An isolated polypeptide targeting MPP8 protein and its application

Abstract

This application relates to an isolated polypeptide targeting the MPP8 protein and its applications, belonging to the field of biomedical technology. The proposed method utilizes small molecule chip synthesis technology and solid-phase polypeptide synthesis technology, combined with an azide-alkyne cycloaddition reaction, to form a bifunctional molecule with high affinity. Its affinity and function were comprehensively verified using methods such as SPR, Western blot, and cell proliferation. Compared with existing technologies, this method not only reduces synthesis costs and increases reaction speed, but also enhances the reliability of quality control and functional verification, ensuring the stability and consistency of the compound. It is suitable for large-scale production and application, and has broad market application prospects.

Description

Technical Field

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

[0002] UNC5246 is a peptide-like ligand targeting the M-phase phosphoprotein 8 (MPP8) chromatin domain. MPP8 is a member of the human silencing center (HUSH) complex and a reader of histone 3-lysine 9-trimethyl (H3K9me3), which is crucial for heterochromatin formation and plays a specific role in cancer metastasis.

[0003] However, UNC5246, as a peptide-like ligand, has a relatively complex structure, requiring multiple synthetic steps and presenting significant purification challenges. This not only increases synthesis costs but may also lead to batch-to-batch quality variations, affecting the stability and consistency of the compound.

[0004] The development technology for UNC5246 compounds has shown significant advantages in screening and identifying high-affinity ligands, but it also suffers from drawbacks such as high cost, slow reaction speed, and high error rate. These drawbacks limit its large-scale application and rapid development to some extent.

[0005] Small molecule microarray (SMM) technology integrates different small molecule drugs (or compounds to be screened) onto a solid support to form a microarray, also known as a drug small molecule 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, easier synthesis and purification process, easier quality control, and lower error rate. Summary of the Invention

[0007] To address the aforementioned issues, this paper presents an isolated peptide targeting the MPP8 protein and its applications. Synthesized using small molecule chip synthesis technology, the peptide with good affinity was screened using fluorescently labeled MPP8 as the target protein. A bifunctional molecule was formed by coupling an alkyne fragment with a pomalidomide azide derivative. The screening results and its function were further confirmed using surface plasmon resonance (SPR), western blot, and cell proliferation methods. This study is of significant importance for drug development targeting the MPP8 protein.

[0008] This application provides a polypeptide targeting the MPP8 protein, wherein the polypeptide has any of the following structures:

[0009] In this application, a separate polypeptide with high efficiency targeting of MPP8 protein was screened by combining chip and fluorescence quantitative detection. It has a strong ability to target MPP8 protein and can be used for the preparation of drug compounds targeting MPP8 protein.

[0010] Optionally, at least one amino acid in the separated polypeptide is modified; The modifications include at least one of glycosylation, phosphorylation, ubiquitination, S-nitrosylation, N-methylation, O-methylation, N-acetylation, C-amidation, N-palmitoylation, N-myristoylation, and lipidation.

[0011] This application provides the application of the isolated peptides targeting MPP8 protein described above in the preparation of drugs targeting MPP8 protein, molecular diagnostics, and protein-protein interaction studies.

[0012] This application provides a polypeptide compound targeting the MPP8 protein, the polypeptide compound targeting the MPP8 protein comprising, in sequence, the above-mentioned isolated polypeptide targeting the MPP8 protein, Lys, an alkyne-containing linker and an azide-treated E3 ligand.

[0013] Optionally, a linker peptide is further provided between the isolated polypeptide targeting the MPP8 protein and the Lys, wherein the linker peptide is a divalent structural fragment selected from one or more of the following groups: C1 alkylene, 6-10 arylene, 5-6 heteroarylene, -(R-)-, carboxyl, -O-, amino acid residues or analogs thereof; wherein 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, and Met.

[0014] Linker peptides are used to link target protein ligand molecules; in this application, they are peptides targeting MPP8 protein and E3 ubiquitin ligases. Their primary function is linking; by adjusting the length and chemical structure of the linker peptide, the degradation efficiency and in vivo half-life of bifunctional degradation molecules can be optimized. The selection of linker peptides is a conventional technique for those skilled in the art and can be chosen as needed.

[0015] Optionally, the amino acid is bAla. It can be selected based on its advantages in flexibility and length, following empirical rules. Furthermore, compared to other amino acids, bAla can effectively increase the length of short peptides without significant side reaction sites.

[0016] Optionally, the alkynyl-containing linker is selected from 4-Pentynoic acid, 6-Heptynoic acid, 3-Butynoic Acid, 8-Nonynoicacid, 3-(2-Propyn-1-yloxy)propanoic Acid, Hex-4-ynoicacid, 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, and 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.

[0017] The aforementioned alkyne-containing linker units all contain alkyne and carboxyl groups, are C54 (4-Pentynoic acid) or analogs thereof, can be linked to azidated E3 ligands and react with the amino group in Lys, thereby achieving covalent linkage between azidated E3 ligands and peptides, forming bifunctional compounds that target MPP8 protein and promote MPP8 degradation.

[0018] Optionally, the E3 ligand is selected from pomalidomide and its derivatives or (S,R,S)-AHPC and its derivatives.

[0019] Pomalidomide and (S,R,S)-AHPC are both common E3 ligands that can undergo azidation and be covalently linked to peptides via alkyne-containing linker units. It should be noted that other E3 ligands or their derivatives besides pomalidomide and (S,R,S)-AHPC can also achieve the same function, exert their effects through similar mechanisms, and achieve the same technical results.

[0020] It should be noted that the choice of alkynyl linker and azidated E3 ligand is one way to link drugs to peptides. In addition, the following alternative strategies can be used: 1) esterification; 2) amidation; 3) thioether bonding; 4) disulfide exchange; 5) ether bonding; 6) olefin metathesis. Those skilled in the art can choose other drugs besides E3 ligands as needed, based on commonly used drug linking methods in the field.

[0021] This application provides a method for preparing the aforementioned polypeptide compound targeting the MPP8 protein, the method comprising the following steps: 1) Sequentially synthesize peptides by linking each element in the peptide compound targeting the MPP8 protein, except for the azide-treated E3 ligand; 2) Prepare the E3 ligand for azidation; 3) The product of step 1) and the product of step 2 are subjected to an addition reaction to obtain the polypeptide compound targeting the MPP8 protein.

[0022] Optionally, when the E3 ligand in the azidated E3 ligand is selected from pomalidomide, step 2) preparing the azidated E3 ligand includes the following reaction: .

[0023] It should be noted that the azidated E3 ligand in this application is synthesized in-house. Of course, purchased azidated E3 ligands can also be used directly. There are no specific limitations on the azidation method of the E3 ligand. Commonly used azidation methods in the field can be used. The above method is only one of many ways to achieve the azidation of E3 ligand.

[0024] This application provides the use of the aforementioned peptide compound targeting the MPP8 protein in the preparation of a drug that degrades the MPP8 protein.

[0025] The beneficial effects of this application include, but are not limited to: 1. According to the present application of the isolated peptide targeting MPP8 protein and its application, although the existing UNC5246 small molecule chip synthesis technology is efficient, it is also expensive. However, the present application uses solid phase peptide synthesis technology, which can reduce the synthesis cost by about 84%.

[0026] 2. According to the present application, the isolated peptide targeting MPP8 protein and its application, the synthesis and purification process of UNC5246 in the prior art is complicated and the quality control is difficult. However, the present application simplifies the synthesis and purification steps, improves the reliability of quality control, and ensures the stability and consistency of the compound. HPLC verification shows that the purity is >98%.

[0027] 3. Regarding the isolated peptide targeting MPP8 protein and its application in this application, although the affinity of UNC5246 has been verified by methods such as surface plasmon resonance (SPR) in the prior art, the functional verification method is relatively simple. However, this invention comprehensively verifies the function of the compound through a variety of experimental methods, such as Western blot and cell proliferation experiments, and verifies the targeting and efficacy from multiple dimensions.

[0028] 4. The isolated peptide targeting the MPP8 protein and its applications, based on this application, offer significant advantages over UNC5246 in terms of synthesis cost, reaction rate, and quality control. The technology developed in this application not only reduces production costs but also improves reaction rate and the reliability of quality control, making it more suitable for large-scale production and application.

[0029] 5. Based on the isolated peptide targeting MPP8 protein and its application, the alkyne fragment is coupled with a pomalidomide azide derivative to form a bifunctional molecule, providing a synergistic targeted degradation effect, and has a variety of potential biological activities and application prospects. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a flowchart illustrating the synthesis of the polypeptide compound targeting the MPP8 protein involved in Example 2 of this application; Figure 2 This is an LC-MC identification result diagram of the polypeptide compound targeting the MPP8 protein involved in Example 2 of this application; Figure 3 This is a graph showing the results of the chip affinity test (fluorescence readings) between the peptide compound targeting the MPP8 protein and the MPP8 protein involved in Test Example 2 of this application. Figure 4 This is a graph showing the results of the degradation of MPP8 protein in molm13 leukemia cells by the compounds of Examples 2-12 involved in Test Example 2 of this application; Figure 5 This is a graph showing the results of the degradation of MPP8 protein in molm13, THP-1 and MV4-11 leukemia cells by the compound of Example 2 involved in Test Example 2 of this application. Figure 6 This is a graph showing the results of the degradation of MPP8 protein in MC38 solid tumor cells by the compound of Example 2 involved in Test Example 2 of this application. Detailed Implementation

[0031] The present application is described in detail below with reference to the embodiments, but 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.

[0032] This application relates to small molecule microarray synthesis technology. The technical principle is as follows: Small molecule microarray (SMM) technology integrates different small molecule drugs (or compounds to be screened) onto a solid support to form a microarray, also known as a drug small molecule chip. This technology can efficiently synthesize and screen a large number of compounds. Specific steps include: 1) Chip preparation: Selecting a suitable solid support, such as polystyrene resin or a gold surface, and immobilizing the small molecule compound on the support. 2) Synthesis: Synthesizing the target compound onto the chip using chemical synthesis methods, such as solid-phase synthesis or solution-phase synthesis. 3) Screening: Incubating the compound on the chip with fluorescently labeled MPP8 protein, and screening for compounds with high affinity for MPP8 by fluorescence signal.

[0033] It also involves solid-phase peptide synthesis technology, the principle of which is to construct peptide chains by progressively adding amino acid units onto a solid support. This technology is simple to operate, has mild reaction conditions, and is easy to purify, making it suitable for large-scale production.

[0034] Specific steps: 1) Solid support preparation: Select a suitable solid support, such as polystyrene resin, and fix the first amino acid onto the support via covalent bonds. 2) Amino acid linkage: Add other amino acids sequentially, and gradually construct the polypeptide chain through deprotection and coupling reactions. 4) Purification: Purify the synthesized polypeptide using methods such as high-performance liquid chromatography (HPLC) to ensure that the purity meets the requirements.

[0035] This application relates to an azide-alkyne cycloaddition reaction. Technical principle: The azide-alkyne cycloaddition is a highly efficient click chemistry reaction used to couple a polypeptide containing an alkynyl group to a pomalidomide azidide derivative to form a bifunctional molecule. This reaction is mild, has high yield, and is easy to control. Specific steps: 1) Synthesis of alkynyl polypeptide: A polypeptide containing an alkynyl group is synthesized using solid-phase polypeptide synthesis technology. 2) Synthesis of azidide derivative: An azidide derivative of pomalidomide is synthesized. 3) Coupling reaction: In a suitable solvent, the alkynyl polypeptide and the azidide derivative are mixed to undergo an azide-alkyne cycloaddition reaction to form a bifunctional molecule. 4) Purification: The coupling product is purified using methods such as HPLC to ensure the required purity.

[0036] It should be noted that this application does not limit the method of polypeptide synthesis. For example, solid-phase synthesis is a commonly used method for polypeptide synthesis, in which amino acids are linked one by one onto a solid support to gradually build a polypeptide chain. This technique has advantages such as simple operation, mild reaction conditions, and easy purification, and can be used to synthesize polypeptide compounds similar to those in Example 2. Liquid-phase synthesis can also be used: Liquid-phase synthesis is a traditional method for polypeptide synthesis, in which amino acids are linked stepwise in solution to build a polypeptide chain. This technique allows for precise control of reaction conditions and is suitable for synthesizing complex polypeptide structures.

[0037] Those skilled in the art can also design multifunctional molecules based on the technical solutions of this application. For example, in addition to coupling the alkynyl fragment with the pomalidomide azidide derivative, other multifunctional designs can 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 performed, such as optimizing the structure of the peptide through computer-aided design and molecular dynamics simulations to improve its stability and affinity, and reduce synthesis costs.

[0038] The abbreviations and their full names involved in this application are shown in Table 1 below.

[0039] Table 1. Abbreviations and full names of the building blocks involved in the solid-state synthesis method of this application.

[0040] Table 1 (continued)

[0041] Regarding Table 1, some building blocks in Table 1 are abbreviations and full names of building blocks participating in the solid-state synthesis method, corresponding to the structural abbreviations after the synthesis reaction in Examples 2-12. Those skilled in the art can determine the structures in Examples 2-12 based on Table 1 and the basic principles of the solid-state synthesis method. For example, LyDDE' and LysM' in Table 1 are the same building blocks. Different numbers are used to distinguish the final products. The LysDDE' building block represents the retention of the Dde protecting group, while the LysM' building block represents the removal of the Dde protecting group and other derivatization.

[0042] The present application solution will be described below through specific embodiments.

[0043] Example 1 1) Synthetic apparatus and materials Linear peptides were synthesized on a solid-phase peptide synthesizer. In the reactor, Fmoc-amino acids were continuously added to a resin support (rink-amide-AM resin) in a known order (from C'-carboxyl terminus to N'-amino terminus). The reaction proceeded, Fmoc was deprotected, and the peptide was finally obtained. Reagents used in the reaction were dissolved using DMF and DCM and added along the tube wall. After shaking, the reaction solution was removed using a vacuum pump at the bottom.

[0044] 2) Reagents required for the reaction: DMF = N,N-dimethylformamide; DCM = dichloromethane; HBTU = benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; HOBT = 1-hydroxybenzotriazole; DIPEA = N,N-diisopropylethylamine; Piperidine = hexahydropyridine; NMM = N-methylmorpholine; Acetic anhydride = acetic anhydride; TIPS = triisopropylsilane; TFA = trifluoroacetic acid; DTT = dithiothreitol; FA = formic acid; ACN = acetonitrile; HEPES = 4-hydroxyethylpiperazine ethanesulfonic acid.

[0045] 3) Peptide chain elongation and condensation step Rink-Amide-AM resin was added to a reaction tube, DCM was added, and the mixture was allowed to stand for 30 min. The DCM was then removed under vacuum to complete resin swelling. 20% Piperidine (in DMF) was added to the reaction tube and shaken for 30 min to remove the protecting Fmoc groups. Then, amino acids, HBTU, HOBT, DMF, and DIPEA (in DMF) were mixed in the specified proportions and added to a solid-phase reaction tube. The mixture was reacted at room temperature for 90 min to carry out the condensation reaction. After washing with DMF, 2% Acetic anhydride / 2% DIPEA (in DMF) was added to protect the unreacted amino acid groups.

[0046] 4) C54 condensation step After LysM was immobilized on the resin, the mixture was first treated with a DMF solution containing 2% hydrazine hydrate for 3 minutes to remove the Dde protecting groups. Subsequently, C54, HBTU, HOBT, DMF, and DIPEA (in DMF) were mixed in the specified proportions and added to the solid-phase reaction tube, and the reaction was carried out at room temperature for 90 minutes to induce a condensation reaction. After washing with DMF, 2% acetic anhydride / 2% DIPEA (in DMF) was added to protect the unreacted amino acid groups.

[0047] 5) Linear peptide dissociation and recovery steps After the desired linear peptide synthesis was completed, the resin was washed once with DMF and dried under vacuum. A cocktail cleavage solution composed of 90% TFA / 5% TIPS / 5% phenol / 5% H2O was added. The mixture was shaken at room temperature for 2–4 h. After the reaction was complete, the cleavage solution was filtered, and the filtrate was added dropwise to 10 volumes of anhydrous diethyl ether at -20°C overnight. The filtered and dried crude peptide was redissolved in FA / DTT (in H2O).

[0048] 6) RP-HPLC purification step The crude peptide solution was separated using a Waters ACQYITY UPLC-C18 column. Mobile phase A: 0.1% FA in H2O; Mobile phase B: 0.1% FA in ACN; Chromatographic conditions: 5% B-100% B, 0.5 ml / min, 3 min. The purified peptides, identified by LC-MC, were then lyophilized under vacuum.

[0049] 7) Preparation steps of pomalidomide azidated derivatives 2-Azide-based acetic acid (6 eq) was dissolved in dichloromethane, and sulfoxide (8 eq) was added dropwise under an ice-water bath. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 hour. Then, pomalidomide (1 eq dissolved in NMP) was added dropwise, and the reaction was continued at room temperature for 14 hours. After the reaction was complete, water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed three times with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and purified by silica gel column chromatography using a gradient elution with dichloromethane / methanol. The eluent containing the target compound was collected. The solvent was removed by rotary evaporation to obtain the purified product.

[0050] 8) Linear peptide coupling with pomalidomide azidated derivatives step The purified peptide (reaction concentration 1 mM) was mixed with pomalidomide azide derivative in DMSO at a molar ratio of 1:1.2. Equal volumes of copper sulfate aqueous solution (1 M) and ascorbic acid aqueous solution (1 M) were mixed (molar ratio to peptide of 1-10) and added to the peptide reaction solution. The reaction was carried out at room temperature for 30 minutes. The crude product was purified again by RP-HPLC and identified by LC-MC.

[0051] Example 2 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-His-Pip-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide.

[0052] Its structural formula is shown below:

[0053] like Figure 1 The diagram shows a flowchart illustrating the preparation of a specific product using the method described in Example 1. The LC-MC identification results are shown below. Figure 2 As shown, the compounds of Examples 3-12 were prepared using the same method disclosed in Example 1, except that the raw materials used were different.

[0054] Example 3 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-Phe-Ala-Phe-LysN-Ser-bAla-bAla-LysM-C54-Pomalidomide.

[0055] Its structural formula is shown below:

[0056] Example 4 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: C21-Phe-Ala-Phe-LysN-Ser-bAla-bAla-LysM-C54-Pomalidomide.

[0057] Its structural formula is shown below:

[0058] Example 5 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-His-Pro-PGly-LysN-TiAla-bAla-bAla-LysM-C54-Pomalidomide.

[0059] Its structural formula is shown below:

[0060] Example 6 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-His-Pro-PGly-LysN-Ser-bAla-bAla-LysM-C54-Pomalidomide.

[0061] Its structural formula is shown below:

[0062] Example 7 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-His-Ile-Phe-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide.

[0063] Its structural formula is shown below:

[0064] Example 8 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-His-Ile-Phe-LysN-TiAla-bAla-bAla-LysM-C54-Pomalidomide.

[0065] Its structural formula is shown below:

[0066] Example 9 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-Phe-Ala-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide.

[0067] Its structural formula is shown below:

[0068] Example 10 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-NAL-Aze-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide.

[0069] Its structural formula is shown below:

[0070] Example 11 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-NAL-Ala-Aze-LysN-PheNt-bAla-bAla-LysM-C54-Pomalidomide.

[0071] Its structural formula is shown below:

[0072] Example 12 This embodiment provides a polypeptide compound that targets the MPP8 protein, with the following molecular formula: Isn-Phe-Ala-Aze-LysN-TiAla-bAla-bAla-LysM-C54-Pomalidomide.

[0073] Its structural formula is shown below:

[0074] Test Example 1 The microarray affinity of the peptides listed in Table 2 to the MPP8 protein was tested, and the results are as follows: Figure 3 As shown in Table 2, the affinity was characterized by fluorescence values, where A represents greater than or equal to 10000; B represents between 7000 and 9999; C represents between 4000 and 6999; and D represents between 3000 and 4000. The fluorescence value scoring results are shown in Table 2 below.

[0075] Table 2. Results of chip affinity tests between peptide sequences and MPP8 protein.

[0076] Table 2 (continued)

[0077] Figure 3 Table 1 shows the results of the chip affinity performance test, while Table 2 shows the fluorescence readings of the fluorescently labeled MPP-8 protein after incubation with the compound chip. The polypeptide sequences in Table 2 all showed good affinity with the MPP8 protein chip.

[0078] The fluorescence reading can be used to preliminarily determine whether there is an interaction between the compounds. Furthermore, by selecting compounds with fluorescence readings significantly higher than the background reading, such as those selected in this application... Figure 3 Compounds with fluorescence readings greater than 3000 underwent further affinity testing. Specifically, the binding affinity of the compounds in Examples 2-12 to MPP8 was detected using SPR technology. SPR technology can detect intermolecular interactions in real time without labeling, providing high-precision affinity data.

[0079] The specific steps include: 1) Chip preparation: Immobilizing the MPP8 protein onto an SPR chip. 2) Sample injection: Injecting the solution of the analyte into the SPR system and incubating it with the MPP8 protein immobilized on the chip. 3) Data acquisition: Monitoring the intermolecular interactions in real time using the SPR system and recording binding and dissociation curves. 4) Data analysis: Analyzing the binding and dissociation curves using SPR software and calculating the affinity constant (Kd).

[0080] The affinity test results are shown in Table 3 below.

[0081] Table 3. Affinity constant test results

[0082] As shown in Table 3, all examples from 2 to 12, except for Example 4, exhibited good affinity results. Specifically, Example 4, verified by SPR, showed no concentration dependence, indicating that the binding was non-specific.

[0083] Test Example 2: Western blot technique Technical principle: The expression and interaction of target proteins are detected through the specific binding of antigens and antibodies.

[0084] Specific steps: 1) Protein extraction: Extract total protein from cells and lyse the proteins using RIPA lysis buffer. 2) Electrophoresis: Perform SDS-PAGE electrophoresis on a polyacrylamide gel to separate proteins of different molecular weights (80V 20min, 120V 60min). 3) Transfer: Transfer the separated proteins to a PVDF membrane (220 mA 100min). 4) Blocking: Block non-specific binding sites on the membrane with blocking buffer (5% skim milk powder) (60min). 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) Colorimetric development: Add an ECL chemiluminescent substrate and detect the chemiluminescent signal using an imaging system.

[0085] Tests showed that Examples 2-12 could degrade MPP8 protein to varying degrees in molm13 cells. The degradation rate was detected using ImageJ grayscale quantification. The degradation rate results of Examples 2-12 are shown in Table 4 below.

[0086] Table 4 Results of MPP8 protein degradation efficiency

[0087] Subsequently, the compound of Example 2 was re-examined for its degradation of MPP8 protein in multiple leukemia cells, including molm-13, THP-1, and MV4-11. The results are as follows: Figure 5 As shown.

[0088] Furthermore, the results of detecting the degradation of MPP8 protein in MC38 solid tumor cells by the compound of Example 2 were as follows: Figure 6 As shown.

[0089] according to Figure 6The results showed that different concentrations of the compound from Example 2 could also degrade MPP8 protein in solid tumor cells such as MC38.

[0090] In summary, this application utilizes small molecule chip synthesis technology and solid-phase peptide synthesis technology, combined with an azide-alkyne cycloaddition reaction, to form a bifunctional molecule with high affinity. Its affinity and function were comprehensively verified using methods such as SPR, Western blot, and cell proliferation. Compared with existing technologies, this approach not only reduces synthesis costs and increases reaction speed but also enhances the reliability of quality control and functional verification, ensuring the stability and consistency of the compound.

[0091] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A polypeptide targeting MPP8 protein, characterized in that, The structure of the polypeptide is shown below: 。 2. The application of the isolated peptide targeting MPP8 protein as described in claim 1 in the preparation of molecular diagnostic products targeting MPP8 protein.

3. A polypeptide compound targeting the MPP8 protein, characterized in that, The peptide compound targeting the MPP8 protein comprises, in sequence, the isolated peptide targeting the MPP8 protein as described in claim 1, Lys, an alkyne-containing linker, and an azide-treated E3 ligand.

4. The polypeptide compound targeting the MPP8 protein according to claim 3, characterized in that, A linker fragment is further provided between the isolated polypeptide targeting the MPP8 protein and the Lys, the linker fragment being a divalent fragment composed of one or more of the following groups: C1 alkylene, 6-10 arylene, 5-6 heteroarylene, carboxyl, -O- or amino acid residues; the amino acid being a natural amino acid or a non-natural amino acid.

5. The polypeptide compound targeting MPP8 protein according to claim 4, characterized in that, The amino acids are selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, and Met.

6. The polypeptide compound targeting the MPP8 protein according to claim 3, characterized in that, The alkynyl-containing connecting 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.

7. The polypeptide compound targeting the MPP8 protein according to claim 3, 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 the polypeptide compound targeting MPP8 protein according to any one of claims 3 to 7, characterized in that, The preparation method includes the following steps: 1) Sequentially synthesize peptides by linking each element in the peptide compound targeting the MPP8 protein, except for the azide-treated E3 ligand; 2) Prepare the E3 ligand for azidation; 3) The product of step 1) and the product of step 2) are subjected to an addition reaction to obtain the polypeptide compound targeting the MPP8 protein.

9. The method for preparing the 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, step 2) preparing the azidated E3 ligand includes the following reaction: 。

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