Chimera for targeted degradation of immune protein as well as preparation method and application of chimera

Abstract

The invention discloses a chimera for targeted degradation of immune protein. The chimera is formed by connecting a ligand of E3 ligase with a ligand of IDO protein through a linker. The invention also discloses a preparation method and application thereof. According to the present invention, a ligand capable of degrading immune-related protein-indoleamine 2, 3-dioxygenase (IDO) is coupled with a ligand of E3 ligase through a linker to construct the PROTAC capable of being used for tumor immunotherapy; the molecule can specifically degrade indoleamine 2, 3-dioxygenase, so that the effects of tumor targeting and immunotherapy are achieved, and the maximum killing effect on tumor cells and tissues is achieved.

Description

Technical Field

[0001] The invention relates to a chimera for targeted degradation of immune proteins and a preparation method and application thereof, belonging to the technical field of targeted protein degradation. Background Art

[0002] The occurrence of tumors is usually caused by malignant genetic mutations of normal cells. Tumor cells have a strong ability to proliferate, so the imbalance between cell death and cell proliferation leads to a rapid increase in the number of tumor cells. Despite great progress in treatment, cancer remains the second leading cause of death in the world after cardiovascular disease. The high incidence and mortality of cancer seriously threaten human health. Based on the close interaction between cancer and the immune system, restarting or enhancing the body's cancer immune cycle so that it can be amplified and spread to achieve the best anti-cancer effect is a key research direction for developing new cancer treatment strategies. After years of hard work, people have developed cancer immunotherapy. Unlike traditional cancer treatments, cancer immunotherapy is a treatment method that activates or promotes the activation of the immune system through drugs to restore or enhance the function of the body's immune system, thereby effectively killing cancer cells.

[0003] Protein hydrolysis targeting chimeras (PROTACs) are a new method in the field of protein targeted degradation. PROTAC is a bifunctional molecule composed of a ligand for an E3 ligase and a ligand for a protein of interest (POI) connected by a chemical linker. PROTAC technology uses heterologous functional chimera molecules to mimic endogenous protein homeostasis mechanisms to achieve post-translational selective degradation of target proteins in eukaryotes. Compared with traditional small molecule inhibitors, PROTAC molecules have the advantages of small dosage, low toxicity, ability to overcome target protein mutations, avoid drug resistance caused by overexpression, and high selectivity, which provides PROTAC molecules with good pharmacological effects, but there are currently few reports on the application of PROTAC technology in the degradation of immune-related proteins. Summary of the invention

[0004] The purpose of the present invention is to provide a chimera for targeted degradation of immune proteins, which is suitable for specific degradation of target proteins and tumor treatment.

[0005] The technical solution adopted by the present invention is: a chimera for targeted degradation of immune proteins, which is formed by connecting the ligand of E3 ligase to the ligand of IDO protein through a linker.

[0006] Preferably, the ligand of the E3 ligase is thalidomide, and the linker is a linker containing a PEG chain.

[0007] Preferably, its chemical structure is shown below:

[0008]

[0009] The invention also discloses the application of the chimera for targeted degradation of immune proteins in the preparation of anti-tumor drugs.

[0010] Preferably, the tumor is breast cancer.

[0011] The present invention also discloses the use of the chimera for targeted degradation of immune proteins in the preparation of drugs for selective degradation of IDO.

[0012] The present invention also discloses a method for synthesizing the chimera for targeted degradation of immune proteins, and the synthesis route thereof is as follows:

[0013]

[0014] The present invention couples the ligand of indoleamine 2,3-dioxygenase (IDO), which is an immune-related protein, with the ligand of E3 ligase through a linker to construct a PROTAC that can be used for tumor immunotherapy. The molecule can specifically degrade indoleamine 2,3-dioxygenase, achieve the effect of tumor targeting and immunotherapy, and have the greatest killing effect on tumor cells and tissues. The present invention uses a chemical method to prepare an immunoproteolysis-targeted chimera and applies it to the research of targeted degradation of proteins in cells and immunotherapy. The immunoproteolysis-targeted chimera is used to induce target proteins and immunotherapy, and the effects of disease research, cancer cell growth inhibition and tumor treatment are achieved by regulating the level of pathogenic proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A chemical synthesis route for the protein targeted degradation chimera IPT.

[0016] Figure 2 The results of IDO expression in SKOV3 cells before and after treatment with the protein targeted degradation chimera IPT.

[0017] Figure 3 The results show that the protein targeted degradation chimera IPT inhibits the proliferation of SKOV3 cells. Specific implementation methods

[0018] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0019] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0020] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0021] Example 1 Structure and Synthesis Method of Protein Targeted Degradation Chimera IPT

[0022] like Figure 1 As shown, a protein targeted degradation molecule compound IPT capable of targeted degradation of the immune-related protein IDO was synthesized using a standard chemical synthesis process.

[0023] The specific steps are as follows: first, the raw material compound 1 (1 eq, purchased from ATK CHEMICAL COMPANY LIMITED, CAS: 2019140-71-5) and mono-Boc ethylenediamine (1 eq) are mixed in a tetrahydrofuran solution, stirred at room temperature for 4 h under TEA to generate compound 2, 20% TFA is added to the reaction solution for BOC protection removal, stirred at room temperature for 2 h, the solvent is removed by distillation under reduced pressure, and column chromatography is used for separation and purification to obtain compound 3; sodium hydroxide aqueous solution is added to compound 3 (1 eq), stirred at room temperature for 2 h, ethyl acetate is extracted and purified by column chromatography to obtain compound 4; compound 5 (1 eq, purchased from MedChemExpress (MCE), CAS: 835616-61-0) is dissolved in anhydrous DMF (0.5 mL), compound 6 (1 eq, purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., CAS: 352439-37-3) and triethylamine (2 eq) are added, and stirred at 90°C for 12 h. Compound 7 was purified by semi-preparative HPLC; Compound 7 (1 eq) was dissolved in anhydrous THF, TEA (1.5 eq) and DMAP (0.2 eq) were added, and after ice bathing for 10 min, p-toluenesulfonyl chloride (2 eq) and DIPEA (1 eq) were added, and the mixture was stirred at 40°C for 24 h, and the solvent was removed by reduced pressure distillation to obtain compound 8. Compound 8 (1 eq) was dissolved in anhydrous DMF, and compound 4 (1.1 eq) and triethylamine (1 eq) were added, and the mixture was stirred at 60°C for 12 h. Compound IPT was purified by semi-preparative HPLC.

[0024] Example 2: Application of protein targeted degradation chimera IPT in targeted IDO degradation in cells

[0025] After adding different concentrations of compounds to SKOV3 cells and incubating for 12 h, Figure 2 As shown, Western blotting detected that the level of IDO in the cells was significantly degraded after the addition of the drug, and the degradation effect increased with the increase of its concentration. The results showed that compound IPT can effectively degrade IDO protein in SKOV3 cells.

[0026] Embodiment 3:

[0027] After adding different concentrations of IPT to SKOV3 cells and incubating for 48 h, Figure 3 As shown, the proliferation rate of cells detected by CCK-8 was significantly downregulated with the increase of incubation concentration, verifying that IPT can achieve targeted protein degradation of IDO and synergize with immunotherapy to inhibit cancer cell proliferation.

[0028] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A chimera for targeted degradation of immune proteins, characterized in that: It is formed by connecting the ligand of E3 ligase to the ligand of IDO protein through a linker.

2. The chimera for targeted degradation of immune proteins according to claim 1, characterized in that: The ligand of E3 ligase is thalidomide, and the linker is a linker containing a PEG chain.

3. The chimera for targeted degradation of immune proteins according to claim 1, characterized in that: Its chemical structure is shown below:

4. Use of the chimera for targeted degradation of immune proteins according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The tumor is breast cancer.

6. Use of the chimera for targeted degradation of immune proteins according to any one of claims 1 to 3 in the preparation of a drug for selective degradation of IDO.

7. The method for synthesizing a chimera for targeted degradation of immune proteins according to any one of claims 1 to 3, characterized in that: Its synthetic route is as follows:

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