An etacrynic acid-based target protein degrader and its application

Abstract

The present invention discloses a target protein degrader based on etacrynic acid and its application, which relates to the technical field of drug development. It includes a target protein degrader based on etacrynic acid, which has the structure shown in the following formula (I): R1LR2 Formula (I); wherein: R1 is etacrynic acid EA; R2 is the ligand JQ1 of bromodomain protein 4; L is a linker chain, and the R1 and R2 are connected by L; for the target protein degrader based on etacrynic acid and its application, the target protein BRD4 is degraded by recruiting the E3 ubiquitin ligase HECTD1 through etacrynic acid, and this process is achieved through the ubiquitin-proteasome, avoiding drug resistance caused by target mutations. It not only provides a new choice for the recruiting molecule of the target protein degrader, but also provides a new E3 ubiquitin ligase, providing a new construction idea for the degrader of difficult-to-target tumor proteins, and having good application in the preparation of anticancer drugs.

Description

Technical Field

[0001] The present invention relates to the technology of drug development, and particularly to a target protein degrader based on etacrynic acid and its application. Background Art

[0002] There are approximately 600 E3 ubiquitin ligases (Rape) in the human genome, and different ligases are specific for different proteins. Currently, only about 1% of E3 ubiquitin ligases have been studied for target protein degradation, 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 specificities and may develop drug resistance after long-term use. 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 ubiquitin enzyme recruiting molecules used have mostly been the marketed drug thalidomide. Subsequently, a number of covalent E3 ubiquitin enzyme recruiting molecules have been developed, 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 for 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 degrader based on etacrynic acid and its application to solve the above deficiencies in the prior art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A target protein degrader based on etacrynic acid, which has the structure shown in the following formula (I):

[0006] R1LR2 Formula (I);

[0007] Wherein:

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

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

[0010] (c) L is a linking chain, and R1 and R2 are linked by L.

[0011] Further, the target protein degrader is:

[0012]

[0013] Further, its preparation method includes synthesizing a target protein degrader, namely EA-JQ1, by connecting the functionalized amine of bromodomain protein 4 ligand JQ1 to the carboxyl terminus of etacrynic acid through an alkyl unit.

[0014] Further, the synthesis conditions are using 1-propylphosphonic anhydride and N,N-diisopropylethylamine as dehydrating condensing agents and dichloromethane as the solvent.

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

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

[0017] Further, the diseases include diseases related to bromodomain protein 4.

[0018] A pharmaceutical composition comprising the target protein degrader based on etacrynic acid.

[0019] A pharmaceutical preparation comprising the target protein degrader based on etacrynic acid and at least one pharmaceutically acceptable drug inactive ingredient.

[0020] Further, the drug inactive ingredient is a carrier, excipient and diluent commonly used in pharmacy.

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

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments recorded in the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings.

[0023] Figure 1 It is a schematic diagram of the mass spectrometry results for verifying the feasible properties of the clinical drug etacrynic acid as a covalent recruitment molecule and the synthesis route of EA-JQ1 provided by the embodiments of the present invention;

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

[0025] Figure 3 Schematic structural diagram of ligand HECTD1 of etacrynic acid provided by the embodiment of the present invention;

[0026] Figure 4 Schematic diagram of the experimental results of EA-JQ1 degrading target protein BRD4 provided by the embodiment of the present invention;

[0027] Figure 5 Schematic diagram of the experimental results of BRD4 degradation through the proteasome provided by the embodiment of the present invention;

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

[0029] Figure 7 Schematic diagram of the experimental results of EA-JQ1 inhibiting BRD4 downstream protein c-MYC provided by the embodiment of the present invention. Detailed implementation manners

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

[0031] Example 1:

[0032] Please refer to Figure 1 , a target protein degrader based on etacrynic acid, which has the following structure of formula (I):

[0033] R1LR2 Formula (I);

[0034] Wherein:

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

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

[0037] (c) L is a linking chain, and R1 and R2 are linked by L.

[0038] The specific implementation manner is, please refer to Figure 1 , through the thiol-reactive quantitative detection technology (QTRP), it is found that the clinical drug etacrynic acid can covalently act on E3 ubiquitin ligase HECTD1_C1368, and this E3 ubiquitin ligase lacks a recruitment molecule.

[0039] The thiol-reactive quantitative 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) separately label the proteomes treated with the control and the compound to be tested with the "clickable" thiol-reactive probe IPM; (2) after enzymatically digesting the IPM-labeled proteomes into peptides, respectively perform copper-catalyzed alkyne-azide cycloaddition reaction ("click chemistry") with "light" and "heavy" isotope-labeled photocleavable azidobiotin to convert the IPM-labeled polypeptides into biotinylated polypeptides; (3) after mixing the "light and heavy" labeled samples, perform avidin enrichment and photocleavage elution; (4) qualitative and quantitative analysis based on LC-MS / MS. Theoretically, if a cysteine residue site is bound by the compound to be tested, it can no longer be labeled by the IPM probe. By quantitatively measuring the change in the labeling efficiency of this probe using the LC-MS / MS method based on "light and heavy isotopes", it is possible to indirectly determine whether this site is the target of the compound to be tested ( Figure 1 a). Using this technique to determine the cysteine targets of the clinical covalent drug etacrynic acid in HEK293T cells, it was found that etacrynic acid can act on the 1368th cysteine of the E3 ubiquitin ligase HECTD1, R = 2.6( Figure 1 b).

[0040] As an oral diuretic, the bioavailability of etacrynic acid is about 100%, and it is more soluble in water. Moreover, the carboxyl group (pKa ~ 3.8) in its molecular structure provides advantages for ligand connection with the target protein. Therefore, etacrynic acid is used as a covalent recruitment molecule for PROTACs.

[0041] Bromodomain-containing protein 4 (BRD4) is an important member of the bromodomain and extra-terminal domain (BET) family. It regulates DNA repair and controls gene expression through histone acetylation, thereby affecting the cell cycle. The effectiveness of BET family members depends on proteasomal degradation. In particular, BRD4 can not only be degraded by the degradant formed by thalidomide and JQ1 through recruiting CRBN, but also be degraded by the degradant 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 terminus of etacrynic acid through an alkyl unit to synthesize a new target protein degrader, which was named EA-JQ1.

[0042] The target protein degrader is:

[0043]

[0044] The specific implementation manner is as follows. Please refer to Figure 1Part b therein, where the green part is etacrynic acid (EA), the blue part is the linker chain, and the red part is the ligand JQ1 of bromodomain-containing protein 4. EA represented by the green part is a compound with significant diuretic effects and is commonly used to treat conditions such as edema and hypertension. Its unique chemical structure endows etacrynic acid with powerful pharmacological activities, enabling it to efficiently promote the excretion of excess water in the body.

[0045] The linker chain depicted by the blue part is the key bridge connecting etacrynic acid (EA) and the BRD4 ligand JQ1. This linker chain not only ensures the stable binding between EA and JQ1 but also cleverly guides this conjugate towards the bromodomain of BRD4.

[0046] The red part is the specific ligand JQ1 of BRD4. As a small molecule inhibitor, JQ1 can precisely 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 the overactivation of BRD4. By inhibiting the function of BRD4, it can inhibit the proliferation of cancer cells and promote apoptosis.

[0047] Its preparation method includes connecting the functionalized amine of the bromodomain-containing protein 4 ligand JQ1 to the carboxyl terminus of etacrynic acid through an alkyl unit to synthesize the target protein degrader, namely EA-JQ1. The synthesis conditions are using 1-propylphosphonic anhydride (T3P) and N,N-diisopropylethylamine (DIEA) as dehydrating condensing agents, dichloromethane (DCM) as the solvent, and the functionalized amine of the bromodomain-containing protein 4 ligand JQ1 as

[0048] The specific implementation method is as follows. Please refer to Figure 1 Part b therein. Using T3P and DIEA as dehydrating condensing agents, DCM as the solvent, adding to EA for reaction to obtain EA-JQ1. The structural formula of EA is

[0049] The application of a target protein degrader based on etacrynic acid in the preparation of drugs for treating tumor diseases related to BRD4 protein.

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

[0051] A pharmaceutical composition comprising a target protein degrader based on etacrynic acid.

[0052] A pharmaceutical preparation comprising an etacrynic acid-based target protein degrader and at least one pharmaceutically acceptable non-drug active ingredient, the non-drug active ingredient being a carrier, excipient and diluent commonly used in pharmacy.

[0053] Specifically, non-drug active ingredients such as carriers, excipients and diluents are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards, so no further elaboration will be made here.

[0054] Example 2:

[0055] Please refer to Figure 2 , this example provides a technical solution on the basis of Example 1: characterizing the target protein degrader by NMR spectrum and mass spectrometry.

[0056] NMR spectrum is a method for structural determination, qualitative and quantitative analysis by using the absorption of radiofrequency radiation by atomic nuclei. Under the action of an external magnetic field, atomic nuclei with nuclear magnetic moments will absorb radiofrequency energy and undergo nuclear spin energy level transitions. The signals generated by this process are recorded to form an NMR spectrum. NMR spectrum is based on the magnetism 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 thus have magnetic moments. When these atomic nuclei are placed in a strong magnetic field, their magnetic moments will re-align and be parallel or anti-parallel to the magnetic field direction, forming two energy levels. If radiofrequency radiation with a specific frequency is used to irradiate these atomic nuclei at this time, when the frequency of the radiofrequency radiation matches the frequency required for the nuclear energy level transition of the atomic nuclei, the atomic nuclei will absorb energy and transition from the low energy level to the high energy level. This phenomenon is called nuclear magnetic resonance, which can reflect the relative positions and distances between atomic nuclei.

[0057] Mass Spectrometry (MS) is a professional technique for identifying compounds by preparing, separating, and detecting gas-phase ions. Its core lies in ionizing the substance to be measured, separating ions according to the mass-to-charge ratio (m / z, i.e., the ratio of mass to charge), and measuring the intensities of various ion peaks for analysis purposes. The sample to be analyzed is sent into the ion source of the mass spectrometer through the injection system. The injection system will send the sample to the appropriate part of the ion source according to the requirements 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 an electric field to form an ion beam. At the same time, a focusing device (such as an ion lens) is used to focus the ion beam to improve its energy and directionality. The accelerated ion beam enters the mass analyzer, which uses the action of an electric field and a magnetic field to cause velocity dispersion of ions with different mass-to-charge ratios, 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 is represented in the form of a bar chart showing the change of the relative peak intensity of ions with m / z, providing the mass numbers and relative peak intensity information of molecular ions and fragment ions, so it can be used to determine the relative molecular mass, compound molecular formula, and structural formula, etc.

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

[0059] Example Three:

[0060] Please refer to Figure 4 , this example provides a technical solution on the basis of Example One: Verification experiment of EA-JQ1 degrading the target protein BRD4:

[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. The expression level of BRD4 in the cells was detected by immunoblotting. The results showed that EA-JQ1 at a concentration as low as 100 nM could cause significant degradation of BRD4 ( Figure 4 a), and at the same time, the expression level of BRD4 in the cells was detected by immunofluorescence, and similar results were obtained ( Figure 4 b), indicating that the degradation of BRD4 by EA-JQ1 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] After incubating HEK293T cells with 1 μM proteasome inhibitor bortezomib (BTZ) for 2 h, 100 nM EA-JQ1 was added to the cells and incubated with the cells for 4 h. The expression of BRD4 in the cells was detected by immunoblotting and immunofluorescence.

[0068] The results showed that after incubation with the proteasome inhibitor BTZ, the degradation of BRD4 caused by EA-JQ1 was attenuated, indicating that EA-JQ1 induced the proteasomal degradation of BRD4 by recruiting the E3 ubiquitin ligase. When the parental drug EA was added to compete in the cells, it could also cause a decrease in the level of EA-JQ1-mediated BRD4 degradation ( Figure 5 a and Figure 5 b), indicating that EA-JQ1 could bind to the same E3 ubiquitin ligase as the parental drug EA.

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

[0070] Example Five:

[0071] Please refer to Figure 3 and Figure 6 In this example, on the basis of Example One, a technical solution is provided: Experiment on the degradation of BRD4 by EA-JQ1 recruiting HECTD1:

[0072] HECTD1, full name is HECT domain containing 1, is a protein containing the HECT domain and has various important functions in biology. As an E3 ubiquitin ligase, HECTD1 participates in the ubiquitination process of proteins. By mediating the ubiquitination of specific proteins, it further regulates 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 an ubiquitin ligase indicates that it may play a role in the occurrence and development of various diseases.

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

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

[0075] Figure 6 In (a), Western blot shows the expression results of BRD4 in HECTD1 wild-type and knockdown HEK293T cells treated with 100 nM EA-JQ1 for 4 h; (b) Confocal laser scanning microscopy shows the expression results of BRD4 in HECTD1 wild-type and knockdown HEK293T cells treated with 100 nM EA-JQ1 for 4 h, and the scale bar is 10 μm.

[0076] Ethacrynic acid with well-defined pharmacokinetic properties can act on cysteine at position 1368 of the E3 ubiquitin ligase HECTD1, R = 2.6 ( Figure 1 a); this enzyme contains 2610 amino acids, among which 53 cysteines are distributed in 4 different domains, including the N-terminal ankyrin repeat sequences, the middle domain MIB / HERC2, and the C-terminal HECT ubiquitin protein ligase domain ( Figure 3 a). The cysteine at position 2579 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 predicted Alpha Fold structure shows that the spatial distance between cysteine at position 1368 targeted by ethacrynic acid and the active site (CYS2579) of this enzyme is 107.5 Å ( Figure 3 b), suggesting that the ubiquitination enzymatic activity of the enzyme itself will not be affected after cysteine at position 1368 of HECTD1 is targeted by ethacrynic acid.

[0077] In addition, the expression level and subcellular localization of the E3 ubiquitin ligase in cells during the design process of PROTACs are also factors that need to be considered. Among the proteome data of 81 cell and tissue types in the Human Protein 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, this protein can be localized in the nucleus and cytoplasm, and HECTD1 can be used as a suitable E3 ubiquitin ligase to design effective degraders.

[0078] Figure 3 In (a), it is the structure 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 predicted Alpha Fold structure of HECTD1, and the yellow line marks the spatial distance between the ethacrynic acid target site CYS1368 and the active site CYS2579 of this enzyme.

[0079] Example Six:

[0080] Please refer to Figure 7 , this example provides a technical solution on the basis of Example One: Experiment on the effect of EA-JQ1 on the expression of BRD4 downstream protein c-MYC:

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

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

[0083] qPCR, namely Real-time Quantitative PCR, is a technology widely used in the field of molecular biology. qPCR quantitatively analyzes the c-MYC sequence by monitoring the change of fluorescence signal during the PCR amplification process. This technology uses the fluorescence signal generated after the fluorescent chemical SYBR Green binds to the PCR product to monitor the change of the product amount after each round of PCR cycle, so as to achieve accurate measurement of the target sequence.

[0084] First, extract the target RNA from the test sample, and then reverse transcribe it into cDNA for subsequent PCR amplification. Design specific primers according to the target sequence. The primers are used to guide DNA polymerase to synthesize a new DNA strand on the template, and the probe is used to generate fluorescence signals

[0085] Then, according to the experimental requirements, configure the PCR reaction system, place the reaction system in a thermal cycler, and perform PCR amplification. The amplification process includes three steps: denaturation, annealing, and extension, which are carried out cyclically. In the denaturation step, double-stranded DNA is unwound into single strands; in the annealing step, the primers bind to specific regions of the template DNA; in the extension step, DNA polymerase synthesizes a new DNA strand under the guidance of the primers. During the PCR amplification process, the fluorescence signal gradually increases with the increase of the product. By monitoring the change of fluorescence signal in each round of cycle with a real-time fluorescence 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 initial template amount, thereby quantitatively analyzing the c-MYC sequence.

[0087] The results showed that drug treatment could lead to an increase in the expression of the c-MYC gene, which was consistent with the results obtained by treating cells with the positive drug MZ1 ( Figure 7 b); Consistent results were also obtained by detecting the expression of c-MYC protein using Western blot ( Figure 7 c).

[0088] Figure 7 In (a) is a schematic diagram of the mode of BRD4 participating in the degradation of c-MYC protein; (b) is a graph of the experimental results of detecting the relative expression of c-MYC gene in HEK293T cells by qPCR after treating with DMSO, EA-JQ1 and the positive control MZ1 for 4 h (n = 3); (c) is a graph of the experimental results of detecting the expression of c-MYC protein in cells by Western blot after treating with DMSO, EA-JQ1 and the positive control MZ1 for 4 h.

[0089] Only some exemplary embodiments of the present invention have been described by way of illustration above. Undoubtedly, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description 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 degrader based on etacrynic acid, characterized in that, It has the following structure of formula (I): R1LR2 Formula (I); Wherein: (a) R1 is etacrynic acid EA; (b) R2 is the ligand JQ1 of bromodomain protein 4; (c) L is a linking chain, and R1 and R2 are linked by L; The target protein degrader is:

2. The targeted protein degrader based on etacrynic acid according to claim 1, wherein, The preparation method includes synthesizing a target protein degrader, namely EA-JQ1, by connecting the functionalized amine of bromodomain protein 4 ligand JQ1 to the carboxyl terminus of etacrynic acid through an alkyl unit; the synthesis conditions are using 1-propylphosphonic anhydride and N,N-diisopropylethylamine as dehydrating condensing agents and dichloromethane as a solvent; the functionalized amine of bromodomain protein 4 ligand JQ1 is 3. Use of a target protein degrader based on etacrynic acid according to any one of claims 1-2 in the preparation of a drug for a disease related to bromodomain protein 4, wherein the disease is lung cancer, breast cancer, acute myeloid lymphoma, liver cancer, gastrointestinal stromal tumor, multiple myeloma and glioblastoma.

4. A pharmaceutical composition, characterized in that, It contains the target protein degrader based on etacrynic acid according to any one of claims 1-2.

5. A pharmaceutical preparation, characterized in that, It contains the target protein degrader based on etacrynic acid according to any one of claims 1-2 and at least one pharmaceutically acceptable drug inactive ingredient.

6. A pharmaceutical preparation according to claim 5, characterized in that, The drug inactive ingredient is a carrier, excipient and diluent commonly used in pharmacy.

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