Protein targeted degradation chimera and its preparation method and application

Abstract

Protein-targeted degradation chimeras, their preparation methods, and applications are shown below: #imgabs0#, where R1 is a polythymidine dendrimer DNA, and R2 is an EGFR / NCL antibody and aptamer drug targeting the tumor cell membrane surface. This invention utilizes scavenger receptors as endocytic receptors for the first time, inducing degradation of target proteins through endocytosis. By regulating the levels of pathogenic proteins, this approach achieves benefits in disease research, cancer cell growth inhibition, and tumor treatment. This invention utilizes chemical methods to synthesize dendrimer DNA-based protein-targeted degradation drugs and applies them to research on the targeted degradation of proteins in cells and the regulation of protein levels in cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein targeted degradation, and in particular relates to a dendritic DNA chimera for protein targeted degradation, and a preparation method and application thereof. Background Art

[0002] Protein regulation plays a crucial role in basic research and disease treatment. Selective targeted protein degradation has opened up new avenues for cancer therapy, offering advantages in targeting undruggable proteins and eliminating proteases and scaffolds. Cell membrane proteins are promising anticancer targets given their crucial role in oncogenic signaling. Selective degradation of cell membrane proteins via the lysosomal pathway offers a promising approach for cancer therapy, but strategies that are easily developed, highly modular, and broadly applicable are still lacking. Summary of the Invention

[0003] Technical problem to be solved: The present invention provides a protein targeted degradation chimera and its preparation method and application, which is suitable for the research of targeted degradation of cell membrane proteins and regulation of protein levels in cells.

[0004] Technical solution: Protein targeted degradation chimera, the structure is as follows:

[0005]

[0006] Among them, R1 is polythymine branched DNA, and R2 is EGFR / NCL antibody and aptamer drug targeting the tumor cell membrane surface.

[0007] The R2 is cetuximab or AS1411.

[0008] The method for preparing a protein-targeted degradation chimera, when R2 is cetuximab, comprises the following steps: first, dissolving a 3'-terminal amino-modified dendritic DNA in DMSO, adding 45-55 eq of dibenzocyclooctyne activated ester thereto, reacting at room temperature for 48 hours, and then purifying the dibenzocyclooctyne-modified dendritic DNA by high-performance liquid chromatography; dissolving cetuximab in a phosphate buffer solution, adding 45-55 eq of Azide-PEG3-NHS thereto; reacting at room temperature overnight, removing Azide-PEG3-NHS by ultrafiltration to obtain azide-modified cetuximab, and then adding 20-30 eq of dibenzocyclooctyne-modified dendritic DNA thereto; and reacting at room temperature for 72 hours, removing the dendritic DNA by ultrafiltration to obtain a protein-targeted degradation chimera.

[0009] When R2 is AS1411, the preparation steps are as follows: first, dissolving the 3'-terminal amino-modified dendritic DNA in DMSO, adding 45-55 eq of dibenzocyclooctyne activated ester thereto, reacting at room temperature for 48 hours, and then purifying by high-performance liquid chromatography to obtain the dibenzocyclooctyne-modified dendritic DNA; dissolving the 3'-terminal amino-modified AS1411 in pure water, adding 45-55 eq of Azide-PEG3-NHS thereto; after reacting at room temperature overnight, removing Azide-PEG3-NHS by ultrafiltration to obtain azide-modified AS1411, and then adding 0.5-2 eq of dibenzocyclooctyne-modified dendritic DNA thereto; after reacting at room temperature for 72 hours, purifying and collecting the product by polyacrylamide gel electrophoresis to obtain a protein-targeted degradation chimera.

[0010] Application of the above protein targeted degradation chimera in the preparation of anti-tumor drugs.

[0011] Anti-tumor drug, the active ingredient of which is the above-mentioned protein targeted degradation chimera.

[0012] Beneficial effects: Cell membrane-associated oncogenic proteins are very promising anti-cancer targets. Although targeted protein degradation has opened up new avenues for cancer treatment by downregulating undruggable proteins, most degraders, such as proteolysis-targeting chimeras (PROTACs), are basically not applicable to membrane proteins that do not have accessible cytoplasmic domains. To overcome this challenge, targeted protein degradation chimeras based on antibodies and nanoantibodies have been developed to achieve lysosomal degradation by hijacking membrane proteins. Recent advances in the construction of bispecific chimeras based on chemical biology methods have demonstrated the possibility of using synthetic ligands to couple to lysosomal transport receptors to degrade membrane proteins. Different lysosomal transport receptors, including the cation-independent mannose-6-phosphate receptor, the liver-specific sialoglycoprotein receptor, and integrins have been used to discover chimeric degraders. The present invention constructs a dendritic DNA chimera, named DENTAC, as a universal and effective strategy for targeted membrane protein degradation. DENTAC features a protein-targeting ligand covalently linked to dendritic DNA. It binds to cell surface scavenger receptors (SRs) and transports target membrane proteins to lysosomes for clearance. We demonstrated the efficacy of DENTAC by targeting nucleolin (NCL) and human epidermal growth factor receptor (EGFR) on the membranes of oncogenic cells. The anticancer application of DENTAC targeting NCL was further validated in a mouse xenograft model of lung cancer. This strategy provides a new avenue for the rapid development of highly effective degraders for membrane proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A method for preparing a chimera for targeted protein degradation;

[0014] Figure 2 The gel electrophoresis and mass spectrometry characterization diagrams of the above drugs;

[0015] Figure 3 EGFR expression in A549 cells after treatment with the above drugs;

[0016] Figure 4 NCL expression in A549 cells after treatment with the above drugs;

[0017] Figure 5 The inhibition of A549 cell proliferation after treatment with the above drugs;

[0018] Figure 6 The growth inhibition of A549 ectopic tumor after treatment with the above drugs;

[0019] Figure 7 is the expression of NCL in A549 ectopic tumor cells after treatment with the above drugs;

[0020] Figure 8 Schematic diagram of the protein targeted degradation chimera of the present invention. DETAILED DESCRIPTION

[0021] Example 1. Preparation of protein-targeted degradation chimeras

[0022] All phosphoramidite monomers and solid phase supports used in nucleic acid synthesis in this method were purchased from Suzhou Genetron Health Co., Ltd.; dibenzocyclooctyne activated ester and Azide-PEG3-NHS were purchased from Xi'an Dianhua Biotechnology Co., Ltd.; and cetuximab was purchased from Aibixin (Shanghai) Biotechnology Co., Ltd. Figure 1 As shown, dendritic DNA (PTDD, 1 eq) with an amino group modification at the 3' end was synthesized by a nucleic acid synthesizer, dissolved in DMSO, and 50 eq of dibenzocyclooctyne activated ester was added thereto. After reacting at room temperature for 48 hours, the dibenzocyclooctyne-modified dendritic DNA was purified by high performance liquid chromatography to obtain.

[0023] For the antibody-dendritic DNA chimera (E-DENTAC), cetuximab (1 eq) was dissolved in phosphate buffered saline, and 50 eq of Azide-PEG3-NHS was added. After reacting overnight at room temperature, the Azide-PEG3-NHS was removed by ultrafiltration to obtain 1 eq of azide-modified cetuximab. 25 eq of dibenzocyclooctyne-modified dendritic DNA was then added to the solution. After reacting for 72 hours at room temperature, the dendritic DNA was removed by ultrafiltration to obtain the protein-targeted degradation chimera.

[0024] For the aptamer-dendritic DNA chimera (N-DENTAC), 1 eq of AS1411 modified with a 3'-terminal amino group was dissolved in pure water, and 50 eq of Azide-PEG3-NHS was added. After reacting overnight at room temperature, the Azide-PEG3-NHS was removed by ultrafiltration to obtain 1 eq of azide-modified AS1411. 1 eq of dibenzocyclooctyne-modified dendritic DNA was then added to the solution. After reacting for 72 hours at room temperature, the product was purified and collected by polyacrylamide gel electrophoresis to obtain the protein-targeted degradation chimera.

[0025] Example 2: Characterization of protein-targeted degradation chimeric drugs

[0026] Separate E-DENTAC / N-DENTAC by electrophoresis on a non-denaturing polyacrylamide gel or a denaturing polyacrylamide gel, and collect the signal by Coomassie staining or GelRed staining. Figure 2 As shown, compared with cetuximab, E-DENTAC has an upward shift in electrophoretic position after the molecular weight increases, and the results of N-DENTAC are consistent with this. The high-resolution mass spectrometry characterization results are shown in Figure 2 .

[0027] Example 3: Application of protein-targeted degradation chimeras in targeted EGFR / NCL degradation in cells

[0028] After adding 25 nM E-DENTAC to A549 cells and incubating for different time periods, the Figure 3 As shown in the flow cytometry results, EGFR in cells was significantly downregulated with increasing incubation time, reaching a maximum degradation rate of about 66% after 60 hours of incubation. Figure 3 As shown in the flow cytometry results, E-DENTAC has the strongest effect on intracellular EGFR degradation at 25nM, and the half-degradation concentration is determined to be approximately 15nM. It was verified that in A549 cells, E-DENTAC has a time-dependent and concentration-dependent effect on EGFR degradation, which indicates that the chimera can achieve targeted EGFR protein degradation.

[0029] After adding 50 nM N-DENTAC to A549 cells and incubating for different time periods, the Figure 4 As shown in the flow cytometry results, NCL in cells was significantly downregulated with increasing incubation time, reaching a maximum degradation rate of about 60% after 48 hours of incubation. Figure 3As shown in the flow cytometry results, the degradation effect of E-DENTAC on intracellular NCL increased with increasing administration concentration, and its half-degradation concentration was determined to be approximately 25nM. This confirmed that in A549 cells, N-DENTAC's degradation of NCL was time-dependent and concentration-dependent, indicating that the chimera can achieve targeted NCL protein degradation.

[0030] After adding 50 nM N-DENTAC to A549 cells and incubating for different time periods, the Figure 5 As shown, the proliferation rate of cells detected by CCK-8 was significantly decreased with the increase of incubation time, verifying that N-DENTAC achieved targeted protein degradation of NCL and had an inhibitory effect on cell proliferation.

[0031] Example 4: Application of protein-targeted degradation chimeras in tumor treatment

[0032] 5×10 6 A549 cells. When the tumor size reaches about 60 mm 3 At the same time, AS1411 (2 mg / kg) or N-DENTAC (5.1 mg / kg) was injected intratumorally every 2 days at the same dose of aptamer. The same dose of control agent (normal saline) was also injected as a negative control. Tumor size and body weight were measured every 2 days. Mice were sacrificed on the 14th day, and tumor tissues and major normal tissues were collected for analysis. The tumor inhibition rate was as follows: Figure 6 As shown in Figure 2, the chimera can achieve tumor growth inhibition. The level of NCL in tumor tissue is as follows Figure 7 As shown, the chimera was verified to achieve targeted NCL protein degradation in vivo.

[0033] The above specific implementation manner does not limit the technical solution of the present invention in any form. Any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A protein targeted degradation chimera, characterized in that: The structure is as follows: Wherein, R1 is polythymidine branched DNA, and R2 is cetuximab or AS1411.

2. The method for preparing the protein targeted degradation chimera according to claim 1, characterized in that: When R2 is cetuximab, the preparation steps are as follows: first, dissolving the 3'-terminal amino-modified dendritic DNA in DMSO, adding 45-55 eq of dibenzocyclooctyne activated ester thereto, reacting at room temperature for 48 hours, and then purifying by high-performance liquid chromatography to obtain the dibenzocyclooctyne-modified dendritic DNA; dissolving the cetuximab in phosphate buffer solution, adding 45-55 eq of Azide-PEG3-NHS thereto; after reacting at room temperature overnight, removing the Azide-PEG3-NHS by ultrafiltration to obtain azide-modified cetuximab, and then adding 20-30 eq of dibenzocyclooctyne-modified dendritic DNA thereto; and after reacting at room temperature for 72 hours, removing the dendritic DNA by ultrafiltration to obtain a protein-targeted degradation chimera.

3. The method for preparing the protein targeted degradation chimera according to claim 1, characterized in that: When R2 is AS1411, the preparation steps are as follows: first, dissolving the 3'-terminal amino-modified dendritic DNA in DMSO, adding 45-55 eq of dibenzocyclooctyne activated ester thereto, reacting at room temperature for 48 hours, and then purifying by high-performance liquid chromatography to obtain the dibenzocyclooctyne-modified dendritic DNA; dissolving the 3'-terminal amino-modified AS1411 in pure water, adding 45-55 eq of Azide-PEG3-NHS thereto; after reacting at room temperature overnight, removing Azide-PEG3-NHS by ultrafiltration to obtain azide-modified AS1411, and then adding 0.5-2 eq of dibenzocyclooctyne-modified dendritic DNA thereto; after reacting at room temperature for 72 hours, purifying and collecting the product by polyacrylamide gel electrophoresis to obtain a protein-targeted degradation chimera.

4. Use of the protein targeted degradation chimera according to claim 1 in the preparation of anti-tumor drugs.

5. An antitumor drug, characterized in that: The active ingredient is the protein targeted degradation chimera according to claim 1.

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