A protac construction based on e3 ubiquitin ligase zer1 and target protein dvl2 and application in heart failure treatment

Abstract

The present application relates to a PROTAC construction based on E3 ubiquitin ligase ZER1 and target protein DVL2 and its application in heart failure treatment. In particular, the present application provides a proteolysis targeting chimera (PROTAC) complex comprising an E3 ubiquitin ligase binding ligand moiety and a target protein binding ligand moiety connected by a linker, wherein the E3 ubiquitin ligase binding ligand is a ZER1 binding ligand, and the target protein binding ligand is a DVL2 binding ligand. The PROTAC molecule in the present application targets DVL2 ubiquitination degradation by recruiting E3 ubiquitin ligase ZER1 to regulate the CaMKII / HDAC4 / MEF2C signaling axis, thereby reducing pathological cardiac hypertrophy and intervening the progression of heart failure, and providing a new therapeutic target for treating cardiac hypertrophy and heart failure caused by pressure overload.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the construction and application of PROTAC molecules based on E3 ubiquitin ligase ZER1 and target protein DVL2. Background Technology

[0002] Myocardial hypertrophy is an adaptive response of the heart to hemodynamic stresses such as hypertension. However, prolonged hypertrophy can lead to pathological remodeling and eventually progress to heart failure (HF). Despite various clinical treatments, the global morbidity and mortality rates of heart failure remain alarmingly high. Therefore, elucidating the pathological mechanisms of heart failure and identifying new therapeutic targets are crucial for developing more effective treatment strategies. Summary of the Invention

[0003] This invention constructs PROTAC molecules by screening and preparing ligands that specifically bind to the E3 ubiquitin ligase substrate receptor ZER1 and the target protein DVL2, for the prevention and treatment of cardiac dysfunctions such as pathological cardiac remodeling and heart failure. The PROTAC molecules of this invention can recruit ZER1 to more extensively target and degrade DVL2, thereby providing cardioprotection, for example, against stress overload-induced myocardial hypertrophy.

[0004] In one aspect, the present invention provides a protein hydrolysis-targeting chimeric (PROTAC) complex comprising an E3 ubiquitin ligase-binding ligand portion and a target protein-binding ligand portion connected by a linker group, wherein the E3 ubiquitin ligase-binding ligand portion is a ZER1-binding ligand portion and the target protein-binding ligand portion is a DVL2-binding ligand portion.

[0005] In another aspect, the present invention provides a pharmaceutical composition comprising the PROTAC complex of the present invention and pharmaceutically acceptable ingredients.

[0006] In another aspect, the present invention provides the use of the PROTAC complex or pharmaceutical composition of the present invention in the preparation of a medicament for the prevention or treatment of cardiac dysfunction.

[0007] In some implementations, this application may include the inventions described below.

[0008] 1. A protein hydrolysis-targeting chimeric (PROTAC) complex comprising an E3 ubiquitin ligase-binding ligand moiety and a target protein-binding ligand moiety linked by a linker group, wherein the E3 ubiquitin ligase-binding ligand moiety is a ZER1-binding ligand moiety and the target protein-binding ligand moiety is a DVL2-binding ligand moiety.

[0009] 2. The PROTAC complex according to Project 1 has the following structure:

[0010] ZER1B-L-DVL2B,

[0011] ZER1B is the ZER1-binding ligand moiety, DVL2B is the DVL2-binding ligand moiety, and L is a linking group.

[0012] The ZER1 binding ligand portion has the following structure:

[0013] .

[0014] 3. The PROTAC complex according to item 1 or 2, wherein the DVL2 binding ligand moiety has the following structure:

[0015] .

[0016] 4. The PROTAC complex according to Item 3, wherein the linking group is selected from:

[0017] -CH2-(CH2OCH2) n -CH2-C(O)-#, where n is an integer selected from 1 to 10, and # represents the linking site with the DVL2 binding ligand moiety.

[0018] 5. The PROTAC complex according to Item 4, wherein n is selected from 1, 2, 3, 4 or 5.

[0019] 6. The PROTAC complex according to item 1 or 5 has the following structure:

[0020] .

[0021] 7. A pharmaceutical composition comprising a PROTAC complex according to any one of items 1-6 and a pharmaceutically acceptable ingredient.

[0022] 8. Use of the PROTAC complex according to any one of items 1-6 or the pharmaceutical composition according to item 7 in the preparation of a medicament for the prevention or treatment of cardiac dysfunction.

[0023] 9. According to the use described in Item 8, wherein the cardiac dysfunction is cardiac remodeling, cardiomyocyte hypertrophy, myocardial fibrosis, myocardial hypertrophy, cardiac hypertrophy, cardiac fibrosis, or heart failure.

[0024] The PROTAC molecule of this invention regulates the CaMKII / HDAC4 / MEF2C signaling axis by recruiting the E3 ubiquitin ligase ZER1 and targeting DVL2 ubiquitination degradation, thereby alleviating cardiac dysfunction such as pathological myocardial hypertrophy and intervening in the progression of heart failure, providing a new therapeutic target for treating myocardial hypertrophy and heart failure caused by stress overload. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the mechanism of action of the PROTAC molecule ZD001 in this invention.

[0026] Figure 2 This invention illustrates a method for screening small molecule ligands for the E3 ubiquitin ligase substrate receptor ZER1 and target protein DVL2; wherein, Figure 2 Part A in the diagram is the ligand screening flowchart; Figure 2 Part B and Figure 2 The C part is related to human ZER1 ( Figure 2 Part B of the text) and human DVL2 ( Figure 2 The results of SPR affinity analysis of the top 50 small molecule compounds interacting with the C part of the protein were obtained by immobilizing human ZER1 or human DVL2 protein on a CM5 sensor chip, injecting small molecule compounds at a concentration of 100 μM for screening, recording sensor maps using the Biacore™ 8K system, and compounds with a reaction unit (RUs) greater than 20 were considered to have a higher binding potential to the corresponding target protein. Figure 2 part D in and Figure 2 The E part in the figure represents ZER1 (based on SPR affinity analysis results and taking into account key drug-like properties). Figure 2 (part D in D) and DVL2 ( Figure 2 A schematic diagram of the chemical structures of the top 5 ligand compounds in Part E of the diagram. Figure 2 The F part in the figure is a two-dimensional interaction diagram of the binding of the finally screened ZER1 ligand HY-114174 with ZER1 (PDB ID: 7XYT); Figure 2 The G section shows a cartoon diagram (left) of the binding of ZER1 ligand HY-114174 with human ZER1 and a three-dimensional structural view of the HY-114174-ZER1 complex (right), in which the carbon skeleton of ZER1 is shown in green, nitrogen atoms in blue, oxygen atoms in red, hydrogen atoms in white, HY-114174 is shown as a light blue rod-shaped model, salt bridges are shown as blue dashed lines, and hydrogen bonds are shown as magenta dashed lines; Figure 2 The H part in the diagram represents the two-dimensional interaction between the final screened DVL2 ligand HY-14800 and DVL2 (PDB ID:8WWR). Figure 2 Part I shows a cartoon diagram (left) of HY-14800 combined with human DVL2 and a three-dimensional structural view of the HY-14800-DVL2 complex (right). The carbon skeleton of DVL2 is shown in green, nitrogen atoms in blue, oxygen atoms in red, and hydrogen atoms in white. HY-14800 is shown as a magenta bar model, and hydrogen bonds are represented by magenta dashed lines. The longer the hydrogen bond, the weaker the interaction between bonds. Figure 2 The JM section contains two-dimensional interaction diagrams, cartoon images, and three-dimensional structural views of the ZER1 ligand compounds HY-125556, HY-137048, HY-P1517, and HY-P0210B obtained through screening. Figure 2 The NQ portion in the diagram represents the two-dimensional interaction diagram, cartoon image, and three-dimensional structural view of HY133894, HY-Q25001, HY-145938, and HY-12657 among the screened DVL2 ligand compounds.

[0027] Figure 3 Further SPR affinity analysis of the ligands screened in this invention with the target proteins is shown; wherein, Figure 3 Part A in the diagram shows the binding affinity results of ligand HY-114174 to the ZER1 protein; Figure 3 Part B of the diagram shows the binding affinity results of ligand HY-14800 to the DVL2 protein.

[0028] Figure 4 The diagram shows the design concept of PROTAC in this invention, illustrating the ligands of ZER1 and DVL2 and the final synthesized PROTAC molecule ZD001.

[0029] Figure 5 This is a synthetic route diagram of the PROTAC molecule ZD001 in this invention, wherein compound 7 is the finally synthesized PROTAC molecule ZD001.

[0030] Figure 6 This diagram illustrates the in vitro degradation effect of the PROTAC molecule ZD001 on DVL2 protein and explores the mechanism by which ZD001 induces DVL2 degradation. (The diagram is included for clarity.) Figure 6 Part A of the image shows Western blot images (left) and quantitative data on DVL2 protein levels in HEK293T cells induced by different concentrations of ZD001 for 24 hours. Figure 6 Part B in the figure is an RT-qPCR analysis of the mRNA level of DVL2 in HEK293T cells after treatment with ZD001; Figure 6Part C in the diagram is a schematic diagram of the effect of ZD001 on cell proliferation detected by the CCK-8 assay. The results show that ZD001 does not affect cell viability within the test concentration range of 0.1 to 50 μM. Figure 6 Part D in the diagram is a schematic diagram of the mechanism by which ZD001 induces DVL2 degradation by pretreating HEK293T cells with the proteasome inhibitor MG132.

[0031] Figure 7 A schematic diagram illustrating the degradation of DVL2 protein by the PROTAC molecule ZD001 in vivo. (The diagram is incomplete and requires further context.) Figure 7 Part A in the figure is the pharmacokinetic curve of C57BL / 6 mice after ZD001 administration; Figure 7 Part B shows the Western blot diagram (left) and the quantitative data of DVL2 protein level (right) in the heart tissue of C57BL / 6 mice 24 h after administration of different concentrations; Figure 7 Section C in the figure shows the RT-qPCR analysis of the mRNA level of DVL2 in cardiac tissue 24 h after ZD001 administration; Figure 7 Part D in the figure shows the Western blot diagram (left) and the quantitative data of DVL2 protein level (right) in the heart tissue of C57BL / 6 mice 48 h after administration of different concentrations; Figure 7 Section E in the figure shows the RT-qPCR analysis of DVL2 mRNA levels in cardiac tissue 48 hours after ZD001 administration.

[0032] Figure 8 This invention demonstrates the therapeutic effect of the PROTAC molecule ZD001 on pathological myocardial hypertrophy induced by pressure overload in TAC modeling. Among other things, Figure 8 Part A in the figure shows the body weight change curve of C57BL / 6 mice after intraperitoneal injection of ZD001 (10 mg / kg, once every other day) for 3 consecutive weeks, 1 week after TAC surgery. Figure 8 Part B in the diagram illustrates the effect of ZD001 on the weight of mouse organs. Figure 8 Section C shows the histological analysis of cardiac sections collected 4 weeks after the TAC experiment or sham surgery control. Gross morphology was assessed by H&E staining (scale bar, 1 mm), cardiomyocyte size changes were evaluated by WGA staining (scale bar, 50 μm), and the degree of myocardial fibrosis was evaluated by MTT staining (scale bar, 50 μm). Figure 8 Section D in the figure shows the ratio of heart weight to body weight (HW / BW) in mice treated with ZD001 or ddH2O after TAC or sham surgery (n=7 independent biological replicates); Figure 8Parts E, F, and G in the figure are echocardiographic assessments of changes in EF, FS, LV Vols levels in mice treated with TAC or sham surgery, ZD001, or ddH2O (n=7 independent biological replicates). Figure 8 The H portion in the image represents a representative M-mode echocardiogram of mice treated with ZD001 or ddH2O after TAC or sham surgery. Figure 8 Parts I, J, and K in the figure represent the mRNA expression levels of ANP, BNP, and β-MHC, markers of heart failure in mice after TAC or sham surgery, at ZD001 or ddH2O, respectively, analyzed by RT-qPCR (n=6 independent biological replicates). Figure 8 The L and M portions in the figure are Western blot images and mRNA expression level detection images of DVL2 in mouse TAC or sham-operated heart groups at ZD001 or ddH2O sites. Detailed Implementation

[0033] The ubiquitin-proteasome system (UPS) is the primary protein degradation mechanism in eukaryotic cells, with substrate selectivity mainly determined by E3 ubiquitin ligases. In most cases, E3 ubiquitin ligases recognize their substrates via short, specific peptide motifs known as degrons. The first degron identified was the N-degron, leading to the discovery of the N-terminal rule pathway, which associates the metabolic stability of proteins with their N-terminal residues. The N-degron pathway regulates a variety of biological processes, including chromosome stability, apoptosis, and the regulation of metabolic enzymes. Dysregulation of the N-degron pathway is associated with impaired cardiovascular development and structural defects in cardiac myofibrils. These findings suggest that N-degron pathway-mediated ubiquitination plays a crucial role in maintaining cardiomyocyte protein homeostasis.

[0034] The inventors' research shows that in a stress overload-induced myocardial hypertrophy model, the protein levels of ZER1 and DVL2 are negatively correlated; increased DVL2 protein levels lead to cardiomyocyte enlargement and exacerbated myocardial fibrosis. ZER1 (Zyg-11 related cell cycle regulator), as the substrate recognition subunit of the Cullin2-RING E3 ubiquitin ligase complex (CRL2), mediates protein degradation by recognizing N-degradation determinants via the Gly / N-degradation determinant pathway and is considered a key regulator of cardiac hypertrophy. DVL2 (disheveled segment polarity protein 2), as a key protein in the Wnt signaling pathway, plays an important role in pathological myocardial remodeling and aligns with ZER1's substrate recognition preference. ZER1 can directly bind to DVL2, promoting its degradation by the proteasome via the Gly / N-degradation determinant pathway and k48-linked polyubiquitination. This degradation inhibits the CaMKII / HDAC4 / MEF2C signaling cascade, thereby alleviating myocardial hypertrophy.

[0035] Protein-targeted degradation (TPD) technology is a breakthrough therapeutic strategy that selectively degrades pathogenic proteins using proteasome and lysosome pathways. Compared to traditional small molecule inhibitors, it shows great promise and multiple advantages in clinical development. Among these, PROTAC is the most widely used technology. PROTAC, a heterobifunctional molecule, is a ternary complex formed by linking an E3 ubiquitin ligase ligand and a target protein (POI) ligand together via a linker. After entering the cell, the PROTAC molecule induces ubiquitination of the target protein (POI) by bringing it closer to the E3 ubiquitin ligase, thereby enabling its degradation by the proteasome. PROTAC, as a key application of TPD, has unique advantages due to its ability to target proteins that are traditionally "undrug-resistant." However, a fundamental limitation of PROTAC technology is its reliance on the recruitment of a limited number of E3 ubiquitin ligases, primarily cereblon (CRBN) and von Hippel-Lindau (VHL), a narrow dependence that presents significant challenges.

[0036] There are currently no reports of ligands that specifically bind to the E3 ubiquitin ligase substrate receptor ZER1 or the DVL2 protein, which has an important influence on myocardial remodeling. There are also no reports of PROTAC molecules that regulate pathological cardiac remodeling by targeting the E3 ubiquitin ligase substrate receptor ZER1 to degrade DVL2.

[0037] This invention develops ligands targeting the E3 ubiquitin ligase substrate receptor ZER1, a ligand targeting the target protein DVL2, and a PROTAC molecule targeting DVL2. By integrating high-throughput virtual screening (HTVS), surface plasmon resonance (SPR) technology, and high-precision molecular docking, ligands for the E3 ubiquitin ligase substrate receptor ZER1 and the target protein DVL2 were screened, and the final PROTAC molecule was synthesized. This invention leverages the ability of the ZER1 ligand to recruit E3 enzymes, achieving the recognition and targeted degradation of the target protein DVL2, thereby intervening in the progression of pathological myocardial hypertrophy and heart failure.

[0038] Unless otherwise specified, the implementation of this application will employ conventional genetic engineering, biological, biochemical, and analytical chemistry techniques in the art.

[0039] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0040] Unless otherwise specified, all laboratory reagents are commercially available products.

[0041] As used herein, the term "ZER1" refers to a subunit of the Cullin 2-RING E3 ubiquitin ligase (CRL2) complex, which primarily recognizes glycine degradation signals at the N-terminus of proteins and mediates their ubiquitination and degradation. ZER1 specifically recognizes unmyristoylated proteins through the crystal structure of the complex it forms with N-terminal glycine. When a protein's N-terminus exposes glycine, ZER1 marks it as a degradation target, triggering the ubiquitination pathway. ZER1 is encoded by the zyg-11 related cell cycle regulator gene.

[0042] As used in this article, the term "DVL2" (disheveled Segment Polarity Protein 2) is a key protein in the Wnt signaling pathway. Members of this protein family contain PDZ and DEP domains. The UniProt ID for human DVL2 is O14641, the Ensembl ID is ENSG00000004975, and the MIM ID is 602151.

[0043] In one aspect, this application provides a ZER1 binding ligand selected from the following structures:

[0044] .

[0045] In one aspect, this application provides a DVL2 binding ligand selected from the following structures:

[0046]

[0047] In one aspect, this application provides a ZER1 binding ligand selected from HY-125556, HY-137048, HY-P1517 and HY-P0210B.

[0048] In one aspect, this application provides a DVL2 binding ligand selected from HY133894, HY-Q25001, HY-145938, and HY-12657.

[0049] In some embodiments, this application provides the use of the ZER1 binding ligand and / or DVL2 binding ligand described above, for example, for the preparation of PROTAC.

[0050] In some implementations, this application provides a method for screening ligands, specifically including the following steps:

[0051] (1) Ligands that target and bind to the active sites of ZER1 and DVL2 were obtained by high-throughput virtual screening;

[0052] (2) Affinity of ligands obtained by virtual screening was detected by surface plasmon resonance (SPR) technique;

[0053] (3) Through molecular docking analysis, the ligand binding characteristics of the further screened drugs were screened again, and finally the ligands that are associated with the substrate recognition subunit ZER1 of E3 ubiquitin ligase and the target protein DVL2 were obtained.

[0054] (4) Finally, the affinity between the ligands and their target proteins was further verified by the SPR experiment.

[0055] In one aspect, this application provides a PROTAC complex comprising an E3 ubiquitin ligase-binding ligand moiety and a target protein-binding ligand moiety linked by a linker group, wherein the target protein-binding ligand is a DVL2-binding ligand. In some embodiments, the E3 ubiquitin ligase-binding ligand may be a ZER1-binding ligand.

[0056] In one aspect, this application provides a PROTAC comprising an E3 ubiquitin ligase-binding ligand portion and a target protein-binding ligand portion linked by a linker group, wherein the E3 ubiquitin ligase-binding ligand is a ZER1-binding ligand. In some embodiments, the target protein-binding ligand may be a DVL2-binding ligand.

[0057] In one aspect, this application provides a protein hydrolysis-targeting chimeric compound (PROTAC) having the following structure: E3B-L-TGB, wherein E3B is the binding ligand portion of the substrate recognition subunit of an E3 ubiquitin ligase (such as CRL2), TGB is the target protein binding ligand portion, and L is a linker group. In some embodiments, E3B can be ZER1B, i.e., the ZER1 binding ligand portion. In some embodiments, TGB can be DVL2B, i.e., the DVL2 binding ligand portion.

[0058] In some implementations, the ZER1 binding ligand and / or the DVL2 binding ligand may be selected from or derived from any of the ZER1 binding ligand and / or the DVL2 binding ligand described above.

[0059] In the PROTAC complex of this application, when the ZER1-binding ligand and / or the DVL2-binding ligand are selected from any of the ZER1-binding ligands and / or the DVL2-binding ligands described above, the ZER1-binding ligand moiety and / or the DVL2-binding ligand moiety can be a group generated by removing a hydrogen atom from the ZER1-binding ligand and / or the DVL2-binding ligand to form the PROTAC complex. For example, the DVL2-binding ligand is selected from HY-14800, and the DVL2-binding ligand moiety has the structure shown below.

[0060] In the PROTAC complex of this application, when the ZER1-binding ligand and / or the DVL2-binding ligand are derived from any of the ZER1-binding ligands and / or the DVL2-binding ligands described above, the ZER1-binding ligand portion and / or the DVL2-binding ligand portion may be a part (group) of the ZER1-binding ligand and / or the DVL2-binding ligand. This part (group) should have an affinity for its target protein (ZER1 or DVL2) or retain a site for interaction with its target protein (ZER1 or DVL2) (e.g., Figure 2 (as shown in the FQ section). For example, the ZER1 binding ligand is selected from HY-114174, and the ZER1 binding ligand portion has the structure shown below.

[0061] In some implementations, the ZER1 binding ligand portion has the following structure:

[0062] .

[0063] In some implementations, the DVL2 binding ligand portion has the following structure:

[0064] .

[0065] In some embodiments, the linking group can be any suitable PROTAC linking group. In some embodiments, the linking group can have the following structure: -CH2-(CH2OCH2) n -CH2-C(O)- #, where n is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and # represents the linking site with TGB (e.g., the DVL2 binding ligand portion). In some embodiments, n is selected from 1, 2, 3, 4, or 5.

[0066] In some embodiments, the linking group can be linked by forming a bond (e.g., an amide bond) with a binding ligand or a portion of a binding ligand.

[0067] In some embodiments, the PROTAC of the present invention has the following structure:

[0068] .

[0069] In one aspect, this application provides a pharmaceutical composition comprising the PROTAC of the present invention and a pharmaceutically acceptable ingredient. In some embodiments, the pharmaceutically acceptable ingredient includes a pharmaceutically acceptable carrier, filler, preservative, solubilizer, transporter, diluent, and / or excipient. Thus, one or more pharmaceutically acceptable ingredients may be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, transporters, diluents, and / or excipients.

[0070] In one aspect, this application provides the use of the PROTAC or pharmaceutical composition of the present invention in the preparation of a medicament for the prevention or treatment of cardiac dysfunction.

[0071] In some implementations, the cardiac dysfunction is cardiac remodeling, cardiomyocyte hypertrophy, myocardial fibrosis, myocardial hypertrophy, cardiac hypertrophy, cardiac fibrosis, or heart failure.

[0072] In some implementations, the cardiac remodeling is a pathological structural remodeling of the heart that occurs under myocardial injury or long-term hemodynamic stress, manifested as myocardial cell hypertrophy, interstitial fibrosis, and abnormal cardiac morphology and function, which is commonly seen in the development of heart failure.

[0073] In some implementations, the cardiac hypertrophy is pressure-induced cardiac hypertrophy.

[0074] The present invention has the following beneficial effects:

[0075] 1. Compared with traditional PROTACs, the PROTAC molecules involved in this invention (such as ZD001) overcome the dependence on a limited number of E3 ubiquitin ligases and provide new targets for E3 ubiquitin ligases to synthesize PROTACs.

[0076] 2. The PROTAC molecules involved in this invention (such as ZD001) are based on the natural connection between ZER1 and DVL2, which overcomes the shortcomings of traditional PROTACs that are prone to a series of adverse reactions and off-target effects when they are based on binding with "novel substrates".

[0077] 3. The PROTAC molecules involved in this invention (such as ZD001) can improve pathological cardiac remodeling induced by stress overload, promote the restoration of cardiomyocytes to normal size and alleviate the symptoms of myocardial fibrosis, and provide a new treatment approach for the intervention of patients with pathological cardiac remodeling and heart failure.

[0078] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0079] Example

[0080] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0081] Example 1. Screening of ZER1 and DVL2 ligands:

[0082] The mechanism of action of PROTAC molecule ZD001 in this invention is as follows: Figure 1 As shown.

[0083] A structure-based high-throughput virtual screening (HTVS) was performed on 74,600 compounds from the Bioactive Compound Library Plus and the Diversity Library to identify potential ligands for human ZER1 (PDB ID: 7XYT) and human DVL2 (PDB ID: 8WWR). The specific flowchart is shown below. Figure 2 As shown in Part A of the diagram. After consecutive docking analyses in standard (SP) and high-precision (XP) modes, 50 compounds were selected for further experimental validation based on their docking scores. Human ZER1 or human DVL2 protein was immobilized on the CM5 sensor chip, and small molecule compounds were injected at a concentration of 100 μM for screening. Sensor maps were recorded using the Biacore™ 8K system. (See diagram for reference.) Figure 2As shown in Parts B and C, in the SPR analysis, compounds with a reaction unit (RU) greater than 20 were considered to have a higher binding potential to their respective target proteins. Based on their chemical structures and taking into account key drug-like properties, including molecular weight, topological polar surface area (TPSA), number of rotatable bonds, and calculated LogP, the top 5 candidate drugs were prioritized, as follows: Figure 2 Parts D and E are shown in the diagram. (Refer to...) Figure 2 Molecular docking simulations of the F and G portions of HY-114174 show that it forms seven hydrogen bonds with ZER1 residues ARG681, ASN679, TRP552, ASN553, ASP556, and GLU600, and also forms two salt bridges with ASP556 and GLU600. Similarly, referring to... Figure 2 Within the H and I portions of HY-14800, three hydrogen bonds are formed between HY-14800 and DVL2 residues ARG338, LEU278, and GLY284. Further SPR experiments were used to determine the binding affinity between the ligands and their target proteins. Figure 3 As shown in section A, HY-114174 exhibits a high binding affinity to ZER1, with a dissociation constant (KD) of 3.81 μM. Similarly, as... Figure 3 As shown in Part B, HY-14800 also has a high binding affinity to DVL2, with a dissociation constant (KD) of 6.52 μM.

[0084] Example 2. Preparation and synthesis of PROTAC molecule ZD001:

[0085] All reagents used in the synthesis of PROTAC were purchased from MedChemExpress (MCE, China). Figure 5 The synthetic route of PROTAC molecule ZD001 is shown, where compound 7 is the final synthesized PROTAC molecule ZD001. The synthesis was performed using LC-MS (Agilent 1290 Infinity II-g6125b, USA), HPLC (Agilent 1260 Infinity II, USA), and... 1The compounds were characterized by ¹H NMR (Bruker AVA, Germany). Compound 1 (HY-14800) (1.14 mmol) was coupled to compound 2 (1.30 mmol) in DMF at room temperature using HATU and DIEA. The crude product was purified by reversed-phase chromatography (TFA system, 45% acetonitrile) to give HM-5010_3 (410 mg, 49%). Compound 3 was deprotected in DCM with TFA and purified by reversed-phase chromatography (60% acetonitrile) to give compound 4 (220 mg, 57%). HY-114174 was purified by removing the FMOC protecting group and adding a protecting group to prevent the amino group of the structural formula from reacting with other groups to give compound 5. Compound 4 (0.298 mmol) was then coupled to compound 5 (0.328 mmol) in DMF using HATU and DIEA. The product was purified by reversed-phase chromatography (40% acetonitrile) to give compound 6 (320 mg, 81%). Finally, compound 6 was deprotected and purified in DCM / TFA to give compound 7 (90 mg, 35%) as a white solid (i.e., target compound ZD001).

[0086] Example 3. Performance test of PROTAC molecule ZD001 in recruiting ZER1 to target and degrade DVL2 protein

[0087] (1) The degradation effect of ZD001 on DVL2 protein in vitro:

[0088] HEK293T cells were treated with ZD001 at concentrations ranging from 0.1 to 50 μM for 24 h to evaluate the effect of ZD001 on the targeted degradation of DVL2. Figure 6 As shown in Parts A and B, intracellular DVL2 levels were significantly reduced at 10 μM, but DVL2 mRNA expression was unaffected. Furthermore, the maximum degradation rate (D...) Max The figure is 65.07%, DC 50 [logC(μM)] is 0.3824, and the half-maximal degradation concentration (DC) is... 50 The value is 2.4 μM.

[0089] (2) CCK-8 cell viability assay:

[0090] The effect of ZD001 on cell proliferation was detected using the CCK-8 assay. Figure 6 As shown in section C, the results indicate that ZD001 does not affect cell viability within the test concentration range of 0.1–50 μM.

[0091] (3) Investigation into the mechanism of ZD001-induced DVL2 degradation:

[0092] HEK293T cells were pretreated with the proteasome inhibitor MG132 to further verify the mechanism by which ZD001 induces DVL2 degradation. (Refer to...) Figure 6 In the D portion of the assay, ZD001 cannot reduce the protein level of DVL2 in the presence of MG132, indicating that ZD001 mediates the degradation of DVL2 via the proteasome pathway.

[0093] (4) In vivo pharmacokinetic assay of ZD001:

[0094] To investigate the in vivo behavior of ZD001, its pharmacokinetic characteristics were evaluated in C57BL / 6 mice. ZD001 was dissolved in double-distilled water at a concentration of 1 mg / mL and administered intraperitoneally (ip) at a dose of 10 mg / kg. Blood samples were collected from the posterior orbital sinus at 0, 20, 40, 80, 120, 240, 480, and 1440 min after administration. The samples were coagulated at room temperature for 1–2 hours and then centrifuged at 3,000 rpm for 15 min at 4 °C. The supernatant was centrifuged again to obtain serum. Serum (40 μL) was mixed with 160 μL of cold methanol:acetonitrile (1:1), vortexed, incubated at 4 °C for 30 min, and then centrifuged at 14,000 rpm for 15 min. The supernatant was vacuum dried, stored at -80 °C, reconstituted with 60 μL of acetonitrile:water (1:1), and analyzed by LC-MS / MS. Standard curves were prepared using ZD001 at concentrations ranging from 0 to 2,000 ng / mL. (Refer to...) Figure 7 Part A of the text, approximately 40 minutes (T) Max The maximum plasma concentration (C) reached 3.18 μg / mL at that time. Max ), terminal half-life (T 1 / 2 The time to plasma concentration was 144 minutes. The area under the plasma concentration-time curve (AUC) was 512.6 μg·min / mL.

[0095] (5) The degradation effect of ZD001 on DVL2 protein in vivo:

[0096] Western blot analysis was used to detect the effect of ZD001 on DVL2 protein levels in mouse heart tissue. It was found that intraperitoneal injection of ZD001 at doses of 10 mg / kg and 15 mg / kg significantly reduced DVL2 protein levels in mouse heart tissue 24 hours later, but did not affect DVL2 mRNA expression. Figure 7 As shown in Parts B and C, there was no statistically significant difference in degradation efficiency between the 15 mg / kg and 10 mg / kg dose groups. Similar degradation effects were also observed 48 hours after administration, such as... Figure 7 Parts D and E are shown in the diagram.

[0097] Example 4. A model of pathological myocardial hypertrophy induced by aortic coarctation (TAC):

[0098] Male C57BL / 6 mice (6–8 weeks old, 22–26 g body weight) were anesthetized via intraperitoneal injection of 2,2,2-tribromoethanol (0.1 ml / 10 g body weight). The animals were then placed in a supine position, and the pectoral muscles were bluntly dissected by cutting the proximal sternum and the first rib, exposing the aortic arch. After separating the aortic arch between the left common carotid artery and the brachiocephalic artery, the aortic arch was ligated twice with 6-0 nylon sutures on a 27-gauge blunt needle for transaortic coarctation (TAC). The needle was immediately removed after ligation, and the ribs and skin were sutured. The sham-operated group received the same interventions as the model group, except for aortic coarctation. To evaluate the therapeutic potential of ZD001 in pathological cardiac hypertrophy, mice with transaortic coarctation (TAC) were treated with 10 mg / kg ZD001 every other day, starting one week post-surgery and continuing for three weeks. At week 4, echocardiographic imaging analysis, histological examination, and related protein and mRNA level detection were performed on the mice.

[0099] Example 5. The therapeutic effect of PROTAC molecule ZD001 on pathological myocardial hypertrophy caused by pressure overload:

[0100] (1) Effects of continuous administration of ZD001 on body weight and other organs

[0101] C57BL / 6 mice were treated with ZD001 (10 mg / kg, intraperitoneally, every other day) for 3 weeks, one week after TAC surgery. Figure 8 As shown in Part A, ZD001 treatment did not affect normal weight gain or liver and kidney organ coefficients in mice. However, as... Figure 8 As shown in Part B, ZD001 significantly reduced the increase in lung and spleen organ coefficients 4 weeks after TAC. Figure 8 As shown in section D, the heart weight to body weight (HW / BW) ratio increases after TAC surgery, while the administration of ZD001 can lead to a significant decrease in the HW / BW ratio after TAC.

[0102] (2) Staining analysis of cardiac tissue after treatment with ZD001

[0103] Slices of mouse heart tissue were stained after the modeling process was completed. For example... Figure 8 As shown in section C, compared to the sham-operated control, H&E staining revealed significant myocardial hypertrophy and increased heart size in mice after TAC surgery; WGA staining showed a significant increase in cardiomyocyte size; and Masson's trichrome staining revealed extensive myocardial fibrosis. In contrast, mice treated with ZD001 showed significantly reduced hypertrophy, decreased cardiomyocyte size, and less myocardial fibrosis deposition.

[0104] (3) Echocardiographic analysis of the heart after treatment with ZD001

[0105] Four weeks after TAC, mice underwent two-dimensional M-mode echocardiography using a Vevo F2 (FUJIFILM, USA) transducer (46 MHz, FUJIFILM, USA). Mice were anesthetized with 2% isoflurane and maintained under 1% isoflurane, with oxygen administered at 0.8 L / min. During echocardiography, body temperature was controlled using warming pads, and electrocardiographic monitoring was performed using limb electrodes. The size and wall thickness of the left ventricle (LV) were measured at least three times from each projection, and cardiac parameters were measured. Left ventricular fractional shortening (FS) and left ventricular ejection fraction (EF) were calculated based on the M-mode measurements. The study and analysis were performed blinded. Echocardiographic analysis was performed using Vevo LAB software. Figure 8 As shown in the EH section, compared with the ddH2O treatment control group, ZD001 treatment preserved cardiac function, as evidenced by a significant reduction in the decrease of ejection fraction (EF) and fractional shortening (FS) and a significant improvement in left ventricular volume (LV Vols) at 4 weeks after TAC.

[0106] (4) Detection of genes related to TAC-induced pathological myocardial hypertrophy at the molecular level using ZD001.

[0107] The evaluation was conducted by qPCR detection of mRNA levels of heart failure marker genes and by detecting DVL2 protein levels in cardiac tissue after ZD001 treatment. Figure 8 As shown in the IK section, the reactivation of fetal gene expression (including ANP, BNP, and β-MHC) induced by stress overload was significantly suppressed in ZD001-treated hearts. Meanwhile, as... Figure 8 As shown in the LM section, ZD001 treatment significantly attenuated the TAC-induced upregulation of DVL2 protein levels without affecting its mRNA expression.

[0108] The above findings indicate that ZD001 can enhance the degradation of DVL2 through the existing E3 ubiquitin ligase substrate receptor ZER1 pathway, thereby alleviating cardiac dysfunction such as pathological cardiac remodeling, demonstrating the therapeutic potential of ZD001 as a PROTAC-based strategy for treating myocardial hypertrophy and heart failure.

[0109] It is understood that although the inventions described in this application are in the specific forms described above, these inventions are not limited to the specific content described in these specific forms. It will be apparent to those skilled in the art that various equivalent changes can be made to the technical features contained in the inventions described herein without departing from the spirit of the inventions described herein, and all such changes should fall within the scope of the inventions.

Claims

1. A PROTAC complex comprising an E3 ubiquitin ligase binding ligand moiety and a target protein binding ligand moiety linked by a linker, wherein the E3 ubiquitin ligase binding ligand moiety is a ZER1 binding ligand moiety and the target protein binding ligand moiety is a DVL2 binding ligand moiety, the PROTAC complex having the structure: 。 2. A pharmaceutical composition comprising the PROTAC complex of claim 1 and a pharmaceutically acceptable ingredient.

3. Use of the PROTAC complex of claim 1 or the pharmaceutical composition of claim 2 for the manufacture of a medicament for the treatment of pathological cardiac hypertrophy.

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