Tumor-specific protein-targeting degraders based on mRNA and click chemistry molecules and uses thereof

Abstract

The application provides a tumor-specific protein targeted degradation agent based on mRNA and a click chemistry molecule and an application, and comprises one or more specific translated mRNA sequences, an azide-coupled target protein ligand and a DBCO-coupled covalent tag ligand; the mRNA sequence enters a tumor cell and specifically expresses a covalent tag-E3 ubiquitin ligase fusion protein; the azide-coupled target protein ligand is connected with the covalent tag-E3 ubiquitin ligase fusion protein through a biological orthogonal click reaction, so that the target protein in the tumor cell is targetedly degraded. The application avoids the treatment range limitation caused by the expression amount difference of E3 ligase in different tumors, and the DBCO-coupled covalent tag ligand constitutes a substrate of the biological orthogonal click reaction, so that the degradation of different target proteins can be realized.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to tumor-specific protein-targeting degraders based on mRNA and click chemistry molecules and their applications. Background Technology

[0002] Proteolytic targeting chimeras (PROTACs) are a novel therapeutic strategy based on the ubiquitin-proteasome system (UPS) in living cells. PROTAC drugs typically consist of two modules: a ligand for the target protein (POI) and a ligand for an E3 ubiquitin ligase. Through the interaction between the E3 ubiquitin ligase and the POI, the POI is ubiquitinated and then degraded by the UPS. This mechanism provides a new approach for targeting proteins that are otherwise untreatable. Currently, most PROTAC drugs in clinical trials utilize the E3 ligases VHL and CRBN to mediate the ubiquitination of target proteins. However, the expression levels of common E3 ubiquitin ligases vary in different tumors, limiting the application and efficacy of PROTAC drugs in cancer treatment. Furthermore, PROTAC molecules lack tumor specificity and can lead to the degradation of target proteins in normal cells, causing damage and toxic side effects. Therefore, there is an urgent need to develop tumor-selective drugs that do not rely on endogenous E3 ligases.

[0003] Covalent tagging technology can covalently couple small molecules to target proteins via chemical bonds, thereby selectively regulating intracellular protein-protein interactions through genetically encoded tag proteins, which is highly consistent with the principle of PROTAC. Existing covalent tagging technologies have advantages such as high connection stability, high specificity, and good bioorthogonality. For example, SNAPTag can specifically recognize and covalently bind to benzylguanine (BG) derivatives, while HaloTag can stably covalently bind to chloroalkanes. Currently, covalent tagging technology has been applied to PROTAC drug design, and techniques such as HaloPROTACs have been developed. However, the reported methods mainly regulate the expression and degradation of exogenous proteins, rather than targeting the degradation of tumor-associated proteins.

[0004] Recent studies have also proposed mRNA therapies, which utilize optimized mRNA sequences, chemical modifications, and efficient delivery systems to activate immunity by encoding antigen proteins or directly produce therapeutic proteins. Furthermore, recent research has explored cell type-specific mRNA translation regulation, such as achieving tumor cell-specific expression through splicing factor mutations or controlling tumor cell-specific protein expression through ADAR-mediated base editing strategies (CellREADR). Therefore, understanding how to selectively and efficiently degrade target proteins in tumor cells via mRNA transfection is of great significance in the development of tumor-specific protein-targeting degraders. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems in related technologies. To this end, the purpose of this application is to propose a tumor-specific protein-targeting degrader based on mRNA and click chemistry molecules and its application. This degrader expresses a covalently tagged E3 ubiquitin ligase fusion protein in tumor cells via mRNA transfection, and functionalizes the covalently tagged E3 ubiquitin ligase fusion protein with a dibenzocyclooctylene (DBCO)-modified covalently tagged ligand. Then, through bioorthogonal click chemistry, the target protein ligand coupled with an azide is linked to the covalently tagged E3 ubiquitin ligase fusion protein, inducing ubiquitination and degradation of the target protein.

[0006] According to the first aspect of this application, a tumor-specific protein-targeting degrader based on mRNA and click chemistry molecules is proposed, comprising one or more specifically translated mRNA sequences, an azide-coupled target protein ligand, and a DBCO-coupled covalently tagged ligand; the mRNA sequence enters tumor cells and specifically expresses a covalently tagged E3 ubiquitin ligase fusion protein; the azide-coupled target protein ligand is linked to the covalently tagged E3 ubiquitin ligase fusion protein via a bioorthogonal click reaction to target and degrade the target protein within the tumor cells.

[0007] In some embodiments, the mRNA includes a 5' sensory recognition region, a 3' protein-coding region, and a stop codon that prevents the translation of the 3' protein-coding region; the 5' sensory recognition region can hybridize with specific mRNAs within the tumor cells to form AC-mismatched double-stranded RNAs, while simultaneously altering the stop codon so that the 3' protein-coding region encodes and expresses the covalently tagged E3 ubiquitin ligase fusion protein.

[0008] In some embodiments, the stop codon is UAG; when the 5' sensor recognition region can hybridize with specific mRNA in the tumor cells to form AC mismatched double-stranded RNA, ADARs-mediated base editing is activated and the stop codon is converted into the UIG codon.

[0009] In some embodiments, the covalently tagged E3 ubiquitin ligase fusion protein includes an N-terminal covalently tagged protein for covalently binding to a covalently tagged protein substrate; a C-terminal E3 ubiquitin ligase mediating the ubiquitination of the target protein; and a linking sequence connecting the covalently tagged protein and the E3 ubiquitin ligase.

[0010] In some embodiments, in different tumor cells, the E3 ubiquitin ligase includes VHL, CRBN, CHIP, MARCH5, NEDD4L, PARKIN, SIAH1, MDM2, BIRC2, DCAF15, DCAF16, RNF4, or RNF114.

[0011] In some embodiments, DBCO is modified onto the covalently tagged protein substrate to form a DBCO-coupled covalently tagged ligand to construct a substrate for a biological orthogonal click reaction.

[0012] In some embodiments, the covalently tagged protein substrate includes benzylguanine derivatives, chloroalkanes, cyclooctyne, or trimethoprim.

[0013] In some embodiments, based on the target protein ligand coupled with the azide, the chain length between the azide group and the target protein ligand is optimized to obtain the optimal target protein ligand coupled with the azide, thereby enabling the target protein ligand coupled with the azide to achieve the highest target degradation level.

[0014] In some embodiments, the tumor cells include adrenocortical cancer cells.

[0015] The application of the degrading agent described in any of the above embodiments in the preparation of tumor therapeutic agents or tumor cell proliferation inhibitors is proposed according to two aspects of this application.

[0016] In some embodiments, the tumor cells are adrenocortical cancer cells.

[0017] This application proposes a tumor-specific protein-targeting degrader based on mRNA and click chemistry molecules and its application, which has the following advantages: the molecular design of the protein hydrolysis-targeting chimeric (PROTAC) in this application does not require E3 enzyme ligands; the PROTAC element in this application has a small molecular weight and high drug bioavailability; in addition, different target proteins can be degraded by changing the target head; at the same time, it specifically kills tumor cells without damaging normal cells, thus avoiding drug side effects.

[0018] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0019] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0020] Figure 1 This is a schematic diagram of the degradation agent proposed in one embodiment of this application;

[0021] Figure 2 This is a structural formula of a small molecule element proposed in an embodiment of this application and an analysis diagram of its degradation ability against the target protein;

[0022] Figure 3 This is a structural optimization diagram of a PROTAC drug proposed in an embodiment of this application;

[0023] Figure 4 This is a protein blot analysis and proteomics analysis diagram for multifunctionality verification proposed in an embodiment of this application;

[0024] Figure 5 This is an analysis diagram of the effect of the degradation agent proposed in one embodiment of this application in treating adrenocortical carcinoma. Detailed Implementation

[0025] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. Rather, the embodiments of this application include all variations, modifications, and equivalents falling within the spirit and scope of the appended claims.

[0026] According to the first aspect of this application, a tumor-specific protein-targeting degrader based on mRNA and click chemistry molecules is proposed, comprising one or more specifically translated mRNA sequences, an azide-coupled target protein ligand, and a DBCO-coupled covalently tagged ligand; the mRNA sequence enters tumor cells and specifically expresses a covalently tagged E3 ubiquitin ligase fusion protein; the azide-coupled target protein ligand is linked to the covalently tagged E3 ubiquitin ligase fusion protein via a bioorthogonal click reaction to target and degrade the target protein within the tumor cells.

[0027] In this application, the tumor-specific protein-targeting degrader is the ClickRNA-PROTAC drug system, which includes one or more specifically translated mRNA sequences, an azide-conjugated target protein ligand, and a DBCO-conjugated covalently tagged ligand. Its mechanism of action is as follows: Figure 1 As shown. One or more specifically translated mRNA sequences,

[0028] like Figure 1The mRNA includes a 5' sensory recognition region, a 3' protein-coding region, and a stop codon located between the 5' sensory recognition region and the 3' protein-coding region. The stop codon prevents the initial translation of the 3' protein-coding region; in this embodiment, the stop codon is UAG. In tumor cells, the 5' sensory recognition region hybridizes with tumor-specific mRNA to form an AC-mismatched double-stranded RNA, activating ADARs-mediated A-to-I base editing, converting the UAG stop codon to the UIG codon. In this way, the translation of the 3' protein-coding region is reactivated. Subsequently, the T2A self-cleaving peptide undergoes self-cleavage to release an effector protein (a covalently tagged E3 ubiquitin ligase fusion protein). Because normal cells lack tumor-specific mRNA, they cannot form double-stranded RNA containing the AC mismatch, nor can they activate AI base editing, thus failing to produce the complete UAG stop codon. Therefore, ClickRNA-PROTAC drugs can only selectively degrade the target protein in tumor cells.

[0029] The covalently tagged E3 ubiquitin ligase fusion protein includes an N-terminal covalently tagged protein for covalently binding to the covalently tagged protein substrate; a C-terminal E3 ubiquitin ligase that mediates the ubiquitination of the target protein; and a linking sequence that connects the covalently tagged protein and the E3 ubiquitin ligase. The SIAH1-SN fusion protein is used as an example for the following explanation.

[0030] The amino acid sequence of the SIAH1-SN fusion protein is shown in SEQ ID NO: 1. Its N-terminus is a SNAPTag covalently tagged protein, which is used to covalently bind to the covalently tagged protein substrate (benzylure derivative); the C-terminus is an E3 ubiquitin ligase SIAH1, which can mediate the ubiquitination of specific proteins and promote their degradation through the ubiquitin-proteasome pathway; the two are connected by a linker sequence (GGGGSGGGGS).

[0031] Where SEQ ID NO: 1 is

[0032] MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVVPAPAAVLGGPEPLM

[0033] QATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAAL

[0034] AGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKP

[0035] GLGGGGGSGGGGSTGSRQTATALPTGTSKCPPSQRVPALTGTTASNNDLASLFECPVCFD

[0036] YVLPPILQCQSGHLVCSNCRPKLTCCPTRGPLGSIRNLAMEKVANSVLFPCKYASSGCEIT

[0037] LPHTEKADHEELCEFRPYSCPCPGASCKWQGSLDAVMPHLMHQHKSITTLQGEDIVFLAT

[0038] DINLPGAVDWVMMQSCFGFHFMLVLEKQEKYDGHQQFFAIVQLIGTRKQAENFAYRLELNGHRRRLTWEATPRSIHEGIATAIMNSDCLVFDTSIAQLFAENGNLGINVTISMCRT.

[0039] In different tumor cells, E3 ubiquitin ligases, including VHL, CRBN, CHIP, MARCH5, NEDD4L, PARKIN, SIAH1, MDM2, BIRC2, DCAF15, DCAF16, RNF4, or RNF114, can all be used in the drug design of this invention to achieve the purpose of ubiquitination of target proteins. Furthermore, different covalently labeled proteins can also be used to achieve the covalent linkage function in this embodiment: such as SNAPTag (covalently bound to benzylguanine derivatives), HaloTag (covalently bound to chloroalkanes), CLIP-Tag (covalently bound to cyclooctyne), and TMP-Tag (covalently bound to trimethoprim), etc.

[0040] In summary, this embodiment utilizes a tumor-specific mRNA response translation regulation strategy based on UAG stop codon AI base editing. ClickRNA-PROTAC drugs can selectively degrade target proteins in tumor cells, while also efficiently degrading them. Furthermore, it offers advantages such as independence from endogenous E3 ubiquitin ligases, tumor specificity, and programmability, providing a new direction for the development and clinical application of PROTAC drugs.

[0041] In this embodiment, the covalently tagged ligand coupled to DBCO is formed by modifying a covalently tagged protein substrate with DBCO, which serves as the substrate for constructing a bioorthogonal click reaction. For example, taking the aforementioned SIAH1-SN fusion protein as an example, DBCO is modified onto the SNAPTag covalently bound substrate benzylguanine to construct BG-DBCO, wherein the chemical structure of BG-DBCO is as follows: Figure 2 As shown in Figure A.

[0042] When using other covalently tagged proteins, DBCO can be coupled to other covalently tagged substrates to construct a bioorthogonal click reaction substrate. For example, covalently tagged protein substrates include benzylguanine derivatives, chloroalkanes, cyclooctyne, or trimethoprim, which can achieve the functions of both covalent tags and bioorthogonal substrates.

[0043] This embodiment utilizes a DBCO bioorthogonal reaction substrate to modify azide molecules onto the target protein ligand, forming an azide-coupled target protein ligand. Based on the azide-coupled target protein ligand, the chain length between the azide group and the target protein ligand is optimized to obtain the optimal azide-coupled target protein ligand, achieving the highest target degradation level. For example, using the target protein BRD4, an azide molecule is coupled to the ligand molecule JQ1 to construct JQ1-N3 (structural formula as shown). Figure 2 (As shown in A). In related technologies, the length of the linker in the PROTAC molecule directly affects the interaction between POI and E3 ubiquitin ligase, thereby affecting the maximum target degradation level (Dmax). Therefore, this application improves Dmax by optimizing the chain length between the azide group and the POI ligand molecule. Figure 3 As shown, this application designed and synthesized 11 JQ1-N3 molecules with different chain lengths, such as... Figure 3 As shown in A, Western blot analysis results indicate that JQ1-N3-C10 is the optimal azide-coupled target protein ligand, as shown in Figure A. Figure 3 As shown in B.

[0044] Furthermore, due to the design of the bioorthogonal click reaction, this application allows for the degradation of different target proteins within tumor cells by changing the target molecule. For example, modifying MRTX849 and NFκB-ODN molecules with azide groups and corresponding linkers can effectively achieve the degradation of KRAS and p65 proteins. Figure 4 A and Figure 4 B demonstrates the multifunctionality of the tumor-specific protein-targeting degrader in this embodiment.

[0045] In some embodiments, tumor cells include adrenocortical cancer cells.

[0046] The mRNA designed and constructed in this embodiment can achieve specific translation in adrenocortical carcinoma. The mRNA sequence is shown in SEQ ID NO: 2. It is divided into four parts from 5' to 3': (1) mCherry sequence, used to verify mRNA expression and screen specific sensor gene sequences; (2) IGF2 recognition sequence, which can enable tumor-specific translation in adrenocortical carcinoma; (3) T2A sequence, which expresses self-cleaving peptide and releases effector protein; and (4) SNAP-SN fusion protein sequence.

[0047]

[0048] Based on the fundamental principles of this application, replacing the IGF2 recognition sequence with recognition sequences of other tumor-specific, highly expressed mRNAs can also be used in the preparation of tumor therapeutic agents or tumor cell proliferation inhibitors. Therefore, this application avoids the therapeutic limitations caused by the varying expression levels of E3 ligase in different tumors by directly encoding an E3 ligase-covalently tagged protein fusion protein. Furthermore, the substrate design of the orthogonal click reaction constructed from the DBCO-coupled covalently tagged ligand enables the degradation of different target proteins, demonstrating the multifunctionality of this tumor-specific protein-targeting degrader. In addition, a tumor-specific single-base editing module is designed into the mRNA sequence to achieve tumor-specific mRNA expression.

[0049] Example 1

[0050] Degradation of intracellular target protein BRD4: The tumor-specific protein-targeting degrader constructed in this embodiment is used for the degradation of the target protein. Taking BRD4 as an example, the specific operation steps are as follows:

[0051] (a) The specific steps for introducing mRNA into cells to express the SIAH1-SN fusion protein are as follows:

[0052] (a1) HEK293FT cells were seeded in DMEM medium supplemented with 10% fetal bovine serum in six-well plates and cultured at 37°C with 5% CO2 for 24 h to achieve a cell density of 80% at the time of transfection.

[0053] (a2) Dilute 400 ng mRNA and 1 μL liposome RNA transfection reagent with 50 μL serum-reduced DMEM medium and incubate at 20°C for 5 min respectively;

[0054] (a3) Mix the diluted mRNA and transfection reagent and incubate at 20°C for 20 min;

[0055] (a4) Add the mixture to the cells in a six-well plate and incubate at 37°C with 5% CO2 for 24 h;

[0056] (a5) Treat the cells with complete medium containing 1 μM BG-DBCO (or other specified concentration) for 30 min, and then replace it with complete medium containing 500 nM JQ1-N3-C10 (or other specified concentration).

[0057] (b) Western blot analysis was performed to assess the expression of the fusion protein and the degradation of the target protein BRD4. The specific steps are as follows:

[0058] (b1) Extraction of total protein from transfected cells. Remove cells, aspirate the culture medium, and wash twice with ice-cold PBS. Add 100 μL of lysis buffer (PMSF) to each well and incubate on ice for 10 min. Gently scrape cells off with a cell scraper and collect the cell debris solution. Perform thorough physical disruption of the cells using an ultrasonic disruptor. Centrifuge (4℃, 14000 rpm, 15 min) and collect the supernatant.

[0059] (b2) Add protein loading buffer and heat at 100°C for 10 min. After removing, load the sample and perform PAGE electrophoresis (150V, 30 min).

[0060] (b3) After removing the gel, transfer it to a membrane. Add blocking buffer (5% skim milk powder), primary antibody anti-BRD4, and secondary antibody (rabbit) in sequence for incubation. Wash with TBST three times between each incubation, 5 min each time. After incubation, add developing buffer and observe the protein bands. The results of Western blot analysis are as follows: Figure 2 B Figure 2 C and Figure 3 B. Verify the degradation effect of the target protein BRD4.

[0061] (c) Proteomic analysis of BRD4, the target protein for drug degradation in this invention, is performed using the following steps:

[0062] (c1) The steps for extracting target proteins from drug-incubated cells are shown in (b1-b2). After electrophoresis, the gel was soaked in Coomassie Brilliant Blue staining solution for 30 min, and then soaked in water for 30 min. The gel bands were cut off for proteomics analysis. The protein samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0063] (c2) During mass spectrometry data analysis, the raw data files were first matched against the Uniprot human protein database using Proteome Discoverer 2.2 software. Precursor and fragment mass tolerances were set to 10 ppm and 0.02 Da, respectively, allowing for the omission of up to two trypsin cleavage sites. Amide methylation (C) was a static modification, while oxidation (M) and acetylation (N-terminus) were variable modifications. The Percolator algorithm was used to determine peptide matching (PSMs) and the false discovery rate of peptide identification, with a confidence level controlled below 1%. Protein enrichment analysis was performed on proteins identified by two or more peptides. Label-free quantification was used for protein abundance analysis. Protein abundance was normalized based on total protein concentration and logarithmically transformed. Missing values ​​were estimated based on the mass spectrometer's detection limit. Enriched proteins were identified by comparing the changes in protein abundance between small molecule-treated samples (experimental group) and untreated samples (control group). Proteins with a fold change greater than 2 and a confidence level less than 0.05 were considered statistically enriched.

[0064] Proteomics results such as Figure 4 As shown in Figure C, the expression of SIAH1-SN and the effective degradation of the target protein BRD4 are demonstrated.

[0065] Example 2

[0066] Compared to the examples that used different target molecules to verify the degradation effect of the target protein, the specific operation steps for verifying the multifunctionality of the tumor-specific protein-targeting degrader are as follows:

[0067] (d) Introduce mRNA into cells to express the SIAH1-SN fusion protein. The specific steps are as follows:

[0068] (d1) HEK293FT cells were seeded in DMEM medium supplemented with 10% fetal bovine serum in six-well plates and cultured at 37°C with 5% CO2 for 24 h to achieve a cell density of 80% at the time of transfection.

[0069] (d2) Dilute 400 ng mRNA and 1 μL liposome RNA transfection reagent with 50 μL serum-reduced DMEM medium and incubate at 20°C for 5 min respectively;

[0070] (d3) Mix the diluted mRNA and transfection reagent and incubate at 20°C for 20 min;

[0071] (d4) The mixture was added to cells in a six-well plate and incubated at 37°C with 5% CO2 for 24 h.

[0072] (d5) Treat cells with complete medium containing 1 μM BG-DBCO (or other specified concentration) for 30 min, then replace with complete medium containing 500 nM azide-coupled ligand molecules (MRTX849-N3-C10 or NFκB-ODN-N3-C10).

[0073] (e) Western blot analysis was performed to assess the expression of the fusion protein and the degradation of the target protein. The specific steps are as follows:

[0074] (e1) Extract total protein from transfected cells. Remove cells, aspirate the culture medium, and wash twice with ice-cold PBS. Add 100 μL of cell lysis buffer (PMSF) to each well and incubate on ice for 10 min. Gently scrape cells off with a cell scraper and collect the cell debris solution. Thoroughly disrupt the cells using an ultrasonic disruptor. Centrifuge (4℃, 14000 rpm, 15 min) and collect the supernatant.

[0075] (e2) Add protein loading buffer and heat at 100°C for 10 min. After removing, load the sample and perform PAGE electrophoresis (150V, 30 min).

[0076] (e3) After removing the gel, transfer it to a membrane. Add blocking buffer (5% skim milk powder), primary antibody, and secondary antibody in sequence for incubation. Wash with TBST three times between each step, 5 min each time. After incubation, add developing buffer and observe the protein bands. The results of Western blot analysis are as follows: Figure 4 A and Figure 4 B, demonstrating the degradation of the target proteins KRAS and p65.

[0077] (f) Proteomic analysis was performed on the drug degradation target proteins KRAS and p65 of this invention, following the same steps as in step (c). The proteomic results are as follows: Figure 4 D、 Figure 4 As shown in Figure E, the expression of SIAH1-SN and the effective degradation of the target proteins KRAS and p65 are demonstrated.

[0078] Example 3

[0079] To verify the differences in BRD4 degradation and cell killing between adrenocortical carcinoma cells and normal adrenal cells, the specific operational steps are as follows:

[0080] (g) Introduce mRNA into cells to express the SIAH1-SN fusion protein. The specific steps are as follows:

[0081] (g1) SW-13 cells and HACC cells were seeded in DMEM medium supplemented with 10% fetal bovine serum in six-well plates and cultured at 37°C with 5% CO2 for 24 h to achieve a cell density of 80% at the time of transfection.

[0082] (g2) Dilute 400 ng mRNA and 1 μL liposome RNA transfection reagent with 50 μL serum-reduced DMEM medium and incubate at 20℃ for 5 min respectively;

[0083] (g3) Mix the diluted mRNA and transfection reagent and incubate at 20°C for 20 min;

[0084] (g4) Add the mixture to the cells in a six-well plate and incubate at 37°C with 5% CO2 for 24 h;

[0085] (g5) Treat the cells with complete medium containing 1 μM BG-DBCO (or other specified concentration) for 30 min, and then replace with complete medium containing 500 nM JQ1-N3-C10.

[0086] (h) Western blot analysis was performed to assess the expression of the fusion protein and the degradation of the target protein. The specific steps are as follows:

[0087] (h1) Extract total protein from transfected cells. Remove cells, aspirate the culture medium, and wash twice with ice-cold PBS. Add 100 μL of lysis buffer (PMSF) to each well and incubate on ice for 10 min. Gently scrape cells off with a cell scraper and collect the cell debris solution. Perform thorough physical disruption of the cells using an ultrasonic disruptor. Centrifuge (4℃, 14000 rpm, 15 min) and collect the supernatant.

[0088] (h2) Add protein loading buffer and heat at 100°C for 10 min. After removing, load the sample and perform PAGE electrophoresis (150V, 30 min).

[0089] (h3) After removing the gel, transfer it to a membrane. Add blocking buffer (5% skim milk powder), primary antibody, and secondary antibody in sequence for incubation. Wash with TBST three times for 5 minutes each time between incubation. After incubation, add developing buffer and observe the protein bands. The results of Western blot analysis are as follows: Figure 5 A, demonstrating the degradation of the target protein BRD4.

[0090] Example 4

[0091] A mouse xenograft model of adrenocortical carcinoma, a mouse model of xenografted adrenocortical carcinoma, and drug treatment. The specific steps are as follows:

[0092] (i1) Female Balb / c nude mice were subcutaneously injected with 100 μL of cell suspension (1 × 10⁻⁶). 7 (cells). The tumor volume reached 100 mm3 on the 9th day after inoculation.

[0093] (i2) Tumor-bearing mice were randomly divided into groups and treated with different drugs via tail vein injection for three cycles, with an interval of 5 days. Simultaneously, 20 μg of mRNA and 10 mg / kg of BG-DBCO were administered, followed by 5 mg / kg of JQ1-N3-C10 12 hours later. Tumor volume and animal body weight were monitored throughout the experiment. Figure 5 B.

[0094] (i3) After the anti-tumor experiment, the main organs (heart, kidney, liver, lung, and spleen) of the mice were subjected to H&E staining, such as... Figure 5 F. Western blot analysis to detect the degradation of BRD4 in tumor tissue, such as... Figure 5 E.

[0095] Based on the above analysis, this embodiment demonstrates that the novel ClickRNA-PROTAC drug, a tumor-specific protein-targeting degrader, can be successfully used for tumor-targeted protein degradation, thereby achieving specific tumor-killing therapy. Compared to traditional PROTAC drugs in related technologies, which often face challenges such as large molecular weight (greater than 1000), poor bioavailability, complex synthesis, limitation by E3 enzyme expression, and lack of tumor targeting, this embodiment achieves targeted degradation of target proteins within tumor cells by expressing a covalently tagged E3 ubiquitin ligase fusion protein, combined with a bioorthogonal click reaction and tumor-targeting RNA single-base editing strategy, thus achieving tumor treatment. Compared to the traditional PROTAC drug dBET1, this embodiment exhibits better tumor-killing effects. Figure 5 C and 5D. Therefore, the ClickRNA-PROTAC drug proposed in this embodiment can be successfully used for the treatment of tumors and has significant advantages compared with PROTAC drugs in related technologies.

[0096] It should be noted that in the description of this application, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Furthermore, in the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0097] Any process or method described in the flowchart or otherwise herein can be understood as representing a module, segment, or portion of code comprising one or more executable instructions for implementing a particular logical function or process, and the scope of the preferred embodiments of this application includes additional implementations in which functions may be performed not in the order shown or discussed, including substantially simultaneously or in reverse order depending on the function involved, as will be understood by those skilled in the art to which embodiments of this application pertain.

[0098] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0099] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A tumor-specific protein-targeting degrader based on mRNA and a click chemistry molecule, characterized in that, include: 1) One or more specifically translated mRNAs, wherein the mRNA is composed of a 5' sensory recognition region, a T2A sequence, and a 3' protein coding region in sequence; in The 3' protein coding region encodes a covalently tagged E3 ubiquitin ligase fusion protein; the covalently tagged E3 ubiquitin ligase fusion protein consists of an N-terminus for covalently binding a covalently tagged protein substrate and a C-terminus for mediating the ubiquitination of the target protein by an E3 ubiquitin ligase. And the connecting peptide that connects the covalently tagged protein and the E3 ubiquitin ligase; The 5' sensory recognition region contains a start codon AUG and a stop codon UAG that prevents the translation of the 3' protein-coding region; the 5' sensory recognition region hybridizes with specific mRNA within tumor cells and forms an AC-mismatched double-stranded RNA at the stop codon UAG; upon activation of ADARs-mediated A-to-I base editing, the UAG stop codon is converted to the UIG codon, thereby enabling the covalently tagged E3 ubiquitin ligase fusion protein encoded by the 3' protein-coding region to be translated and expressed within tumor cells; The T2A sequence releases the covalently tagged E3 ubiquitin ligase fusion protein encoded by the 3' protein coding region; 2) A DBCO-conjugated covalently tagged ligand, which is formed by covalently modifying DBCO onto the substrate of the covalently tagged protein; the substrate of the covalently tagged protein recognizes and covalently binds to the covalently tagged protein; 3) The target protein ligand coupled to the azide is formed by coupling the target protein ligand to the azide group, wherein the target protein recognizes and connects with the target protein ligand; the azide and DBCO are connected to form the substrate of the bioorthogonal click reaction.

2. The degrading agent according to claim 1, characterized by, The E3 ubiquitin ligase is VHL, CRBN, CHIP, MARCH5, NEDD4L, PARKIN, SIAH1, MDM2, BIRC2, DCAF15, DCAF16, RNF4, or RNF114.

3. The degrading agent according to claim 1, wherein The covalently tagged protein substrate is a protein substrate modified with benzylguanine, chloroalkanes, cyclooctyne, or trimethoprim.

4. The degrading agent according to claim 1, characterized in that, The tumor cells were adrenocortical cancer cells.

5. The method for optimizing the degrading agent according to any one of claims 1-4, characterized in that, Based on the target protein ligand coupled with the azide, the chain length between the azide group and the target protein ligand is optimized to obtain the optimal target protein ligand coupled with the azide, so that the target protein ligand coupled with the azide reaches the highest target degradation level.

6. The use of the degrading agent according to any one of claims 1-3 in the preparation of tumor therapeutic agents or tumor cell proliferation inhibitors.

7. The application according to claim 6, characterized in that, The tumor cells were adrenocortical cancer cells.

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