Target protein degradation agent based on ethacrynic acid and application thereof

Abstract

The invention discloses a target protein degradation agent based on ethacrynic acid and application thereof, relates to the technical field of drug development, and comprises a target protein degradation agent based on ethacrynic acid, which has a structure as shown in the following formula (I): R1LR2 formula (I), wherein R1 is ethacrynic acid EA; r2 is a ligand JQ1 of a bromodomain protein 4; l is a connecting chain, and R1 and R2 are connected through L; according to the target protein degradation agent based on ethacrynic acid and the application thereof, E3 ubiquitinase HECTD1 is recruited through ethacrynic acid to realize degradation of target protein BRD4, and the process is realized through ubiquitin-proteasome, so that drug resistance caused by target mutation is avoided, a new choice is provided for recruiting molecules of the target protein degradation agent, and the target protein degradation agent has the advantages that the target protein degradation agent is simple and convenient to operate, and the application prospect is wide. Meanwhile, a new E3 ubiquitination enzyme is also provided, a new construction thought is provided for a degradation agent of tumor protein difficult to target, and the E3 ubiquitination enzyme has good application in preparation of anti-cancer drugs.

Description

Technical Field

[0001] The present invention relates to drug development technology, and in particular to an ethacrynic acid-based target protein degradation agent and application thereof. Background Art

[0002] There are about 600 E3 ubiquitin ligases (Rape) in the human genome, and different ligases are specific to different proteins. Currently, only about 1% of E3 ubiquitin ligases have been studied in the degradation of target proteins, and most bifunctional molecules (PROTACs) achieve target protein degradation by recruiting E3 ubiquitin ligases cereblon (CRBN) and von Hippel-Lindau (VHL). However, such E3 ubiquitin ligases show different substrate specificity, and long-term use may produce drug resistance. Therefore, it is necessary to discover other targetable E3 ubiquitin ligases with differentiated characteristics to achieve global target protein degradation.

[0003] In addition, since the rise of PROTACs technology in 2010, the E3 ubiquitinase recruitment molecules used are mostly the marketed drug thalidomide. On this basis, subsequent research and development have led to the development of multiple covalent E3 ubiquitinase recruitment molecules, such as the natural product nimbolide and the small molecule fragment KB02. However, the pharmacokinetic properties of natural products and synthetic fragments have not been explored, which poses certain challenges to the optimization of the pharmacokinetic properties of the bifunctional small molecules constructed later. Summary of the invention

[0004] The purpose of the present invention is to provide a target protein degradation agent based on ethacrynic acid and its application to solve the above-mentioned deficiencies in the prior art.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a target protein degradation agent based on ethacrynic acid, which has the structure described in the following formula (I):

[0006] R1LR2 Formula (I);

[0007] in:

[0008] (a) R1 is ethacrynic acid EA;

[0009] (b) R2 is the ligand JQ1 of bromodomain protein 4;

[0010] (c) L is a connecting chain, and the R1 and R2 are connected via L.

[0011] Furthermore, the target protein degradation agent is:

[0012]

[0013] Furthermore, the preparation method includes connecting the functionalized amine of the bromodomain protein 4 ligand JQ1 to the carboxyl end of ethacrynic acid through an alkyl unit to synthesize a target protein degrader, namely EA-JQ1.

[0014] Furthermore, the synthesis conditions are: 1-n-propyl phosphoric anhydride and N,N-diisopropylethylamine as dehydration condensation agents, and dichloromethane as solvent.

[0015] Furthermore, the functionalized amine of the bromodomain protein 4 ligand JQ1 is

[0016] Application of an ethacrynic acid-based target protein degrader in the preparation of drugs for tumor-related diseases.

[0017] Furthermore, the disease includes a disease associated with bromodomain protein 4.

[0018] A pharmaceutical composition comprising the ethacrynic acid-based target protein degrader.

[0019] A pharmaceutical preparation comprises the ethacrynic acid-based target protein degrader and at least one pharmaceutically acceptable inactive ingredient.

[0020] Furthermore, the inactive ingredients of the drug are carriers, excipients and diluents commonly used in pharmacy.

[0021] Compared with the prior art, the present invention provides a target protein degrader based on ethacrynic acid and its application. The target protein degrader recruits E3 ubiquitinase HECTD1 through ethacrynic acid, a clinical covalent drug with clear pharmacokinetic properties, to achieve degradation of target protein BRD4, and the process is achieved through ubiquitin-proteasome, avoiding drug resistance caused by target mutations. It not only provides a new choice for the recruitment molecules of target protein degraders, but also provides a new E3 ubiquitinase, providing a new construction idea for degraders of difficult-to-target tumor proteins, and has good application in the preparation of anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0023] Figure 1 Mass spectrometry results for verifying the feasibility of the clinical drug ethacrynic acid as a covalent recruitment molecule and a schematic diagram of the synthesis route of EA-JQ1 provided in an embodiment of the present invention;

[0024] Figure 2 NMR spectrum and mass spectrum characterization diagram of EA-JQ1 provided in the embodiment of the present invention;

[0025] Figure 3 A schematic diagram of the structure of HECTD1, a ligand of ethacrynic acid provided in an embodiment of the present invention;

[0026] Figure 4 A schematic diagram of the experimental results of EA-JQ1 degradation of the target protein BRD4 provided in the embodiments of the present invention;

[0027] Figure 5 A schematic diagram of the experimental results of proteasome degradation of BRD4 provided in an embodiment of the present invention;

[0028] Figure 6 A schematic diagram of the experimental results of EA-JQ1 recruiting HECTD1 to degrade BRD4 protein provided in an embodiment of the present invention;

[0029] Figure 7 Schematic diagram of the experimental results of EA-JQ1 inhibiting BRD4 downstream protein c-MYC provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] Embodiment 1:

[0032] See also Figure 1 , a target protein degradation agent based on ethacrynic acid, which has the following formula (I):

[0033] R1LR2 formula (I);

[0034] in:

[0035] (a) R1 is ethacrynic acid EA;

[0036] (b) R2 is the ligand JQ1 of bromodomain protein 4;

[0037] (c) L is a connecting chain, and R1 and R2 are connected through L.

[0038] For specific implementation, please refer to Figure 1 Through the quantitative detection technology of thiol reactivity (QTRP), it was found that the clinical drug ethacrynic acid can covalently act on the E3 ubiquitin ligase HECTD1_C1368, and this E3 ubiquitin ligase lacks recruitment molecules.

[0039] The quantitative thiol-reactive technique (QTRP) is specifically used to identify the targets of thiol-reactive substances at the site level. This technique can be divided into the following four main steps: (1) labeling the control and test compound-treated proteomes with the "clickable" thiol-reactive probe IPM; (2) after the IPM-labeled proteome is enzymatically hydrolyzed into peptides, the IPM-labeled peptides are subjected to copper-catalyzed alkynyl-azide cyclization reaction ("click chemistry") with "light" and "heavy" isotope-labeled photolyzed biotin to convert the IPM-labeled peptides into biotinylated peptides; (3) after mixing the "light" and "heavy" labeled samples, avidin enrichment and photolysis elution are performed; (4) qualitative and quantitative analysis based on LC-MS / MS. In theory, if a cysteine ​​residue site is bound by the test compound, it can no longer be labeled by the IPM probe. By quantitatively measuring the change in probe labeling efficiency based on the "light and heavy isotope" labeled LC-MS / MS method, it can be indirectly determined whether the site is the target of the test compound ( Figure 1 a). This technique was used to determine the cysteine ​​target of the clinical covalent drug ethacrynic acid in HEK293T cells, and it was found that ethacrynic acid can act on the 1368th cysteine ​​of the E3 ubiquitin ligase HECTD1, with R = 2.6 ( Figure 1 b).

[0040] As an oral diuretic, ethacrynic acid has a bioavailability of approximately 100% and is more soluble in water. The carboxyl group (pKa ~ 3.8) in its molecular structure provides advantages when connecting to the ligand of the target protein. Therefore, ethacrynic acid is used as a covalent recruitment molecule for PROTACs.

[0041] Bromodomain protein 4 (BRD4) is an important member of the bromodomain and extra-terminal (BET) family. It regulates DNA repair and controls gene expression through histone acetylation, thereby affecting the cell cycle. The effectiveness of BET family members' degradation depends on proteasomes, especially BRD4, which can be degraded not only by the degradosome formed by thalidomide and JQ1 through recruiting CRBN, but also by the degradosome formed by the natural product nimbolide and JQ1 through recruiting RNF114. Therefore, the ligand JQ1 of BRD4 protein was selected, and its functionalized amine was connected to the carboxyl end of ethacrynic acid through an alkyl unit to synthesize a new target protein degrader, which was named EA-JQ1.

[0042] The target protein degraders are:

[0043]

[0044] For specific implementation, please refer to Figure 1Part b in the figure shows that the green part is ethacrynic acid EA, the blue part is the connecting chain, and the red part is the ligand JQ1 of bromodomain protein 4. EA represented by the green part is a compound with significant diuretic effect, which is usually used to treat diseases such as edema and hypertension. Its unique chemical structure gives ethacrynic acid a strong pharmacological activity and can effectively promote the excretion of excess water in the body.

[0045] The connecting chain depicted in blue is the key bridge connecting ethacrynic acid EA and BRD4 ligand JQ1. This connecting chain not only ensures the stable binding between EA and JQ1, but also cleverly guides the complex to the bromodomain of BRD4.

[0046] The red part is JQ1, a specific ligand of BRD4. As a small molecule inhibitor, JQ1 can accurately recognize and bind to the bromodomain of BRD4, thereby blocking the interaction between BRD4 and acetylated chromatin and inhibiting the abnormal transcription of its downstream genes. This mechanism makes JQ1 show great potential in the field of cancer treatment, especially for those tumor types that rely on overactivation of BRD4. By inhibiting the function of BRD4, it inhibits the proliferation of cancer cells and promotes cell apoptosis.

[0047] The preparation method comprises the following steps: connecting the functionalized amine of the bromodomain protein 4 ligand JQ1 to the carboxyl end of ethacrynic acid through an alkyl unit to synthesize a target protein degradation agent, namely EA-JQ1; the synthesis conditions are: using 1-n-propylphosphoric anhydride (T3P) and N, N-diisopropylethylamine (DIEA) as dehydration condensation agents, using dichloromethane (DCM) as a solvent, and the functionalized amine of the bromodomain protein 4 ligand JQ1 is

[0048] For specific implementation, please refer to Figure 1 In part b, T3P and DIEA are used as dehydration condensation agents, DCM is used as solvent, and EA is added Reaction was performed to obtain EA-JQ1, the structural formula of EA is

[0049] Application of an ethacrynic acid-based target protein degrader in the preparation of drugs for tumor diseases associated with BRD4 protein.

[0050] The specific implementation method is that the expression of BRD4 protein is closely related to the occurrence and development of various tumor diseases, including lung cancer, breast cancer, acute myeloid lymphoma (AML), liver cancer, gastrointestinal stromal tumor (GIST), multiple myeloma (MM) and glioblastoma (GBM).

[0051] A pharmaceutical composition comprising an ethacrynic acid-based target protein degrader.

[0052] A pharmaceutical preparation comprises an ethacrynic acid-based target protein degrader and at least one pharmaceutically acceptable inactive pharmaceutical ingredient, wherein the inactive pharmaceutical ingredient is a carrier, excipient and diluent commonly used in pharmacy.

[0053] The specific implementation method is that non-drug active ingredients such as carriers, excipients and diluents are well known in the art, and those skilled in the art can determine that they meet clinical standards, so no further details are given here.

[0054] Embodiment 2:

[0055] See also Figure 2 This embodiment provides a technical solution based on the first embodiment: characterizing the target protein degrader by NMR spectrum and mass spectrum.

[0056] NMR spectroscopy is a method that uses the absorption of radio frequency radiation by atomic nuclei to determine structure, and to perform qualitative and quantitative analysis. Under the action of an external magnetic field, atomic nuclei with nuclear magnetic moments absorb radio frequency energy and undergo nuclear spin energy level transitions. The signals generated by this process are recorded to form an NMR spectrum. NMR spectroscopy is based on the magnetic properties of atomic nuclei and their behavior in a magnetic field. Certain atomic nuclei (such as 1 H. 13 C. 15 N, etc.) have spin phenomena and therefore have magnetic moments. When these atomic nuclei are placed in a strong magnetic field, their magnetic moments will be rearranged and parallel or antiparallel to the direction of the magnetic field, forming two energy levels. If these atomic nuclei are irradiated with radio frequency radiation of a specific frequency at this time, when the frequency of the radio frequency radiation matches the frequency required for the energy level transition of the atomic nuclei, the atomic nuclei will absorb energy and transition from low energy levels to high energy levels. This phenomenon is called nuclear magnetic resonance, which reflects the relative position and distance information between the atomic nuclei.

[0057] Mass spectrometry (MS) is a professional technology that identifies compounds by preparing, separating and detecting gas-phase ions. Its core is to ionize the substance to be tested, separate it according to the mass-to-charge ratio (m / z, i.e. the ratio of mass to charge), and measure the intensity of various ion peaks to achieve the purpose of analysis. The sample to be analyzed is sent into the ion source of the mass spectrometer through the injection system, and the injection system will send the sample to the appropriate part of the ion source according to the needs of the ionization mode; in the ion source, the sample molecules are ionized into charged ions; the ions generated after ionization are accelerated under the action of the electric field to form an ion beam, and the ion beam is focused by a focusing device (such as an ion lens) to improve its energy and directionality; the accelerated ion beam enters the mass analyzer, which uses the action of the electric field and magnetic field to cause ions of different mass-to-charge ratios to disperse in velocity, so as to focus them separately and obtain a mass spectrum; the ions separated by the mass analyzer are received by the detector and converted into electrical signals for recording. The detector usually uses highly sensitive detection elements such as electron multipliers or microchannel plates to ensure that weak ion signals can be accurately detected and recorded. The recorded mass spectrum shows the change of relative kurtosis of ions with m / z in the form of a bar graph, providing the mass number and relative kurtosis information of molecular ions and fragment ions, which can be used to determine relative molecular mass, molecular formula and structural formula of compounds, etc.

[0058] Figure 2 (a) is a characterization diagram of the degrader EA-JQ1 by NMR spectroscopy; (b) is a characterization diagram of the degrader EA-JQ1 by mass spectrometry; the successful synthesis of the target protein degrader EA-JQ1 is demonstrated.

[0059] Embodiment three:

[0060] See also Figure 4 This embodiment provides a technical solution based on the first embodiment: EA-JQ1 degradation target protein BRD4 verification experiment:

[0061] Human embryonic kidney cells (HEK293T) were treated with different concentrations of the degrader EA-JQ1 for 4 h, and then the cells were lysed and the expression level of BRD4 in the cells was detected by immunoblotting; the results showed that EA-JQ1 concentration as low as 100 nM could cause significant degradation of BRD4 ( Figure 4 a), and similar results were obtained by immunofluorescence detection of BRD4 expression levels in cells ( Figure 4 b), indicating that EA-JQ1-induced BRD4 degradation requires the participation of the target protein.

[0062] HEK293T cells were then treated with 100 nM EA-JQ1 for different periods of time, and the expression level of BRD4 in the cells was detected by immunoblotting. The results showed that EA-JQ1 could cause BRD4 degradation when incubated with cells for 2 h, and BRD4 degradation was more obvious when the incubation time reached 4 h or longer ( Figure 4 c); The fluorescence intensity of BRD4 was detected by immunofluorescence when EA-JQ1 was incubated with cells for different time periods, the BRD4 targeted degrader MZ1 was used as a positive control, and the ligand of BRD4 protein JQ1 was used as a negative control; the results showed that EA-JQ1 incubated with cells for 2 hours could cause a decrease in the fluorescence intensity of BRD4, which was consistent with the result obtained when the positive drug MZ1 was incubated with cells, while JQ1 alone could not cause BRD4 degradation ( Figure 4 d).

[0063] EA-JQ1-induced changes in protein expression were determined by quantitative proteomics experiments based on stable isotope dimethyl labeling. The results showed that similar to other JQ1-based covalent degraders, EA-JQ1 can only cause degradation of BRD4, but not BRD4 homologous proteins BRD2 and BRD3. EA-JQ1 treatment can also reduce the expression of another bromodomain-containing protein BAZ2A (zinc finger domain protein 2A). Among the 5423 quantified proteins, some non-BET proteins also underwent significant degradation ( Figure 4 e).

[0064] Figure 4 (a) is a western blotting showing the expression results of BRD4 in HEK293T cells after treatment with different concentrations of EA-JQ1 for 4 hours; (b) is a confocal laser scanning microscope showing the expression results of BRD4 in HEK293T cells after treatment with different concentrations of EA-JQ1 for 4 hours; the cell nucleus is stained with DAPI (blue); (c) is a western blotting showing the expression results of BRD4 in HEK293T cells after treatment with 100nM EA-JQ1 for different time periods; (d) is a confocal laser scanning microscope showing the expression results of BRD4 in HEK293T cells after treatment with 100nM EA-JQ1 for different time periods; the cell nucleus is stained with DAPI (blue), MZ1 is the positive control, and JQ1 is the negative control; (d) is a volcano plot of proteome changes in HEK293T cells after treatment with 5μM EA-JQ1 for 4 hours.

[0065] Embodiment 4:

[0066] See also Figure 5 This embodiment provides a technical solution based on the first embodiment: BRD4 degradation experiment through proteasome:

[0067] HEK293T cells were incubated with 1 μM proteasome inhibitor bortezomib (BTZ) for 2 h, and then EA-JQ1 at a concentration of 100 nM was added thereto. After incubation with the cells for 4 h, the expression of BRD4 in the cells was detected by immunoblotting and immunofluorescence.

[0068] The results showed that incubation of cells with the proteasome inhibitor BTZ attenuated the degradation of BRD4 caused by EA-JQ1, indicating that EA-JQ1 recruits E3 ubiquitin ligases to cause BRD4 degradation through the proteasome. When the parent drug EA was added to the cells to compete, the level of BRD4 degradation mediated by EA-JQ1 was also reduced ( Figure 5 a and Figure 5 b), indicating that EA-JQ1 can bind to the same E3 ubiquitin ligase as the parent drug EA.

[0069] Figure 5 (a) is a picture showing the expression of BRD4 protein in HEK293T cells by immunoblotting; (b) is a picture showing the expression of BRD4 in HEK293T cells by laser confocal microscopy, with a scale bar of 10 μm.

[0070] Embodiment five:

[0071] See also Figure 3 and Figure 6 This embodiment provides a technical solution based on the first embodiment: Experiment on EA-JQ1 recruiting HECTD1 to degrade BRD4:

[0072] HECTD1, the full name of which is HECT domain containing 1, is a protein containing a HECT domain that has multiple important functions in biology. As an E3 ubiquitin ligase, HECTD1 participates in the ubiquitination process of proteins, mediating the ubiquitination of specific proteins and regulating the degradation, localization and activity of these proteins; HECTD1 may participate in the regulation of the cell cycle and cell proliferation by regulating the ubiquitination of certain key cell cycle proteins or proliferation-related proteins; although there are currently few studies on the direct association between HECTD1 and specific diseases, its role as a ubiquitin ligase suggests that it may play a role in the occurrence and development of a variety of diseases.

[0073] HECTD1 was knocked down in HEK293T cells using small interfering RNA, and then the degradation of BRD4 in wild-type and knockdown cells was detected by western blotting and immunofluorescence.

[0074] The experimental results showed that both siRNA sequences could lead to a decrease in the expression of HECTD1 in cells. Compared with wild-type cells, no degradation of BRD4 was observed in cells where HECTD1 was knocked down ( Figure 6 a and Figure 6 b), indicating that EA-JQ1 can recruit the E3 ubiquitination ligase HECTD1, thereby leading to BRD4 degradation.

[0075] Figure 6 (a) is a western blotting showing the expression results of BRD4 in wild-type and knockdown HEK293T cells of HECTD1 for 4 hours after treatment with EA-JQ1 at a concentration of 100 nM; (b) is a laser confocal microscopy showing the expression results of BRD4 in wild-type and knockdown HEK293T cells of HECTD1 for 4 hours after treatment with EA-JQ1 at a concentration of 100 nM, with a scale bar of 10 μm.

[0076] Ethacrynic acid, which has clear pharmacokinetic properties, can act on the 1368th cysteine ​​of the E3 ubiquitin ligase HECTD1, with R = 2.6 ( Figure 1 a); The enzyme contains 2610 amino acids, of which 53 cysteines are distributed in four different domains, including the N-terminal ankyrin repeat sequence, the middle domain MIB / HERC2 and the C-terminal HECT ubiquitin protein ligase domain ( Figure 3 a), Cysteine ​​2579 located in the HECT domain can form a thioester bond with the ubiquitin chain, and then transfer the ubiquitin chain to the substrate to exert its enzymatic activity. The Alpha Fold predicted structure shows that the spatial distance between Cysteine ​​1368 targeted by ethacrynic acid and the active site of the enzyme (CYS2579) is 107.5A ( Figure 3 b), suggesting that targeting cysteine ​​1368 of HECTD1 by ethacrynic acid does not affect the ubiquitination activity of the enzyme itself.

[0077] In addition, the expression level and subcellular localization of E3 ubiquitin ligase in cells are also factors that need to be considered during PROTACs design. In the proteomic data of 81 cells and tissues in the human proteome atlas, HECTD1 can be detected in all cell and tissue types, and is highly expressed in urothelial cells and skeletal muscle. At the same time, the protein can be located in the nucleus and cytoplasm. HECTD1 can be used as a suitable E3 ubiquitin ligase to design an effective degrader.

[0078] Figure 3 (a) is the structural diagram of the E3 ubiquitin ligase HECTD1; C2579 (marked in blue) is the active site of HECTD1, and C1368 (marked in red) is the targetable site of ethacrynic acid; (b) is the Alpha Fold predicted structure of HECTD1, and the yellow line marks the spatial distance between the ethacrynic acid targeting site CYS1368 and the active site CYS2579 of the enzyme.

[0079] Embodiment six:

[0080] See also Figure 7 This embodiment provides a technical solution based on the first embodiment: Experiment on the effect of EA-JQ1 on the expression of BRD4 downstream protein c-MYC:

[0081] c-MYC is a widely present and highly studied proto-oncogene in the human genome. The protein it encodes plays a key role in cell biology. It is a transcription factor that participates in regulating cell growth, proliferation, metabolism, and tissue development. In cancer, overexpression of c-MYC is often closely related to the occurrence and development of tumors. BRD4 can phosphorylate the 58th tyrosine of c-MYC protein, leading to ubiquitination of c-MYC and degradation through the proteasome ( Figure 7 a).

[0082] After EA-JQ1 and HEK293T cells were co-incubated for 4 h, the cells were collected and the expression of c-MYC in the cells was detected by qPCR.

[0083] qPCR, or real-time quantitative polymerase chain reaction (qPCR), is a technology widely used in the field of molecular biology. qPCR quantitatively analyzes the c-MYC sequence by real-time monitoring of changes in fluorescent signals during PCR amplification. This technology uses the fluorescent signal generated by the combination of the fluorescent chemical SYBR Green and the PCR product to monitor changes in the amount of the product after each round of PCR cycles, thereby achieving accurate measurement of the target sequence.

[0084] First, the target RNA is extracted from the sample to be tested, and then reverse transcribed into cDNA for subsequent PCR amplification. Specific primers are designed according to the target sequence. The primers are used to guide DNA polymerase to synthesize new DNA chains on the template, while the probe is used to generate fluorescent signals.

[0085] Then, according to the experimental requirements, the PCR reaction system is configured and placed in a thermal cycler for PCR amplification. The amplification process includes three steps: denaturation, annealing, and extension, which are carried out in cycles. In the denaturation step, the double-stranded DNA is unwound into a single strand; in the annealing step, the primer binds to the specific region of the template DNA; in the extension step, the DNA polymerase synthesizes a new DNA chain under the guidance of the primer. During the PCR amplification process, the fluorescent signal gradually increases with the increase of the product. By monitoring the changes in the fluorescent signal in each cycle through a real-time fluorescent PCR instrument, an amplification curve can be drawn.

[0086] According to the amplification curve and the set fluorescence threshold, the Ct value (cycle threshold), that is, the number of PCR cycles required for the fluorescence signal to reach the threshold, is calculated. The Ct value is inversely proportional to the amount of the starting template, thereby performing a quantitative analysis of the c-MYC sequence.

[0087] The results showed that drug treatment can lead to increased expression of the c-MYC gene, which is consistent with the results obtained by treating cells with the positive drug MZ1 ( Figure 7 b); The expression of c-MYC protein was detected by immunoblotting, and the same results were obtained ( Figure 7 c).

[0088] Figure 7 (a) is a schematic diagram of BRD4 involved in the degradation of c-MYC protein; (b) is an experimental result diagram of the relative expression of c-MYC gene in HEK293T cells after being treated with DMSO, EA-JQ1 and positive control MZ1 for 4 hours, detected by qPCR (n=3); (c) is an experimental result diagram of the expression of c-MYC protein in HEK293T cells after being treated with DMSO, EA-JQ1 and positive control MZ1 for 4 hours, detected by immunoblotting.

[0089] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A target protein degradation agent based on ethacrynic acid, characterized in that: It has the structure described by the following formula (I): R1LR2 formula (I); in: (a) R1 is ethacrynic acid EA; (b) R2 is the ligand JQ1 of bromodomain protein 4; (c) L is a connecting chain, and the R1 and R2 are connected via L.

2. The target protein degradation agent based on ethacrynic acid according to claim 1, characterized in that: The target protein degradation agent is:

3. The target protein degradation agent based on ethacrynic acid according to claim 1, characterized in that: The preparation method comprises connecting the functionalized amine of the bromodomain protein 4 ligand JQ1 to the carboxyl end of ethacrynic acid through an alkyl unit to synthesize a target protein degrader, namely EA-JQ1.

4. The target protein degradation agent based on ethacrynic acid according to claim 3, characterized in that: The synthesis conditions are as follows: 1-n-propyl phosphoric anhydride and N,N-diisopropylethylamine are used as dehydration condensation agents, and dichloromethane is used as solvent.

5. The target protein degradation agent based on ethacrynic acid and its use according to claim 3, characterized in that: The functionalized amine of the bromodomain protein 4 ligand JQ1 is 6. Use of an ethacrynic acid-based target protein degrader as claimed in any one of claims 1 to 5 in the preparation of drugs for tumor-related diseases.

7. The use according to claim 6, characterized in that: The diseases include diseases associated with bromodomain protein 4.

8. A pharmaceutical composition, characterized in that It comprises the ethacrynic acid-based target protein degrader according to any one of claims 1 to 5.

9. A pharmaceutical preparation, characterized in that It comprises the ethacrynic acid-based target protein degrader according to any one of claims 1 to 5 and at least one pharmaceutically acceptable inactive ingredient.

10. A pharmaceutical preparation according to claim 9, characterized in that: The inactive ingredients of the medicine are carriers, excipients and diluents commonly used in pharmacy.

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