PROTAC oral medicine targeting PCSK9 as well as preparation method and application of PROTAC oral medicine
By designing an oral PROTAC drug that targets PCSK9 and degrades PCSK9 via the ubiquitin-proteasome pathway, the shortcomings of existing PCSK9-targeting technologies have been overcome. This approach achieves highly efficient reduction of plasma LDL-C and restoration of LDLR function, making it suitable for the treatment of hypercholesterolemia and atherosclerosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-03
AI Technical Summary
Currently, there are no highly efficient and specific PROTAC molecules targeting PCSK9 for the treatment of hypercholesterolemia and cardiovascular disease. Traditional inhibitors are insufficient in overcoming drug resistance and targeting 'undruggable' proteins.
A PROTAC oral drug targeting PCSK9 was designed, comprising a PCSK9-binding peptide, an E3 ubiquitin ligase ligand, a cell-penetrating peptide, and a flexible linker. It degrades PCSK9 via the ubiquitin-proteasome pathway and is prepared using a solid-phase synthesis method. It is combined with the E3 ligase to form a ternary complex to improve degradation efficiency.
It significantly reduces plasma LDL-C levels, restores the cholesterol clearance capacity of LDLR, enhances lipid-lowering efficacy in synergistic effect with statins, has good safety profile, is suitable for the treatment of hypercholesterolemia and atherosclerosis, and demonstrates the ability to degrade PCSK9 across species.
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Figure CN121775154A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an oral PROTAC drug targeting PCSK9, its preparation method, and its application. Background Technology
[0002] Elevated serum low-density lipoprotein cholesterol (LDL-C) levels are a major risk factor for cardiovascular disease. LDL receptor (LDL receptor)-mediated clearance of low-density lipoprotein (LDL) is the primary determinant of circulating LDL-C levels. After binding to LDLR on the cell surface, LDL is internalized and enters the cell via classic clathrin-coated vesicles. In acidic endosomes, LDLR dissociates from LDL and circulates back to the cell surface; the released LDL is then transported to lysosomes for degradation.
[0003] PCSK9 (proprotein convertase subtilisin type 9) is a protein primarily synthesized and secreted by the liver. It plays a crucial role in lipid metabolism, particularly in regulating low-density lipoprotein cholesterol (LDL-C) levels. PCSK9 inhibitors reduce LDL-C clearance by inhibiting PCSK9 function, thereby lowering plasma LDL-C levels and are used to treat hypercholesterolemia and reduce the risk of cardiovascular disease. Currently, PCSK9 inhibitors mainly include monoclonal antibodies, small interfering RNA, and antisense oligonucleotides.
[0004] The mechanism of action differs from traditional small molecule inhibitors. Traditional inhibitors suppress the function of target proteins by occupying their active sites (site-driven), while PROTACs utilize the cell's natural ubiquitin-proteasome system (UPS) to induce ubiquitination and subsequent degradation of target proteins (event-driven). A PROTAC molecule is a bifunctional chimera composed of three parts: a target protein ligand—specifically binds to the target protein (e.g., PCSK9); an E3 ubiquitin ligase ligand—recruits E3 ligases (e.g., VHL, CRBN); and a linker—optimizes spatial conformation to improve degradation efficiency. Compared to small molecule inhibitors that only inhibit specific functions of target proteins (e.g., kinase activity), PROTACs can completely eliminate target proteins, thereby blocking their kinase and non-kinase-dependent functions, offering significant advantages in overcoming drug resistance and targeting "undruggable" proteins.
[0005] Currently, although PROTAC technology has made progress in several disease areas (such as oncology and neurodegenerative diseases), there are no reports of PROTAC molecules targeting PCSK9. Therefore, developing a highly efficient and specific PCSK9-degrading PROTAC compound holds promise for providing a novel intervention strategy for the treatment of cardiovascular diseases. Summary of the Invention
[0006] The purpose of this invention is to provide an oral PROTAC drug targeting PCSK9, its preparation method and application, which specifically targets the proprotein convertase subtilisin 9 (PCSK9) for the treatment of hypercholesterolemia and related cardiovascular diseases.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a PROTAC oral drug targeting PCSK9, the drug comprising the following structure: PCSK9 binding peptide, E3 ubiquitin ligase ligand, cell-penetrating peptide and flexible linker.
[0008] Preferably, the amino acid sequence of the PCSK9 binding peptide is CTSWEEYLDWV.
[0009] Preferably, the amino acid sequence of the E3 ubiquitin ligase ligand is ALAPYIP.
[0010] Preferably, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR.
[0011] Preferably, the flexible connector is GSGS.
[0012] Preferably, the structural formula of the drug is shown in Formula I: Formula I.
[0013] Preferably, the drug further includes a fluorescent tag, wherein the fluorescent tag is Rhodamine.
[0014] Preferably, the drug is prepared by solid-phase synthesis.
[0015] The present invention also provides the use of the drug in the preparation of products for treating lipid metabolism disorders.
[0016] Preferably, the lipid metabolism disorder is hypercholesterolemia or atherosclerosis.
[0017] This invention provides a PCSK9-targeting oral PROTAC drug, its preparation method, and its application. The drug comprises the following structure: a PCSK9-binding peptide, an E3 ubiquitin ligase ligand, a cell-penetrating peptide, and a flexible linker. The PCSK9-binding peptide of this invention is a high-affinity peptide sequence designed using SZNS4, which can specifically bind to the PCSK9 active site. The E3 ubiquitin ligase ligand can link to the ALAPYIP motif for recruiting the VHL E3 ubiquitin ligase complex. The cell-penetrating peptide increases cell penetration ability. The flexible linker uses a GSGS linker to maintain the spatial conformation of the functional domains.
[0018] The drug provided by this invention can be used to prepare products for treating lipid metabolism disorders such as hypercholesterolemia and atherosclerosis. Furthermore, the drug of this invention can be used in combination with statins to synergistically enhance lipid-lowering efficacy.
[0019] This invention is based on the PCSK9 crystal structure (PDB: 4NMX), and optimizes the peptide sequence through molecular docking and kinetic simulation; it screens for candidate peptides with low binding energy and stable structure, significantly improving degradation efficiency. Simultaneously, it combines PCSK9 with E3 ligase to form a ternary complex, which degrades PCSK9 via the ubiquitin-proteasome pathway. Attached Figure Description
[0020] Figure 1 Orally administered PROTAC molecule SZNS4 enters liver LX-2 cells; Figure 2 The oral administration of the PROTAC molecule SZNS4 degrades PCSK9 levels in liver LX-2 cells; Figure 3 To detect the binding of orally administered PROTAC molecule SZNS4 to PCSK9 in the CESTA assay; Figure 4 To detect the reversal effect of oral PROTAC molecule SZNS4 on PCSK9 protein degradation in MG132; Figure 5 Oral administration of the PROTAC molecule SZNS4 restored LDLR levels in liver LX-2 cells; Figure 6 The oral administration of the PROTAC molecule SZNS4 degraded the level of PCSK9 in the liver of HFD mice; Figure 7 The oral administration of the PROTAC molecule SZNS4 reduced TC and LDL-C levels in HFD mice; Figure 8 The oral administration of the PROTAC molecule SZNS4 stabilized and controlled LDL-C levels in HFD mice; Figure 9Effects of oral administration of the PROTAC molecule SZNS4 on ALT / AST in HFD mice; Figure 10 SZNS4 degrades human liver PCSK9 protein. Detailed Implementation
[0021] This invention provides a PROTAC oral drug targeting PCSK9, the drug comprising the following structure: PCSK9 binding peptide, E3 ubiquitin ligase ligand, cell-penetrating peptide and flexible linker.
[0022] In this invention, the amino acid sequence of the PCSK9 binding peptide is CTSWEEYLDWV (SEQ.ID NO.1).
[0023] In this invention, the amino acid sequence of the E3 ubiquitin ligase ligand is ALAPYIP (SEQ.ID NO.2).
[0024] In this invention, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR (SEQ.ID NO.3).
[0025] In this invention, the flexible connector is GSGS (SEQ.ID NO.4).
[0026] In this invention, the structural formula of the drug is shown in Formula I: Formula I.
[0027] In this invention, the drug further includes a fluorescent tag, wherein the fluorescent tag is Rhodamine.
[0028] In this invention, the drug is prepared by solid-phase synthesis.
[0029] The present invention also provides the use of the drug in the preparation of products for treating lipid metabolism disorders.
[0030] In this invention, the lipid metabolism disorder is hypercholesterolemia or atherosclerosis.
[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0032] Example 1: Drug Design and Preparation
[0033] 1. To obtain a binding peptide that efficiently induces target protein ubiquitination and subsequent degradation, a target protein sequence targeting PCSK9 was designed using computer-aided drug design (CADD) technology, which can specifically bind to the PCSK9 active site. An E3 ubiquitin ligase ligand was used to recruit the VHL E3 ubiquitin ligase complex. A cell-penetrating peptide facilitated the transmembrane delivery of the degradation agent. A flexible linker, the GSGS linker, was used to maintain the spatial conformation of the functional domains. A fluorescent tag was added to facilitate in vitro and in vivo experimental verification of PROTAC efficacy. The specific structure is shown in Formula I.
[0034] Formula I.
[0035] 2. The drug of the present invention comprises a PCSK9 binding peptide, an E3 ubiquitin ligase ligand, a cell-penetrating peptide, and a flexible linker, wherein: PCSK9 binding peptide: A high-affinity peptide sequence designed using SZNS4 (preferred sequence: CTSWEEYLDWV-GSGS-ALAPYIPC (disulfide bond)) specifically binds to the PCSK9 active site; E3 ubiquitin ligase ligand: ligates the ALAPYIP motif and is used to recruit the VHL E3 ubiquitin ligase complex; Cell-penetrating peptide (CPP): A YGRKKRRQRRR sequence peptide that enhances cell penetration. LX-2 cells were treated with synthetic SZNS4, and penetration was observed under a microscope. It was observed that the penetrating peptide promoted the transmembrane delivery of degradative agents with increasing concentration and time. Figure 1 ); Flexible connectors: GSGS connectors are used to maintain the functional domain spatial configuration.
[0036] 3. The preparation method of the drug includes the following steps: (1) Weigh 2-CTC resin, soak it in DCM for 1 hour, and wash it with DMF 3 times; (2) Take 1 eq of the first protected amino acid, 1.5 eq of DIEA, and DMF as solvent, and react with the resin for 2 hours; (3) Drain the resin, clean it with DMF 3 times, add methanol + DIEA to the end cap for 1 hour; (4) Clean the resin, remove Fmoc with 20% piperidine DMF solution, and react for 10 min × 2 times; (5) Wash the resin, add 3 eq of the second amino acid + 3 eq of HOBT + 3 eq of DIC, using DMF as solvent, and react for 1.5 hours; (6) Repeat steps 4-5 until the last amino acid residue at the N-terminus is attached and remove the Fmoc at the N-terminus; (7) Add 3 eq of Rhodamine + 3 eq of HBTU + 3 eq of DIEA, using DMF as solvent, and react for 4 hours; (8) Wash the resin and dry it. (9) 95% TFA + 2% Tis + 2% EDT + 1% H2O cleaves the resin and polypeptide side chain protecting groups, and reacts for 2 hours; (10) Filter the resin, wash the filtrate with ice-cold ether, centrifuge and keep the precipitate, which is the crude product; (11) Dissolve the crude product in acetonitrile and water, adjust the pH to 7.5-8 with ammonium bicarbonate solution, react at room temperature, monitor the oxidation by mass spectrometry and liquid chromatography, and purify by liquid chromatography after complete oxidation, and freeze dry.
[0037] Example 2 Design Innovation Points
[0038] 1) CADD-driven rational design: Based on the PCSK9 crystal structure (PDB: 4NMX), peptide sequences were optimized through molecular docking and kinetic simulations. Candidate peptides with low binding energy and stable structures were screened, significantly improving degradation efficiency. The specific method is as follows: LX-2 cells were treated with SZNS4 from 0 nM to 100 nM for 4 h, and cell proteins were extracted. PCSK9 in the lysate was examined using a fully automated quantitative microblotting system. The results showed that SZNS4 at a concentration of 100 nM achieved a PCSK9 degradation rate >90%. Figure 2 ).
[0039] 2) Dual mechanism of action: Simultaneously, it combines with PCSK9 and E3 ligases to form a ternary complex (verified by CETSA). Figure 3 PCSK9 is degraded via the ubiquitin-proteasome pathway (verified by the MG132 inhibitor). Figure 4 The procedure was as follows: LX-2 cells were treated with 20 nm SZNS4 for 4 hours, then collected into test tubes and centrifuged to precipitate the cells. The supernatant was carefully removed and the cells were resuspended in PBS. The test tubes were placed at a specified temperature (seven temperature endpoints between 37 and 55°C) for 3 minutes (Veriti thermal cycler, Applied Biosystems), followed by cooling at room temperature for 3 minutes. Proteins were extracted from the cell pellet using lysis buffer, and PCSK9 was detected using a fully automated microblotting system. The results showed that SZNS4 significantly reduced the denaturation rate of PCSK9 under high temperature conditions, indicating the existence of a specific binding interaction. Furthermore, LX-2 cells were treated with 50 nm SZNS4 and 5 μm MG132, and the extracted proteins were detected for PCSK9. The results showed that MG132 effectively blocked SZNS4-induced PCSK9 degradation, further confirming the proteasome-dependent degradation mechanism.
[0040] Relieving PCSK9's inhibition of low-density lipoprotein receptor (LDLR) and restoring the liver's cholesterol clearance capacity. Figure 5 The specific steps are as follows: Liver LX-2 cells were treated with different concentrations of SZNS4 for 4 hours, and cell proteins were extracted. The expression of LDLR was detected by a quantitative fully automated microblotting system. The results showed that the expression of LDLR increased with increasing SZNS4 concentration.
[0041] 3) Liver-targeting properties: In a self-fed high-fat diet (HFD) mouse model, SZNS4 was orally administered for 3 consecutive days. Subsequently, the livers of the mice were harvested, and the levels of PCSK9 and LDLR were detected. The results showed that SZNS4 significantly reduced hepatic PCSK9 expression and upregulated LDLR levels. Figure 6 ).
[0042] Example 3: Effect Verification
[0043] (1) Highly effective cholesterol reduction: In a high-fat diet (HFD) mouse model, serum total cholesterol (TC) decreased by 30%, and low-density lipoprotein cholesterol (LDL-C) decreased by 30%. Figure 7 The specific steps are as follows: HFD mice were orally administered SZNS4 at a dose of 50 mg / kg three times consecutively on days 1, 3, and 5. Whole blood samples were collected from the retro-orbital venous plexus on days 3 and 7, and serum was separated. The concentrations of TC and LDL-C in the serum were quantitatively detected using a total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) assay kit. The results showed that the metabolic changes in the high-fat diet mice were significantly reversed.
[0044] (2) Stability: Long-term (23 days) oral administration every other day (50 mg / kg) can reduce LDL-C by approximately 40%. Figure 8 The specific steps are as follows: HFD mice were orally administered SZNS4 once every other day for a total of 23 days. Whole blood samples were collected from the posterior orbital venous plexus and serum was separated to detect LDL-C levels.
[0045] (3) Safety: No significant effect on liver function (ALT / AST) Figure 9 The treatment had no effect on mouse body weight, food intake, or liver function indicators (ALT and AST levels). The procedure was as follows: HFD mice were orally administered 50 mg / kg SZNS4 three times consecutively on days 1, 3, and 5. Whole blood samples were collected from the retro-orbital venous plexus, and serum was separated to detect ALT and AST levels. The results showed that SZNS4 treatment had no significant effect on mouse body weight, food intake, or liver function indicators (ALT and AST levels), indicating its excellent safety profile.
[0046] (4) Cross-species applicability: Validation of PCSK9 degradation and LDLR upregulation in human liver tissue ( Figure 10 The specific steps are as follows: Human liver samples removed during surgery were soaked in 50 mg / kg SZNS4 solution for 4 hours, and proteins were extracted. The expression of PCSK9 and LDLR was detected by Western blotting. The results showed that the peptide effectively degraded PCSK9 and significantly upregulated LDLR expression.
[0047] As shown in the above embodiments, the present invention provides a PCSK9-targeting PROTAC oral drug, its preparation method, and its application. The drug comprises the following structure: a PCSK9-binding peptide, an E3 ubiquitin ligase ligand, a cell-penetrating peptide, and a flexible linker. The drug provided by the present invention can be used to prepare products for treating lipid metabolism disorders such as hypercholesterolemia and atherosclerosis. Furthermore, the drug of the present invention can be used in combination with statins to synergistically enhance lipid-lowering efficacy.
[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. An oral PROTAC drug targeting PCSK9, characterized in that, The drug comprises the following structure: PCSK9 binding peptide, E3 ubiquitin ligase ligand, cell-penetrating peptide, and flexible linker.
2. The drug according to claim 1, characterized in that, The amino acid sequence of the PCSK9 binding peptide is CTSWEEYLDWV.
3. The drug according to claim 1, characterized in that, The amino acid sequence of the E3 ubiquitin ligase ligand is ALAPYIP.
4. The drug according to claim 1, characterized in that, The amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR.
5. The drug according to claim 1, characterized in that, The flexible connector is GSGS.
6. The drug according to claim 1, characterized in that, The structural formula of the drug is shown in Formula I: Formula I.
7. The drug according to any one of claims 1 to 6, characterized in that, The drug also includes a fluorescent tag, which is Rhodamine.
8. The medicament according to claim 7, characterized in that, The drug is prepared by solid-phase synthesis.
9. The use of the drug according to any one of claims 1 to 8 in the preparation of a product for treating lipid metabolism disorders.
10. The application according to claim 9, characterized in that, The lipid metabolism disorder mentioned refers to hypercholesterolemia or atherosclerosis.