.

[0037] It should be noted that the synthesis method of the polypeptide is not limited in this application. For example, solid-phase synthesis technology can be adopted: Solid-phase synthesis technology is a commonly used polypeptide synthesis method. By connecting amino acids one by one on a solid-phase carrier, the polypeptide chain is gradually constructed. This technology has the advantages of simple operation, mild reaction conditions, easy purification, etc., and can be used to synthesize polypeptide compounds similar to those in Example 2. Liquid-phase synthesis technology can also be adopted: Liquid-phase synthesis technology is a traditional polypeptide synthesis method. By gradually connecting amino acids in solution, the polypeptide chain is constructed. This technology can precisely control the reaction conditions and is suitable for synthesizing complex polypeptide structures.

[0038] Those skilled in the art can also carry out multifunctional molecular design based on the technical solution of this application. For example, in addition to coupling the alkyne fragment with the azide derivative of pomalidomide, other multifunctional designs can also be considered, such as introducing other bioactive groups or targeting groups to enhance the multifunctionality and application prospects of the compound. Structural optimization can also be carried out. For example, through computer-aided design and molecular dynamics simulation, the structure of the polypeptide can be optimized to improve its stability and affinity and reduce the synthesis cost.

[0039] The abbreviations and their full names involved in this application are shown in Table 1 below.

[0040] Table 1 Abbreviations and full names of building blocks involved in the solid-phase synthesis method of this application

[0041] Continued Table 1

[0042] Regarding the description of Table 1, some of the building blocks in Table 1 are the abbreviations and full names of the building blocks participating in the solid-phase synthesis method, corresponding to the structural abbreviations after the completion of the synthesis reactions in Examples 2 to 12. Those skilled in the art can determine the structures in Examples 2 to 12 based on Table 1 and the basic principles of the solid-phase synthesis method. For example, LyDDE’ and LysM’ in Table 1 are the same building block, and different numbers are selected to distinguish the final products. The LysDDE’ building block represents the retention of the Dde protecting group, and the LysM’ building block represents the removal of the Dde protecting group and other derivatizations.

[0043] The solution of this application will be described below through specific examples.

[0044] Example 1 1) Synthesis equipment and materials The linear polypeptide is synthesized on a solid-phase polypeptide synthesizer. In the reactor, Fmoc-amino acids are continuously added to the resin support (rink-amide-AM resin) in a known order (from the C'-terminal carboxyl end to the N'-terminal amino end), reacted, and the Fmoc group is deprotected. Finally, the polypeptide is obtained through operations. DMF and DCM are used to dissolve the reagents for the reaction, and they are added along the wall of the reaction tube. After shaking the reaction, the reaction solution is removed by a vacuum pump on the lower side.

[0045] 2) Reagents required for the reaction: DMF = N,N-dimethylformamide; DCM = dichloromethane; HBTU = benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBT = 1-hydroxybenzotriazole; DIPEA = N,N-diisopropylethylamine; Piperidine = piperidine; NMM = N-methylmorpholine; Acetic anhydride = acetic anhydride; TIPS = triisopropylsilane; TFA = trifluoroacetic acid; DTT = dithiothreitol; FA = formic acid; ACN = acetonitrile; HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0046] 3) Polypeptide chain elongation and condensation steps Add the Rink-Amide-AM resin to the reaction tube, add DCM and let it stand for 30 min, and then vacuum out the DCM to complete the resin swelling. Add 20% Piperidine (in DMF) to the reaction tube and shake for 30 min to remove the protecting Fmoc group. Then, mix the amino acid, HBTU, HOBT, DMF, and DIPEA (in DMF) in proportion and add them to the solid-phase reaction tube, and react at room temperature for 90 min for the condensation reaction. After washing with DMF, add 2% Acetic anhydride / 2% DIPEA (in DMF) to protect the unreacted amino acid groups.

[0047] 4) C54 condensation step After LysM is immobilized on the resin, first treat it with a DMF solution containing 2% hydrazine hydrate for 3 minutes to remove the Dde protecting group. Subsequently, mix C54, HBTU, HOBT, DMF, and DIPEA (in DMF) in proportion and add them to the solid-phase reaction tube, and react at room temperature for 90 min for the condensation reaction. After washing with DMF, add 2% Acetic anhydride / 2% DIPEA (in DMF) to protect the unreacted amino acid groups.

[0048] 5) Linear peptide liberation and recovery steps After the synthesis of the required linear peptide is completed, wash the resin once with DMF and then dry it by suction. Add a solution composed of 90% TFA / 5% TIPS / 5% phenol / 5% H2 The cocktail cutting fluid compounded with O. Shake it at room temperature for 2 - 4 h. After the reaction is completed, filter the cutting fluid by suction, and add the filtrate dropwise to 10 times the volume of anhydrous ether at -20 °C overnight. The crude peptide after suction filtration and drying is redissolved in FA / DTT (in H 2 O).

[0049] 6) RP-HPLC purification step Separate the crude peptide solution using a Waters ACQYITY UPLC-C18 column. Mobile phase A: 0.1% FA in H 2 O; Mobile phase B: 0.1% FA in ACN; The chromatographic conditions are 5% B - 100% B, 0.5 ml / min for 3 min. The pure peptide identified by LC-MC is freeze-dried under vacuum.

[0050] 7) Preparation step of pomalidomide azide derivative Dissolve 2-azidoacetic acid (6 e.q.) in dichloromethane, add thionyl chloride (8 e.q.) dropwise under an ice-water bath. After the addition is complete, react at room temperature for 1 hour. Then add pomalidomide (1 e.q. dissolved in NMP) dropwise. After the addition is complete, continue to react at room temperature for 14 hours. After the reaction is completed, add water, extract twice with ethyl acetate, combine the organic phases, wash three times with saturated sodium chloride aqueous solution, and dry over anhydrous sodium sulfate. Concentrate under reduced pressure, purify the crude reaction product by silica gel column chromatography, use dichloromethane / methanol as the eluent, elute by gradient, and collect the eluate containing the target compound. Rotate and evaporate to remove the solvent to obtain the purified product.

[0051] 8) Step of coupling linear peptide with pomalidomide azide derivative Mix the pure peptide (reaction concentration 1 mM) with pomalidomide azide derivative in a molar ratio of 1:1.2 in DMSO. Mix an aqueous copper sulfate solution (1 M) and an aqueous ascorbic acid solution (1 M) in equal volume (the molar ratio to the polypeptide is 1 - 10), and then add it to the polypeptide reaction solution. React at room temperature for 30 minutes. The crude product is purified again by RP-HPLC and identified by LC-MC.

[0052] Example 2 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-His-Pip-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide.

[0053] Its structural formula is as shown below:

[0054] AsFigure 1 As shown, it is a flowchart for specifically preparing specific products using the preparation method of Example 1, and the LC-MC identification results are as follows Figure 2 As shown, the compounds of Examples 3 to 12 were also prepared by the method disclosed in Example 1, except that different raw materials were used.

[0055] Example 3 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-Phe-Ala-Phe-LysN-Ser-bAla-bAla-LysM-C54-Pomalidomide.

[0056] Its structural formula is as follows:

[0057] Example 4 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: C21-Phe-Ala-Phe-LysN-Ser-bAla-bAla-LysM-C54-Pomalidomide.

[0058] Its structural formula is as follows:

[0059] Example 5 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-His-Pro-PGly-LysN-TiAla-bAla-bAla-LysM-C54-Pomalidomide.

[0060] Its structural formula is as follows:

[0061] Example 6 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-His-Pro-PGly-LysN-Ser-bAla-bAla-LysM-C54-Pomalidomide.

[0062] Its structural formula is as follows:

[0063] Example 7 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-His-Ile-Phe-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide。

[0064] Its structural formula is as follows:

[0065] Example 8 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-His-Ile-Phe-LysN-TiAla-bAla-bAla-LysM-C54-Pomalidomide。

[0066] Its structural formula is as follows:

[0067] Example 9 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-Phe-Ala-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide。

[0068] Its structural formula is as follows:

[0069] Example 10 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-NAL-Aze-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide。

[0070] Its structural formula is as follows:

[0071] Example 11 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-NAL-Ala-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide。

[0072] Its structural formula is as follows:

[0073] Example 12 This example provides a polypeptide compound targeting the MPP8 protein, and its molecular formula is as follows: Isn-Phe-Ala-Aze-LysN-TiAla-bAla-bAla-LysM-C54-Pomalidomide.

[0074] Its structural formula is as shown below:

[0075] Test Example 1 The polypeptide in Table 2 below was tested for chip affinity performance with the MPP8 protein, and the test results are as Figure 3 shown, and the fluorescence value was used to characterize the affinity, where A represents greater than or equal to 10000; B represents between 7000 and 9999; C represents between 4000 and 6999; D represents between 3000 and 4000, and the fluorescence value scoring results are shown in Table 2 below.

[0076] Table 2 Chip Affinity Test Results of Polypeptide Sequences with MPP8 Protein

[0077] Continued Table 2

[0078] Figure 3 are the chip affinity performance test results, while the data shown in Table 2 are the fluorescence readings after the fluorescence-labeled MPP-8 protein was incubated with the compound chip. All the polypeptide sequences in Table 2 showed good affinity with the MPP8 protein chip.

[0079] Based on this fluorescence reading, it can be preliminarily judged whether there is an interaction between compounds. In addition, by further selecting compounds with fluorescence readings significantly higher than the background reading, for example, in this application, compounds Figure 3 with fluorescence readings greater than 3000 in were selected for further affinity tests. Specifically, the SPR technology was used to detect the binding ability of the compounds in Examples 2 to 12 to MPP8. The SPR technology can detect the intermolecular interaction in real time and without labels, providing high-precision affinity data.

[0080] The specific steps include: 1) Chip preparation: Fix the MPP8 protein on the SPR chip. 2) Sample injection: Inject the test compound solution into the SPR system and incubate it with the MPP8 protein fixed on the chip. 3) Data acquisition: Real-time monitor the intermolecular interaction through the SPR system and record the binding and dissociation curves. 4) Data analysis: Use SPR software to analyze the binding and dissociation curves and calculate the affinity constant (Kd).

[0081] The results of the affinity test are shown in Table 3 below.

[0082] Table 3 Results of the affinity constant test

[0083] According to the results in Table 3, except for Example 4, Examples 2 to 12 all showed good affinity results. Among them, Example 4 was verified by SPR and showed no concentration dependence, indicating that this binding belongs to non-specific binding.

[0084] Test Example 2 Western blot technique Technical principle: Detect the expression and interaction of target proteins through the specific binding of antigens and antibodies.

[0085] Specific steps: 1) Protein extraction: Extract total protein from cells and lyse the protein using RIPA lysis buffer. 2) Electrophoresis: Perform SDS-PAGE electrophoresis on the protein samples in a polyacrylamide gel to separate proteins of different molecular weights (80V for 20 min, 120V for 60 min). 3) Transfer: Transfer the separated proteins to a PVDF membrane (220 mA for 100 min). 4) Blocking: Block the non-specific binding sites on the membrane with a blocking solution (5% skim milk powder) (60 min). 5) Primary antibody incubation: Add a specific primary antibody against MPP8 or the test compound and incubate overnight at 4°C. 6) Secondary antibody incubation: Add a secondary antibody that specifically binds to the primary antibody and incubate at room temperature for 1 hour. 7) Color development: Add an ECL chemiluminescent substrate and detect the chemiluminescent signal through an imaging system.

[0086] After detection, Examples 2 to 12 could degrade MPP8 protein to varying degrees in molm13 cells, and the degradation rate was detected using the ImageJ gray scale quantification method. The degradation rate results of Examples 2 to 12 are shown in Table 4 below.

[0087] Table 4 Results of the MPP8 protein degradation efficiency

[0088] Subsequently, the results of the degradation of MPP8 protein by the compound of Example 2 were detected again in multiple leukemia cells such as molm-13, THP-1, and MV4-11, and the results are as Figure 5 shown.

[0089] Furthermore, the results of the degradation of MPP8 protein by the compound of Example 2 were detected in MC38 solid tumor cells, and the results are as Figure 6 shown.

[0090] According to Figure 6The results show that the compounds of Example 2 at different concentrations can also degrade the MPP8 protein in solid tumor cells such as MC38.

[0091] In summary, the solution of this application utilizes the small molecule chip synthesis technology and solid-phase peptide synthesis technology, combines the azide-alkyne cycloaddition reaction to form a bifunctional molecule with high affinity, and comprehensively verifies its affinity and function through methods such as SPR, Western blot, and cell proliferation. Compared with the existing technical solutions, this technical solution not only reduces the synthesis cost, improves the reaction speed, but also enhances the reliability of quality control and function verification, ensuring the stability and consistency of the compounds.

[0092] As described above, the above are only the embodiments of this application. The protection scope of this application is not limited by these specific embodiments, but is determined by the claims of this application. For those skilled in the art, various changes and modifications can be made to this application. Any modifications, equivalent replacements, improvements, etc. made within the technical idea and principle of this application shall be included within the protection scope of this application.

Claims

1. An isolated polypeptide targeting MPP8 protein, characterized in that: The polypeptide has any of the following structures: 。 2. The isolated polypeptide targeting MPP8 protein according to claim 1, characterized in that At least one amino acid in the isolated polypeptide is modified; The modification includes at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification, C-amidation modification, N-palmitoylation modification, N-myristoylation modification and lipidation modification.

3. Use of the isolated polypeptide targeting MPP8 protein as claimed in claim 1 or 2 in the preparation of drugs targeting MPP8 protein, molecular diagnosis and protein interaction research.

4. A polypeptide compound targeting MPP8 protein, characterized in that: The polypeptide compound targeting the MPP8 protein comprises the isolated polypeptide targeting the MPP8 protein according to claim 1 or 2, Lys, an alkyne-containing linking unit and an azidated E3 ligand which are sequentially connected.

5. The polypeptide compound targeting MPP8 protein according to claim 4, characterized in that: A connecting short peptide is also provided between the isolated polypeptide targeting the MPP8 protein and the Lys, and the connecting short peptide is selected from a divalent structural fragment consisting of one or more of the following groups: a C1 alkylene group, a 6-10-membered arylene group, a 5-6-membered heteroarylene group, -(R-)-, a carboxyl group, -O-, a residue of an amino acid or its analog; the amino acid is a natural amino acid or a non-natural amino acid; Optionally, the amino acid is selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met.

6. The polypeptide compound targeting MPP8 protein according to claim 4, characterized in that: The alkynyl-containing connecting unit is selected from 4-Pentynoic acid, 6-Heptynoic acid, 3-Butynoic Acid, 8-Nonynoicacid, 3-(2-Propyn-1-yloxy)propanoic Acid, Hex-4-ynoic acid, Hexadec-15-ynoic acid, Dec-9-ynoic acid, Dodec-11-ynoic acid, Tridec-12-ynoic acid, Undec-10-ynoic acid, 2,2-Dimethylbut-3-ynoic acid, 2,2-Dimethylpent-4-ynoic acid, 3,6,9,12-Tetraoxapentadec-14-ynoic acid, 3,6,9,12,15-Pentaoxaoctadec-17-ynoic acid acid, Propargyl-peg6-acid, Propargyl-peg7-acid, Octadec-17-ynoic acid, 13-Tetradecynoic Acid, (E)-Pent-2-en-4-ynoic acid, 4-Oxooct-7-ynoic acid, 4,4-Dimethylnon-8-ynoic acid.

7. The polypeptide compound targeting MPP8 protein according to claim 4, characterized in that: The E3 ligand is selected from pomalidomide and its derivatives or (S, R, S)-AHPC and its derivatives.

8. The method for preparing a polypeptide compound targeting MPP8 protein according to any one of claims 4 to 7, characterized in that: The preparation method comprises the following steps: 1) Sequentially synthesize peptide connections of various elements in the peptide compound targeting the MPP8 protein except the azidated E3 ligand; 2) preparing the azidated E3 ligand; 3) subjecting the product of step 1) to an addition reaction with the product of step 2 to obtain the polypeptide compound targeting the MPP8 protein.

9. The method for preparing a polypeptide compound targeting MPP8 protein according to claim 8, characterized in that: When the E3 ligand in the azidated E3 ligand is selected from pomalidomide, the step 2) preparing the azidated E3 ligand comprises the following reaction: 。 10. Use of the polypeptide compound targeting MPP8 protein according to any one of claims 4 to 7 in the preparation of a drug for degrading MPP8 protein.

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