WO2023168881A1

WO2023168881A1 - Polypeptide protac molecule, and preparation method therefor and use thereof

Info

Publication number: WO2023168881A1
Application number: PCT/CN2022/106922
Authority: WO
Inventors: 王浩; 安红维; 张薿元; 侯大勇
Original assignee: 国家纳米科学中心
Priority date: 2022-03-11
Filing date: 2022-07-21
Publication date: 2023-09-14
Also published as: CN114560908A

Abstract

Disclosed are a polypeptide nanoprotein ubiquitination degradation agent PROTAC molecule, and a preparation method therefor and the use thereof. The polypeptide nanoprotein ubiquitination degradation agent is a proteolysis-targeting chimera based on a ubiquitination-proteasome system, wherein the polypeptide nanoprotein ubiquitination degradation agent comprises a module molecule comprising a module molecule A and a module molecule B; the module molecule A comprises a coupling group A, a self-assembly group, and an E3 ligase recognition group; the module molecule B comprises a coupling group B, a self-assembling group, and a targeting binding group; and the coupling group A and the coupling group B are used for linking the module molecule A and the module molecule B. The polypeptide nanoprotein ubiquitination degradation agent can achieve dose-dependent degradation, can be specifically triggered in a tumor region, significantly reduces off-target toxicity, and is a protein ubiquitination degradation agent having selectivity and specificity.

Description

A polypeptide PROTAC molecule and its preparation method and application

Technical field

This application belongs to the field of biotechnology, and specifically relates to a polypeptide PROTAC molecule and its preparation method and application.

Background technique

The occurrence and development of most diseases are related to the abnormal expression or aggregation of proteins. To address the pathological mechanisms of such diseases, the traditional drug development idea is to develop small molecule inhibitors or protein inhibitors, which occupy and block the binding sites of target proteins. Active site of action, inhibits the functional activity of the protein. In the traditional drug development idea, the target protein needs to have active pockets and binding sites. At the same time, this occupancy-driven model requires maintaining a high drug concentration for a period of time to exert a good therapeutic effect, but higher Drug concentration can easily cause off-target effects and adverse reactions. In addition, small molecule inhibitors can easily cause compensatory increases in proteins or genetic mutations, leading to the development of drug resistance.

Targeted protein degradation technology is a new technology that uses the inherent protein degradation mechanism in eukaryotic cells to regulate protein homeostasis to interfere with protein function. The rise of the targeted protein degradation technology has solved to a certain extent the problems faced by small molecule inhibitors. Dilemma. Currently, the most mature technology in this field is the proteolysis-targeting chimera (PROTAC) technology based on the ubiquitination-proteasome system.

PROTAC is a heterobifunctional small molecule or peptide compound that uses a linker to connect the target protein binding domain (PBD) and the E3 ligase ligand to obtain PROTAC. By shortening the distance between the target protein and E3 ubiquitin ligase, a "target protein-PROTAC-E3 ligase" ternary complex is formed, and then the target protein is added with a ubiquitination tag, and finally the ubiquitin-proteasome system is used Degrade target protein.

PROTAC technology has the following two main advantages: (1) PROTAC only needs to have binding activity to the target protein and does not need to directly inhibit the functional activity of the target protein. Therefore, it can target traditional "undruggable" targets; (2) PROTAC Through the "event-driven" mode of degradation of target proteins, only a chemical dose of PROTAC molecules is required to transiently combine with the target protein through non-covalent force to achieve degradation of the target protein. Therefore, it has high efficiency and toxic side effects. Characteristics of weak and strong tolerance.

However, PROTAC molecules still face many potential problems and challenges: (1) Small molecule PROTAC has a hook effect (HOOK effect), that is, it tends to bind to a single E3 ligase or target protein at high concentrations, rather than binding to both at the same time. protein, and thus cannot function to bring the target protein and E3 ligase closer together. This dose-independent protein degradation greatly limits the clinical translation of PROTAC molecules. (2) Potential off-target effects cause the distribution of PROTAC molecules in non-targeted normal tissues and organs, resulting in toxicity, which limits the clinical translation of PROTAC molecules.

Therefore, it is of great significance to develop a dose-dependent and selective protein ubiquitination degrader.

Contents of the invention

This application provides a polypeptide PROTAC molecule and its preparation method and application. The polypeptide nanoprotein ubiquitination degradation agent provided by this application is a proteolysis-targeting chimera based on the ubiquitination-proteasome system. , PROTAC). The polypeptide nanoprotein ubiquitination degradation agent provided by this application can achieve dose dependence and is a selective protein ubiquitination degradation agent. The polypeptide nanoprotein ubiquitination degradation agent includes two module molecules. After entering the tumor cells, the two module molecules obtain heterobifunctional molecules through enzyme or GSH activation coupling reaction in the tumor cells. The heterobifunctional molecules are The molecule has the ability to bind E3 ligase and target protein, thereby performing the function of protein ubiquitination. The critical assembly concentration of the heterobifunctional molecule is reduced, and an assembly structure is formed in situ within the cell. At the same time, due to the distance effect, the distance between the E3 ligase and the target protein is brought closer to meet the requirements of ubiquitination. The long-lasting retention assembly formed in cells has the potential to ubiquitinate and degrade proteins. More importantly, the self-assembly ability of the heterobifunctional molecules formed in situ effectively resists the degradation of small molecule PROTACs under high concentration conditions. The resulting hook effect.

In a first aspect, the present application provides a polypeptide nanoprotein ubiquitination degradation agent, the polypeptide nanoprotein ubiquitination degradation agent includes module molecules, and the module molecules include module molecule A and module molecule B;

Wherein, the module molecule A includes a coupling group A, a self-assembly group and an E3 ligase recognition group;

The module molecule B includes a coupling group B, a self-assembly group and a targeting binding group; and

The coupling group A and the coupling group B are used to connect module molecule A and module molecule B.

In this application, the polypeptide nanoprotein ubiquitination degradation agent activates a coupling reaction under the conditions of enzymes or GSH highly expressed in tumor cells. The molecules after the coupling reaction have the ability to bind E3 ligase and target proteins, thereby exerting The function of protein ubiquitination degradation agent is to connect module molecule A and module molecule B through coupling group A and coupling group B, and then shorten the spatial distance between the target protein and E3 ligase to achieve Degradation of target proteins. At the same time, due to the extension of the topological structure of the reacted molecules, the critical assembly concentration is reduced, thereby triggering intracellular in-situ assembly. The assembly can achieve long-lasting, concentration-dependent protein degradation in the cell.

Preferably, the self-assembly group includes a self-assembly unit, and the self-assembly unit includes the amino acid sequence shown in SEQ ID NO. 1 to 22, preferably the amino acid sequence shown in SEQ ID NO. 22.

In this application, the self-assembly unit has nucleation-dependent and protein-mediated assembly capabilities. The amino acid sequence of the self-assembly unit is as follows:

SEQ ID NO.1: KLVFFAE;

SEQ ID NO.2: KLVFF;

SEQ ID NO.3: FF;

SEQ ID NO.4: YFFGNNQQNY;

SEQ ID NO.5:GSNKGAIIGLM;

SEQ ID NO.6: GKVQIINKKLDL;

SEQ ID NO.7: SYSSYGQS;

SEQ ID NO.8:GNQQQNY;

SEQ ID NO.9: GNQQQQY;

SEQ ID NO.10: GEWTYD;

SEQ ID NO.11: WTVNYS;

SEQ ID NO.12: FESNFN;

SEQ ID NO.13: HLFNLT;

SEQ ID NO.14: NQFIIS;

SEQ ID NO.15: YQLIWQ;

SEQ ID NO.16: NQFNLM;

SEQ ID NO.17: NQNNFN;

SEQ ID NO.18: YNNYNN;

SEQ ID NO.19: QNLLWQ;

SEQ ID NO.20: STWIYE;

SEQ ID NO.21: YYQNYQ; or

SEQ ID NO.22: GNNQQNY.

Preferably, the E3 ligase recognition group includes an E3 ligase recognition unit, and the structure of the E3 ligase recognition unit includes at least one of the structures represented by formula (I) or formula (II):

In this application, the E3 ligase recognized by the E3 ligase recognition group includes VHL and CRBN. The structure shown in formula (I) targets the E3 ligase VHL Ligand 1, and the structure shown in formula (II) targets E3 ligase CRBN.

Preferably, the targeted binding group includes a targeted binding unit, and the targeted binding unit includes at least one of a small molecule chemical drug or a small molecule polypeptide.

Preferably, the small molecule chemical drug includes any one or a combination of at least two of gefitinib derivatives, enzalutonium derivatives, BMS-1 derivatives or ER estrogen receptor inhibitor derivatives.

Preferably, the amino acid sequence of the small molecule polypeptide includes the amino acid sequence shown in SEQ ID NO. 23:

SEQ ID NO.23:LARLLT.

Preferably, the structure of the targeting binding unit includes any one or a combination of at least two of the structures represented by formula (III), formula (IV), formula (V) or formula (VI):

In this application, the targeting molecules recognized by the targeted binding unit include androgen receptor (AR), estrogen receptor (ER), epithelial growth factor cell proliferation and signaling receptor (EGFR) or cell programmed receptor. Any one or a combination of at least two of death-ligand 1 (PD-L1).

Preferably, the coupling group A in the module molecule A includes a catalytic unit and a reaction unit A.

Preferably, the catalytic unit and reaction unit A in the module molecule A are connected through an amide bond.

Preferably, the coupling group B in the module molecule B includes a catalytic unit and a reaction unit B.

Preferably, the catalytic unit and the reaction unit B in the module molecule B are connected through an amide bond.

Preferably, the catalytic units in the coupling group A and the coupling group B both include GHK(Cu ²⁺ ), and the structure of the GHK(Cu ²⁺ ) includes the structure represented by formula (VII):

In this application, after GHK (Cu ²⁺ ) is reduced by GSH in tumor cells, the catalytic activity is activated to form catalytically active Cu ⁺ . Its design purpose is to achieve specific response only in tumor cells, solving the problem of PROTACs The molecules have poor specificity in the body and have side effects.

Preferably, the reaction unit A in the module molecule A contains an azide group, and the structure of the reaction unit A includes the structure shown in formula (VIII):

Where n=1～10, n is an integer, for example, it can be 1, 2, 4, 6, 8 or 10.

Preferably, the reaction unit B in the module molecule B includes an alkynyl group, and the structure of the reaction unit B includes the structure represented by formula (IX):

Where m=1～10, m is an integer, for example, it can be 1, 2, 4, 6, 8 or 10.

In this application, the azide group and the alkynyl group are catalyzed by the activated catalyst Cu ⁺ , and a click reaction occurs, thereby realizing the coupling of module molecule A and module molecule B in the polypeptide nanoprotein ubiquitination degrader, in situ in tumor cells Module molecule A and module molecule B are assembled into a polypeptide nanoprotein ubiquitination degradation agent.

In this application, the coupling group is a reactive group that undergoes a bioorthogonal coupling reaction with or without a catalyst, and the reaction includes: azide-alkyne cycloaddition reaction (SPAAC ) and azide and alkynyl cycloaddition reaction (CuAAC) under Cu ⁺ catalysis; Diels-Alder reaction based on nitroso (Diels-Alder reaction); trans ring based on ring tension IEDDA bioorthogonal reaction of octene dienophile and tetrazine compound (diene compound); cycloaddition reaction of 2-cyanobenzo thiazole (CBT) and thiol group. When the coupling group is coupled using a cycloaddition reaction of azide and alkynyl groups under Cu ⁺ catalysis, Cu ²⁺ in the catalytic reaction unit GHK(Cu ²⁺ ) is reduced to Cu ⁺ by GSH, The azido group in reaction unit A and the alkynyl group in reaction unit B undergo a cycloaddition reaction, thereby connecting module molecule A and module molecule B.

Preferably, the module molecule A further includes a connecting unit A, which connects the coupling group A, the self-assembly unit and the E3 ligase recognition unit through amide bonds respectively.

Preferably, the module molecule B further includes a connecting unit B, which connects the coupling group B, the self-assembly unit and the targeting binding unit through amide bonds respectively.

Preferably, both the connecting unit A and the connecting unit B include amino acid derivatives.

In this application, the structures of the amino acid derivatives selected for the connecting unit A and the connecting unit B may be the same or different.

Preferably, the structure of the amino acid derivative includes any one or a combination of at least two of the structures represented by formula (X), formula (XI), formula (XII), or formula (XIII):

Wherein, in formula (X), n=1～5, n is an integer, for example, it can be 1, 2, 3, 4 or 5; m=1～5, m is an integer, for example, it can be 1, 2, 3, 4 or 5;

In formula (XI), n=1～5, n is an integer, for example, it can be 1, 2, 3, 4 or 5; m=1～5, m is an integer, for example, it can be 1, 2, 3, 4 or 5 ;

In formula (XII), n=1～5, n is an integer, for example, it can be 1, 2, 3, 4 or 5;

In formula (XIII), n=1 to 5, n is an integer, for example, it can be 1, 2, 3, 4 or 5.

Preferably, the amino acid derivative is optionally connected to an amino acid repeating unit, and the amino acid repeating unit includes a glycine repeating unit Gn, a serine repeating unit Sn, a repeating unit (GS)n of glycine and serine or GGGS (SEQ ID NO. 24) Any one or a combination of at least two.

In this application, the types of amino acid repeating units connected to the amino acid derivatives may be the same or different.

Preferably, n=1˜5 in the glycine repeat unit Gn, n is an integer, for example, it can be 1, 2, 3, 4 or 5.

Preferably, n=1˜5 in the serine repeating unit Sn, n is an integer, for example, it can be 1, 2, 3, 4 or 5.

Preferably, n in the repeating unit (GS) n of glycine and serine is 1 to 5, and n is an integer, such as 1, 2, 3, 4 or 5.

Preferably, the connecting unit is an amino acid derivative represented by formula (XII), n=4.

Preferably, the connection sequence of the module molecule A includes a catalytic unit, a reaction unit, a connection unit and an assembly unit in sequence, wherein an E3 ligase recognition unit is also connected to the connection unit.

Preferably, the connection sequence of the module molecule B includes a catalytic unit, a reaction unit, a connection unit and an assembly unit in sequence, wherein a targeting binding unit is also connected to the connection unit.

Preferably, the structure of the E3 ligase recognition unit in the module molecule A is formula (I), and the structure of the module molecule A is as shown in formula (XIV):

Preferably, the structure of the targeting binding unit in the module molecule B is formula (III), and the structure of the module molecule B is as shown in formula (XV):

Preferably, the structure of the targeting binding unit in the module molecule B is formula (IV), and the structure of the module molecule B is as shown in formula (XVI):

In this application, the structural framework of the module molecule in the polypeptide nanoprotein ubiquitination degradation agent is GHK(Cu ²⁺ )R group-X-GNNQQNY (SEQ ID NO. 22), where R represents the group containing stack A reactive group of a nitrogen-based or alkynyl derivative; and X represents a targeting unit.

Among them, the molecule with the structure shown in formula (XII) is GHK(Cu ²⁺ )K(N ₃ )-E-(VHL-1)-GNNQQNY (SEQ ID NO. 22);

The molecule with the structure shown in formula (IV) is GHK(Cu ²⁺ )Pra-K-(Enza)-GNNQQNY (SEQ ID NO. 22);

The molecule with the structure shown in formula (IV) is GHK(Cu ²⁺ )Pra-K-(Gefi)-GNNQQNY (SEQ ID NO. 22).

In the above molecular formula, in GHK, G (glycine), H (histidine), K (lysine); K (N ₃ ) represents a lysine derivative containing an azide group, E (glutamic acid), VHL-1 represents the targeting molecule targeting E3 ligase VHL. In GNNQQNY (SEQ ID NO.22), G (glycine), N (asparagine), Q (glutamine), Y (tyrosine) ;Pra represents an alkynyl group; Enza represents an enzalutonium derivative; Gefi represents a gefitinib derivative. In this application, the targeted binding units of Enza (enzalutonium derivative) and Gefi (gefitinib derivative) can respectively achieve the effects of androgen receptor (AR) or epithelial growth factor cell proliferation and signaling. Binding of the intracellular domain of the receptor (EGFR); VHL-1 can achieve binding to the E3 ligase VHL Ligand 1; therefore, the polypeptide nanoprotein ubiquitination degradation agent constructed in situ has the ability to bind the target protein and the E3 ligase at the same time ability.

In this application, after the polypeptide nanoprotein ubiquitination degradation agent enters the tumor cells, the catalytic unit GHK (Cu ²⁺ ) in the coupling group A and the coupling group B is catalyzed by the highly expressed GSH in the tumor cells. , thereby allowing a coupling reaction between module molecule A and module molecule B to achieve selective construction of heterobifunctional molecules for ubiquitination and degradation of polypeptide nanoproteins in tumor cells. In the heterobifunctional molecule for ubiquitination and degradation of polypeptide nanoproteins, the target protein target and the E3 ligase target are located on the same side of the nanoprotein ubiquitination degradation agent monomer, which can bring the target protein and the E3 ligase closer together. the spatial distance between.

In this application, the heterobifunctional molecule accelerates the assembly process after binding to the target protein and E3 ligase, and self-assembles in situ in cells to form a polypeptide nanoprotein ubiquitination degradation agent with a nanofiber structure, which can exert long-lasting, Dose-dependent protein degradation function to achieve long-term degradation of target proteins in cells.

The polypeptide nanoprotein ubiquitination degradation agent with the nanofiber structure has the ability to simultaneously bind the target protein and E3 ligase, and has a large specific surface area, which can provide more protein binding sites for the target protein and E3 ligase. Due to the shortened spatial distance, the ubiquitination process of the target protein by the E3 ligase is better realized. The binding of the target protein to the E3 ligase is adjustable and adaptive. Therefore, the target protein formed is increased. -The stability of the degradation agent-E3 ligase ternary complex improves the degradation efficiency of the target protein.

The polypeptide nanoprotein ubiquitination degradation agent with a nanofiber structure has a concentration-dependent target protein degradation effect. Traditional small molecule PROTACs molecules will form binary complexes as the concentration increases, instead of forming ternary complexes that can perform degradation functions, that is, the hook effect. However, the polypeptide nanoprotein ubiquitination degradation agent with a nanofiber structure does not tend to form a binary complex at high concentrations due to the assembly structure of the nanofiber, and therefore can achieve concentration-dependent protein degradation. This assembly strategy can resist the hook effect produced by PROTAC molecules that only bind to a single protein at high concentrations.

The polypeptide nanoprotein ubiquitination degradation agent described in this application provides a universal Linker design strategy. By assembling the nanofiber-shaped polypeptide nanoprotein ubiquitination degradation agent in tumor cells, the surface effect of the nanoassembly provides It has multiple binding sites for target proteins and E3 ligases. The target proteins and E3 ligases can be adaptively combined to sites with appropriate distance and steric hindrance to exert the ubiquitination degradation function. The design strategy of the polypeptide nanoprotein ubiquitination degradation agent described in this application represents a general type of PROTAC design strategy. In the design strategy, the molecular design using catalytic reaction coupling assembly can be used as a universal polypeptide nanoprotein ubiquitination degradation agent. The degradation agent design strategy can also expand the types of E3 ligase targets and target protein targets, and prepare more peptide nanoprotein ubiquitination degraders that target other target proteins.

In a second aspect, the present application provides a method for preparing the polypeptide nanoprotein ubiquitination degradation agent described in the first aspect, the preparation method comprising the following steps:

The coupling group, self-assembly group, E3 ligase recognition group and targeted binding group are synthesized through a polypeptide solid-phase synthesis method, and the coupling group, self-assembly group and E3 ligase recognition group are combined. Connect, connect the coupling group, the self-assembly group and the targeted binding group to obtain the polypeptide nanoprotein ubiquitination degradation agent.

Preferably, the preparation method of the polypeptide nanoprotein ubiquitination degradation agent includes the following specific steps:

(1) Fix the C-terminal of the first amino acid on the resin, and use Fmoc to protect the N-terminal;

(2) Remove the N-terminal protection of the first amino acid in the deprotection solution, use a detection reagent to perform deprotection detection, add the pretreated amino acid to the deprotected resin for reaction, and sequentially connect the amino acids to form a fixed Resin polypeptides; and

(3) Couple the polypeptide fixed on the resin in step (2) with the E3 ligase recognition unit or targeting binding unit respectively through an amide condensation reaction, and obtain module molecule A or module molecule B respectively after cleavage and purification.

Preferably, in step (1), the resin includes Wang resin with a modified density of 0.3-0.35mM, and the modified density can be, for example, 0.30mM, 0.31mM, 0.33mM or 0.35mM, etc.

Preferably, in step (2), the deprotection solution includes a dimethylformamide solution containing hexahydropyridine.

Preferably, the volume fraction of hexahydropyridine in the deprotection solution is 18-22%, for example, it can be 18%, 19%, 20%, 21% or 22%.

Preferably, in step (2), the detection reagent includes ninhydrin test solution.

Preferably, in step (2), the pretreated amino acid is prepared by the following method: combining the amino acid to be connected with benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate Mix and dissolve with N-methylmorpholine and dimethylformamide to obtain pretreated amino acids.

Preferably, in step (3), the molar ratio of the polypeptide fixed on the resin to the E3 ligase recognition unit or targeting binding unit is independently 1:(3-5), for example, it can be 1:3, 1 ∶4 or 1:5 etc.

Preferably, in step (3), the amide condensation reaction includes the following steps:

The E3 ligase recognition unit or target binding unit is mixed with benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, respectively, to obtain a mixed solution, which is mixed with N-methylmorpholine and dimethylformamide are dissolved, and the lysine with the protective group removed and the polypeptide fixed on the resin are added respectively for reaction.

Preferably, in step (3), the lysis solution used in the lysis includes an aqueous solution of trifluoroacetic acid and triisopropylsilane.

Preferably, the volume fraction of trifluoroacetic acid in the lysis solution is 92.5-95%, for example, it can be 92.5%, 93%, 94% or 95%, etc., and the volume fraction of triisopropylsilane is 2-95%. 2.5%, for example, it can be 2%, 2.1%, 2.3% or 2.5%.

Preferably, in step (3), the purification uses a preparative reversed-phase high performance liquid chromatograph.

As the preferred technical solution of the present application, the preparation method of the polypeptide nanoprotein ubiquitination degradation agent includes the following specific steps:

(1) Fix the C-terminus of the first amino acid on Wang resin with a modification density of 0.3-0.35mM, and use Fmoc to protect the N-terminus;

(2) Remove the N-terminal protection of the first amino acid in the deprotection solution. The reaction reagent is a dimethylformamide solution containing hexahydropyridine, in which the volume fraction of hexahydropyridine is 18 to 22%. Use Use the ninhydrin test solution for deprotection detection; mix the amino acid to be connected with benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, and use N-methylmorpholine and Dimethylformamide is dissolved to obtain pretreated amino acids; the pretreated amino acids are added to the deprotected resin for reaction, and the amino acids are sequentially connected into polypeptides fixed on the resin; and

(3) Couple the polypeptide fixed on the resin in step (2) with the E3 ligase recognition unit or the targeting binding unit through an amide condensation reaction. The molar ratio of the binding units is independently 1:(3~5); respectively, the E3 ligase recognition unit or the targeting binding unit and the benzotriazole-N, N, N', N'-tetramethylurea Hexafluorophosphate is mixed to obtain a mixed solution, the mixed solution is dissolved with N-methylmorpholine and dimethylformamide respectively, and the lysine from which the protective group is removed and the polypeptide fixed on the resin are added respectively. Reaction; lysis is carried out using a lysis solution, which includes an aqueous solution of trifluoroacetic acid and triisopropylsilane. The volume fraction of trifluoroacetic acid in the lysis solution is 92.5 to 95%, and the volume fraction of triisopropylsilane is 92.5% to 95%. The concentration is 2 to 2.5%, and then purified using a preparative reversed-phase high-performance liquid chromatograph to obtain module molecule A or module molecule B respectively.

In a third aspect, the present application provides a pharmaceutical composition, which includes the polypeptide nanoprotein ubiquitination degrading agent described in the first aspect.

Preferably, the administration method of the pharmaceutical composition includes at least one of intravenous administration or infusion administration.

Preferably, the dosage concentration of the pharmaceutical composition is 100 μM or less, for example, it can be 100 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM or 10 μM, etc., preferably 10 to 50 μM.

In this application, the pharmaceutical composition can specifically degrade target proteins in tumor cells and inhibit tumor growth.

In the fourth aspect, the present application provides the use of at least one of the polypeptide nanoprotein ubiquitination degrading agent described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of drugs for treating tumors.

Preferably, the tumor includes at least one of prostate tumor or lung tumor.

The numerical range described in this application not only includes the point values listed above, but also includes any point value between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, this application will not exhaustively list the ranges. Specific point values included.

Compared with the existing technology, this application has the following beneficial effects:

(1) The polypeptide nanoprotein ubiquitination degradation agent provided in this application provides a new idea for solving the structure-activity relationship limitations in the development process of small molecule PROTACs. Traditional PROTACs rely on Linkers to connect target proteins and E3 ligase targets to build ternary complexes to trigger protein degradation due to close proximity. The length, conformation and modification sites of the Linker will greatly affect the degradation efficiency of the target protein. In the development of PROTACs for new targets and the optimization of PROTACs degradation efficiency, there is currently no universally applicable Linker design strategy. The polypeptide nanoprotein ubiquitination degradation agent provided in this application provides a universal Linker design strategy. By assembling the nanofiber-shaped polypeptide nanoprotein ubiquitination degradation agent in tumor cells, the surface effect of the nanoassembly provides Multiple binding sites for target proteins and E3 ligases. The target proteins and E3 ligases can be adaptively combined to sites with appropriate distance and steric hindrance to exert ubiquitination and degradation functions.

(2) Small molecule PROTACs generally face the problem that at high concentrations, they will preferentially form a binary complex between PROTAC and the target protein or E3 ligase instead of a ternary complex, which affects the degradation efficiency of the protein; in this application, the The structure of the nanofiber-shaped peptide nanoprotein ubiquitination degrader breaks the concentration-independent protein degradation of small molecule PROTACs; at high concentrations, the topology of the assembly is further extended, providing a larger surface area for target binding. proteins and E3 ligases to achieve efficient and stable ternary complex formation.

(3) The polypeptide nanoprotein ubiquitination degradation agent can be specifically triggered in the tumor area, has specificity and selectivity, and significantly reduces off-target toxicity. This strategy can efficiently degrade target proteins at the cellular and animal levels, thereby inducing apoptosis of tumor cells and thereby inhibiting tumor growth. The polypeptide nanoprotein ubiquitination degradation agent will not produce obvious side effects in the body and has good biocompatibility.

Description of the drawings

Figure 1 is a schematic diagram of the formation process of the nanofiber-shaped polypeptide nanoprotein ubiquitination degradation agent in Example 1.

Figure 2 is a structural diagram of module molecule A in Example 1.

Figure 3 is a structural diagram of module molecule B in Example 1.

Figure 4 is a structural diagram of module molecule B in Example 2.

Figure 5 is a structural diagram of module molecule A in Example 3.

Figure 6 is a structural diagram of module molecule A in Example 4.

Figure 7 is a structural diagram of module molecule B in Example 4.

Figure 8 is a structural diagram of module molecule A in Example 5.

Figure 9 is a structural diagram of module molecule B in Example 5.

Figure 10 is a picture of the Western Blot detection results in test example 1.

Figure 11 is a diagram of imaging results of mice in Test Example 2.

Figure 12 is a graph of statistical results of mouse tumor volume in Test Example 3.

Detailed ways

The technical solutions of the present application will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

The sources of experimental instruments and materials in the specific implementation are as shown in the following table:

试剂/仪器Reagents/Instruments	厂家factory	货号/型号Item number/model
Fmoc-Tyr(tBu)-Wang ResinFmoc-Tyr(tBu)-Wang Resin	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	4200142001
Fmoc-谷氨酰胺(Fmoc-Gln(Trt)-OH)Fmoc-Glutamine (Fmoc-Gln(Trt)-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3630136301
Fmoc-甘氨酸(Fmoc-Gly-OH)Fmoc-Glycine (Fmoc-Gly-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3530135301
Boc-甘氨酸(Boc-Gly-OH)Boc-Glycine (Boc-Gly-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3070130701
Fmoc-组氨酸(Fmoc-His(Trt)-OH)Fmoc-histidine (Fmoc-His(Trt)-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3670136701
Fmoc-赖氨酸(Fmoc-Lys(Boc)-OH)Fmoc-Lys(Fmoc-Lys(Boc)-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3680236802
Fmoc-赖氨酸(Fmoc-Lys(Dde)-OH)Fmoc-Lys(Fmoc-Lys(Dde)-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3688436884
Fmoc-天冬酰胺(Fmoc-Asn(Trt)-OH)Fmoc-Asparagine(Fmoc-Asn(Trt)-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3510235102
Fmoc-酪氨酸(Fmoc-Tyr(Trt)-OH)Fmoc-tyrosine(Fmoc-Tyr(Trt)-OH)	吉尔生化上海有限公司Gill Biochemical Shanghai Co., Ltd.	3690136901
多肽固相合成管Peptide solid phase synthesis tube	重庆欣维尔玻璃有限公司Chongqing Xinwell Glass Co., Ltd.	P120010CP120010C

Preparation of test solution:

Deprotection solution: Mix hexahydropyridine and dimethylformamide (DMF) at a volume ratio of 1:4. The volume fraction of hexahydropyridine in the deprotection solution is 20%.

Reaction solution: Mix NMM and DMF at a volume ratio of 1:24.

Lysis solution: Mix TFA, TIS and H ₂ O. After mixing, the volume fraction of each solution is: 92.5% TFA, 2.5% TIS and 2.5% H ₂ O.

Ninhydrin test solution: one drop each of ninhydrin, vitamin C and phenol.

Other common reagents used in specific embodiments include: dimethylformamide (DMF), piperidine, dichloromethane (DCM), benzotriazole-N,N,N',N'-tetramethylurea Hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, copper acetate.

Example 1

This embodiment provides a polypeptide nanoprotein ubiquitination degradation agent. The polypeptide nanoprotein ubiquitination degradation agent can activate the catalytic click reaction process under the reduction of GSH in tumor cells, trigger the assembly process at the same time, and then initiate the original process in the cell. A schematic diagram of the formation process of the nanofiber-shaped polypeptide nanoprotein ubiquitination degradation agent is shown in Figure 1.

The module molecule A of the polypeptide nanoprotein ubiquitination degradation agent targets and recognizes the E3 ligase VHL Ligand 1, and the module molecule B targets and recognizes AR; the module molecule A is GHK(Cu ²⁺ )K(N ₃ )- E-(VHL-1)GNNQQNY (SEQ ID NO. 22), the structure of the module molecule A is shown in Figure 2; the module molecule B is GHK(Cu ²⁺ )Pra-K-(Enza)GNNQQNY ( SEQ ID NO. 22), the structure of the module molecule B is shown in Figure 3.

In module molecule A and module molecule B, in GHK, G (glycine), H (histidine), K (lysine); K (N ₃ ) represents lysine containing an azide group, E (glutamine) acid), VHL-1 represents the targeting molecule targeting E3 ligase VHL Ligand 1. In GNNQQNY (SEQ ID NO.22), G (glycine), N (asparagine), Q (glutamine), Y (Tyrosine); Pra represents an alkynyl group; Enza represents an enzalutonium derivative.

The preparation method of the modular molecule A includes the following steps:

(1) Fix the C-terminus of the first amino acid on Wang resin (0.35mM modification density), and use Fmoc to protect the N-terminus.

(2) Remove the N-terminal protection of the first amino acid in the deprotection solution, use a detection reagent to perform deprotection detection, add the pretreated amino acid to the deprotected resin for reaction, and sequentially connect the amino acids to form a fixed Resin polypeptides;

Wherein, the polypeptide fixed on the resin is prepared by the following steps:

Fmoc (fluorenemethoxycarbonyl) deprotection: weigh 0.1g Wang resin and put it into the peptide solid-phase synthesis tube, add DMF to swell for 30 minutes. Remove the DMF, use deprotection solution to perform Fmoc deprotection reaction, and place on a shaker for 10 minutes. Remove the deprotection solution and wash 3 times with DMF and DCM. Take 10mg Wang resin from the polypeptide solid-phase synthesis tube into a test tube and wash 2 times with ethanol. If the ninhydrin method detects dark blue, it is a positive result. Prepare Connect the first amino acid (R) to enter the amino acid condensation reaction;

Amino acid condensation: Take 10 times the equivalent of amino acid and HBTU according to the amino acid sequence of module molecule A, dissolve it in 7 mL of reaction solution, put it into the peptide solid-phase synthesis tube, and stir the reaction; after 1 hour, take 10 mg from the peptide solid-phase synthesis tube Wang resin was placed in a test tube and washed twice with ethanol. After the ninhydrin method detected no discoloration (that is, a negative result), the condensation reaction was successful. Aspirate the liquid in the peptide solid-phase synthesis tube and wash it twice with DMF and DCM each to obtain the peptide resin after the first amino acid condensation;

Repeat the above "Fmoc deprotection-amino acid condensation" reaction steps on the obtained peptide resin until the last amino acid Boc-glycine reaction is completed, and the polypeptide fixed on the resin is obtained.

(3) Couple the polypeptide fixed on the resin in step (2) with the E3 ligase recognition unit through an amide condensation reaction, and the molar ratio of the polypeptide fixed on the resin to the E3 ligase recognition unit or targeted binding unit Each independently is 1:3. After cleavage and purification, module molecule A is obtained respectively;

The amide condensation reaction coupling and cleavage purification include the following steps:

Amide condensation reaction: Use 2% hydrazine hydrate to remove the Dde protection of the lysine side chain; mix the E3 ligase recognition unit with HBTU to obtain a mixed solution, dissolve the mixed solution with NMM and DMF, and add the deprotected lysine side chain. React the lysine with the protective group removed and the polypeptide fixed on the resin;

Lysis: After the reaction is completed, wash the resin 3 times with DMF and DCM each, 2 times with methanol, and continue to drain for 20 minutes. Take out the synthesized peptide resin from the peptide solid-phase synthesis tube, put the lysis solution in ice bath for 20 minutes, and then lyse it in the lysis solution for 2 hours at room temperature. After filtering the resin, evaporate it to dryness on a rotary evaporator, and wash it three times with anhydrous ether under ice bath conditions. Anion exchange is then performed to exchange trifluoroacetate ions into acetate ions, and then Cu ²⁺ coordination is performed under the condition of pH=8 to obtain crude peptide;

Purification: Use preparative reverse-phase HPLC to purify the crude peptide. After purification, module molecule A is obtained, which is then lyophilized and stored at -20°C until use. HPLC detects the purity (purity >95%), uses mass spectrometry (MS, electrospray) to identify the pure peptide obtained, and the molecular weight result of the identification measurement is the same as the target molecular weight;

The preparation method of the module molecule B refers to the preparation method of the module molecule A.

Example 2

This embodiment provides a polypeptide nanoprotein ubiquitination degradation agent. The module molecule A of the polypeptide nanoprotein ubiquitination degradation agent is consistent with the module molecule A in Example 1, and the module molecule B targets and recognizes EGFR; Module molecule A is GHK(Cu ²⁺ )K(N ₃ )-E-(VHL-1)-GNNQQNY (SEQ ID NO. 22); said module molecule B is GHK(Cu ²⁺ )Pra-K-( Gefi)-GNNQQNY (SEQ ID NO. 22), the structure of the module molecule B is shown in Figure 4; in the module molecule B, Gefi represents a gefitinib derivative. The preparation method of the polypeptide nanoprotein ubiquitination degradation agent refers to the preparation method of Example 1.

Example 3

This embodiment provides a polypeptide nanoprotein ubiquitination degradation agent. The module molecule A of the polypeptide nanoprotein ubiquitination degradation agent targets and recognizes E3 ligase CRBN, and the module molecule B is consistent with the module molecule B in Example 1. ; The module molecule A is GHK(Cu ²⁺ )K(N ₃ )-E-(CRBN)-GNNQQNY (SEQ ID NO. 22). The structure of the module molecule A is shown in Figure 5.

In module molecule A, CRBN represents a targeting molecule targeting E3 ligase CRBN. The preparation method of the polypeptide nanoprotein ubiquitination degradation agent refers to the preparation method of Example 1.

Example 4

This embodiment provides a polypeptide nanoprotein ubiquitination degradation agent. The module molecule A of the polypeptide nanoprotein ubiquitination degradation agent targets to recognize E3 ligase VHL Ligand 1, and the module molecule B targets to recognize the target protein EGFR; so The module molecule A is GHK(Cu ²⁺ )K(N ₃ )-E-(VHL)-GSGS(SEQ ID NO.25)-GNNQQNY(SEQ ID NO.22), and its connecting unit A is also connected with amino acids. Repeating unit GSGS (SEQ ID NO.25), the structure of the module molecule A is shown in Figure 6; the module molecule B is GHK(Cu ²⁺ )Pra-K-(Gefi)GSGS (SEQ ID NO.25 )-GNNQQNY (SEQ ID NO. 22), the connecting unit B is also connected to the amino acid repeating unit GSGS (SEQ ID NO. 25). The structure of the module molecule B is shown in Figure 7. The preparation method of the polypeptide nanoprotein ubiquitination degradation agent refers to the preparation method of Example 1.

Example 5

This embodiment provides a polypeptide nanoprotein ubiquitination degradation agent. The module molecule A of the polypeptide nanoprotein ubiquitination degradation agent targets to recognize E3 ligase VHL Ligand 1, and the module molecule B targets to recognize the target protein EGFR; so The module molecule A is GHK(Cu ²⁺ )K(N ₃ )-E-(-(OEG) ₃ -VHL)-GNNQQNY (SEQ ID NO. 22), and the structure of its connecting unit A is as shown in formula (XI) is shown, where m is equal to 3 and n is equal to 4; the structure of the module molecule A is shown in Figure 8, and the module molecule B is GHK(Cu ²⁺ )Pra-K-(-(OEG) ₃ -Enza)- GNNQQNY (SEQ ID NO. 22), the structure of its connecting unit B is shown in formula (X), where m is equal to 3 and n is equal to 4. The structure of the module molecule B is shown in Figure 9. The preparation method of the polypeptide nanoprotein ubiquitination degradation agent refers to the preparation method of Example 1.

Test example 1

This test example conducts a cell-level protein degradation effect verification experiment on the polypeptide nanoprotein ubiquitination degradation agent prepared in Example 2.

The cell line selected in this test example is the lung cancer A549 cell line with high EGFR expression. The polypeptide nanoprotein ubiquitination degrading agent in Example 2 was used to incubate A549 cells in a concentration-dependent and time-dependent manner, and intracellular proteins were extracted; the intracellular EGFR expression level was verified by Western Blot.

The Western Blot detection results are shown in Figure 10. A549 cells showed a concentration-dependent decrease in the expression level of EGFR protein under the combined action of module molecule A and module molecule B at concentrations of 10 μM, 20 μM, and 50 μM. The results show that the polypeptide nanoprotein ubiquitination degradation agent has a concentration-dependent protein degradation effect that resists the HOOK effect.

Test example 2

This test example conducts animal-level specific recognition and long-term retention experiments on the polypeptide nanoprotein ubiquitination degradation agent prepared in Example 2.

Preparation of polypeptide-Cy probe: Connect the polypeptide nanoprotein ubiquitination degradation agent prepared in Example 2 to the Cy probe. The Cy probe is connected to Cy through the sulfhydryl group of cysteine. The Cy probe is connected to facilitate Observe the retention of the polypeptide nanoprotein ubiquitination degradation agent.

Construction of mouse subcutaneous tumor model: Lung cancer cells were used to establish mouse subcutaneous tumors. 1×10 ⁶ A549 cells were injected into the subcutaneous tissue of the right leg of the mouse. After 2 weeks, the tumor formed, and a mouse subcutaneous tumor model was obtained.

The peptide-Cy probe was used to conduct specific recognition and long-term retention experiments at the animal level. The animals selected for the experiments were mice. The peptide-Cy probe was injected into the tail vein of mice. Three mice were used. The small animal in vivo imager (IVIS Spectrum) was used for imaging. The imaging results of the mice are shown in Figure 11. The results show that the polypeptide -Cy probe has obvious signal accumulation in tumor tissue and can retain for up to 48 hours.

Test example 3

In this test example, an animal tumor growth inhibition experiment was conducted on the polypeptide nanoprotein ubiquitination degradation agent prepared in Example 2.

The polypeptide nanoprotein ubiquitination degradation agent described in Example 2 was injected into the tail vein of mice (as the experimental group), and the physiological saline group was used as the control group. Imaging was performed with a small animal in vivo imaging device (IVIS Spectrum), and the tumor statistics were calculated. Volume, the results of the statistical curve chart of mouse tumor volume are shown in Figure 12. Comparing the experimental group and the simultaneous administration and normal saline group, it can be found that after the experimental group was injected with the peptide nanoprotein ubiquitination degrader, the tumor growth rate was significantly The tumor volume in the mice was significantly lower than that in the normal saline group, indicating that the polypeptide nanoprotein ubiquitination degradation agent has obvious tumor inhibitory effect.

In summary, the polypeptide nanoprotein ubiquitination degradation agent provided in this application achieves good protein degradation effect at the animal tissue level and achieves good tumor inhibition effect in mice. The polypeptide nanoprotein ubiquitination degradation agent provided by this application can activate the catalytic click reaction process under the reduction of GSH in tumor cells, trigger the assembly process at the same time, and then form nanofiber-like polypeptide nanoprotein ubiquitination degradation in situ in the cell. Agent, the nanofiber-shaped polypeptide nanoprotein ubiquitination degradation agent has the ability to adapt to the adjustable binding of target proteins and E3 ligases, thereby mediating the degradation of ubiquitination proteasome caused by close distance; as the concentration increases, The increased size and surface area of the assembly can provide more sites to form a stable target protein-assembly-E3 ligase ternary complex, thereby allowing the system to achieve concentration-dependent protein degradation that is different from that of small molecules. At the same time, since the nanofiber-shaped polypeptide nanoprotein ubiquitination degradation agent will achieve long-term retention in cells, it can achieve long-term degradation of target proteins in cells.

The applicant declares that the above are only specific implementation modes of the present application, but the protection scope of the present application is not limited thereto. Persons skilled in the technical field should understand that any person skilled in the technical field will not disclose any information disclosed in this application. Within the technical scope, changes or substitutions that can be easily imagined fall within the protection scope and disclosure scope of this application.

Claims

A polypeptide nanoprotein ubiquitination degradation agent, which includes module molecules, and the module molecules include module molecule A and module molecule B;

Wherein, the module molecule A includes a coupling group A, a self-assembly group and an E3 ligase recognition group;

The module molecule B includes a coupling group B, a self-assembly group and a targeting binding group; and

The coupling group A and the coupling group B are used to connect module molecule A and module molecule B.
The polypeptide nanoprotein ubiquitination degradation agent according to claim 1, wherein the self-assembly group includes a self-assembly unit, and the self-assembly unit includes the amino acid sequence shown in SEQ ID NO. 1 to 22, preferably The amino acid sequence shown in SEQ ID NO.22.
The polypeptide nanoprotein ubiquitination degradation agent according to claim 1, wherein the E3 ligase recognition group includes an E3 ligase recognition unit, and the structure of the E3 ligase recognition unit includes formula (I) or formula ( II) At least one of the structures shown:
The polypeptide nanoprotein ubiquitination degradation agent according to claim 1, wherein the targeted binding group includes a targeted binding unit, and the targeted binding unit includes at least one of a small molecule chemical drug or a small molecule polypeptide. kind;

Preferably, the small molecule chemical drug includes any one or a combination of at least two of gefitinib derivatives, enzalutonium derivatives, BMS-1 derivatives, and ER androgen receptor inhibitor derivatives;

Preferably, the amino acid sequence of the small molecule polypeptide includes the amino acid sequence shown in SEQ ID NO. 23;

Preferably, the structure of the targeting binding unit includes any one or a combination of at least two of the structures represented by formula (III), formula (IV), formula (V) or formula (VI):
The polypeptide nanoprotein ubiquitination degradation agent according to claim 1, wherein the coupling group A in the module molecule A includes a catalytic unit and a reaction unit A;

Preferably, the catalytic unit and reaction unit A in the module molecule A are connected through an amide bond;

Preferably, the coupling group B in the module molecule B includes a catalytic unit and a reaction unit B;

Preferably, the catalytic unit and reaction unit B in the module molecule B are connected through an amide bond;

Preferably, the catalytic units in the coupling group A and the coupling group B both include GHK(Cu 2+ ), and the structure of the GHK(Cu 2+ ) includes the structure represented by formula (VII):

Preferably, the reaction unit A in the module molecule A contains an azide group, and the structure of the reaction unit A includes the structure shown in formula (VIII):

Where n=1～10, n is an integer; preferably, the reaction unit B in the module molecule B contains an alkynyl group, and the structure of the reaction unit B includes the structure represented by formula (IX):

Among them, m=1～10, m is an integer.
The polypeptide nanoprotein ubiquitination degradation agent according to any one of claims 1 to 5, wherein the module molecule A further includes a connecting unit A, and the connecting unit A respectively connects the coupling group A through an amide bond. , the self-assembly unit is connected to the E3 ligase recognition unit;

Preferably, the module molecule B also includes a connecting unit B, which connects the coupling group B, the self-assembly unit and the targeting binding unit through amide bonds respectively;

Preferably, both the connecting unit A and the connecting unit B include amino acid derivatives;

Preferably, the structure of the amino acid derivative includes any one or a combination of at least two of the structures represented by formula (X), formula (XI), formula (XII), or formula (XIII):

Among them, in formula (X), n=1~5, n is an integer, m=1~5, m is an integer;

In formula (XI), n=1~5, n is an integer, m=1~5, m is an integer;

In formula (XII), n=1～5, n is an integer;

In formula (XIII), n=1～5, n is an integer.
The polypeptide nanoprotein ubiquitination degradation agent according to claim 6, wherein the amino acid derivative is optionally connected to an amino acid repeating unit, and the amino acid repeating unit includes a glycine repeating unit Gn, a serine repeating unit Sn, glycine and Any one or a combination of at least two of the repeating units of serine (GS)n or GGGS (SEQ ID NO. 24);

Preferably, n=1˜5 in the glycine repeating unit Gn, n is an integer;

Preferably, n=1˜5 in the serine repeating unit Sn, n is an integer;

Preferably, n in the repeating unit (GS) n of glycine and serine is 1 to 5, and n is an integer;

Preferably, the connecting unit is an amino acid derivative represented by formula (XII), n=4;

Preferably, the connection sequence of the module molecule A includes a catalytic unit, a reaction unit, a connection unit and an assembly unit in sequence, wherein the connection unit is also connected to an E3 ligase recognition unit;

Preferably, the connection sequence of the module molecule B includes a catalytic unit, a reaction unit, a connection unit and an assembly unit in sequence, wherein a targeting binding unit is also connected to the connection unit.
The polypeptide nanoprotein ubiquitination degradation agent according to any one of claims 1 to 7, wherein the structure of the E3 ligase recognition unit in the module molecule A is formula (I), and the structure of the module molecule A As shown in formula (XIV):

Preferably, the structure of the targeting binding unit in the module molecule B is formula (III), and the structure of the module molecule B is as shown in formula (XV):

Preferably, the structure of the targeting binding unit in the module molecule B is formula (IV), and the structure of the module molecule B is as shown in formula (XVI):
A method for preparing the polypeptide nanoprotein ubiquitination degradation agent according to any one of claims 1-8, which includes the following steps:

The coupling group, self-assembly group, E3 ligase recognition group and targeted binding group are synthesized through a polypeptide solid-phase synthesis method, and the coupling group, self-assembly group and E3 ligase recognition group are combined. Connect, connect the coupling group, the self-assembly group and the targeted binding group to obtain the polypeptide nanoprotein ubiquitination degradation agent.
The preparation method of the polypeptide nanoprotein ubiquitination degradation agent according to claim 9, wherein the preparation method of the polypeptide nanoprotein ubiquitination degradation agent includes the following steps:

(1) Fix the C-terminal of the first amino acid on the resin, and use Fmoc to protect the N-terminal;

(2) Remove the N-terminal protection of the first amino acid in the deprotection solution, use a detection reagent to perform deprotection detection, add the pretreated amino acid to the deprotected resin for reaction, and sequentially connect the amino acids to form a fixed Resin polypeptides;

(3) Couple the polypeptide fixed on the resin in step (2) with the E3 ligase recognition unit or targeting binding unit respectively through an amide condensation reaction, and obtain module molecule A or module molecule B respectively through cleavage and purification.
The method for preparing a polypeptide nanoprotein ubiquitination degradation agent according to claim 10, wherein in step (1), the resin includes Wang resin with a modified density of 0.3-0.35mM;

Preferably, in step (2), the deprotection solution includes a dimethylformamide solution containing hexahydropyridine;

Preferably, the volume fraction of hexahydropyridine in the deprotection solution is 18 to 22%;

Preferably, in step (2), the detection reagent includes ninhydrin test solution;

Preferably, in step (2), the pretreated amino acid is prepared by the following method: combining the amino acid to be connected with benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate Mix and dissolve with N-methylmorpholine and dimethylformamide to obtain pretreated amino acids.
The method for preparing a polypeptide nanoprotein ubiquitination degradation agent according to claim 10 or 11, wherein in step (3), the molar ratio of the polypeptide fixed on the resin to the E3 ligase recognition unit or the targeted binding unit Each independently is 1:(3～5);

Preferably, in step (3), the amide condensation reaction includes the following steps:

The E3 ligase recognition unit or target binding unit is mixed with benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, respectively, to obtain a mixed solution, which is mixed with N-methylmorpholine and dimethylformamide are dissolved, and the lysine with the protective group removed and the polypeptide fixed on the resin are added respectively to react;

Preferably, in step (3), the lysis solution used for the lysis includes an aqueous solution of trifluoroacetic acid and triisopropylsilane;

Preferably, the volume fraction of trifluoroacetic acid in the lysis solution is 92.5-95%, and the volume fraction of triisopropylsilane is 2-2.5%;

Preferably, in step (3), the purification uses a preparative reversed-phase high performance liquid chromatograph.
A pharmaceutical composition comprising the polypeptide nanoprotein ubiquitination degradation agent according to any one of claims 1-8;

Preferably, the administration method of the pharmaceutical composition includes at least one of intravenous administration or infusion administration;

Preferably, the dosage concentration of the pharmaceutical composition is 100 μM or less, preferably 10 to 50 μM.
The application of at least one of the polypeptide nanoprotein ubiquitination degrading agent according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 13 in the preparation of drugs for treating tumors;

Preferably, the tumor includes at least one of prostate tumor or lung tumor.