PROTAC medicine targeting PCSK9 as well as preparation method and application of PROTAC medicine

Abstract

The invention relates to the technical field of biological medicine. The invention provides a PCSK9-targeted PROTAC medicine as well as a preparation method and application thereof. The medicine comprises the following structures: a PCSK9 binding peptide, an E3 ubiquitin ligase ligand, a cell penetrating peptide and a flexible linker. The medicine provided by the invention can be used for preparing products for treating lipid metabolism disorder diseases such as hypercholesteremia and atherosclerosis. In addition, the medicine disclosed by the invention can also be combined with statins to synergistically enhance the lipid-lowering curative effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a PROTAC drug targeting PCSK9 and a preparation method and application thereof. BACKGROUND

[0002] Elevated serum low density lipoprotein cholesterol (LDL-C) levels are a major risk factor for the development of cardiovascular disease. Low density lipoprotein receptor (LDLR)-mediated clearance of low density lipoprotein (LDL) is the main factor determining the level of LDL-C in the circulation. After binding to the LDLR on the cell surface, LDL is internalized into the cell through the classic clathrin-coated vesicles. In the acidic endosome, LDLR dissociates from LDL and recycles back to the cell surface, and the released LDL is transported to the lysosome for degradation.

[0003] PCSK9 (proprotein convertase subtilisin / kexin type 9) is a protein mainly synthesized and secreted by the liver, which plays an important role in lipid metabolism, especially in regulating the level of low density lipoprotein cholesterol (LDL-C). PCSK9 inhibitors reduce the clearance of LDL-C by inhibiting the function of PCSK9, thereby lowering the plasma LDL-C level, for the treatment of hypercholesterolemia and reducing the risk of cardiovascular disease. Currently, PCSK9 inhibitors mainly include monoclonal antibodies, small interfering RNAs, antisense oligonucleotides, etc.

[0004] The mechanism of action is different from that of traditional small molecule inhibitors. Traditional inhibitors inhibit the function of target proteins by occupying the active site (occupancy-driven), while PROTAC utilizes the natural ubiquitin-proteasome system (UPS) in cells to induce ubiquitination and subsequent degradation of target proteins (event-driven). PROTAC molecules are bifunctional chimeras, consisting of the following three parts: target protein ligand - specifically binds to the target protein (such as PCSK9); E3 ubiquitin ligase ligand - recruits E3 ligase (such as VHL, CRBN, etc.); Linker - optimizes the spatial conformation to improve degradation efficiency. Compared with small molecule inhibitors that can only inhibit the specific function of target proteins (such as kinase activity), PROTAC can completely eliminate target proteins, thereby blocking their kinase and non-kinase dependent functions, and has significant advantages in overcoming drug resistance, targeting "undruggable" proteins, etc.

[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 a 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 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 TVFTSWEEYL, VFTSWEEYLD, HICRKLKIGY, or TSWEEYLDWV.

[0009] Preferably, the E3 ubiquitin ligase ligand is ALAPYIP.

[0010] Preferably, the cell-penetrating peptide is YGRKKRRQRRR.

[0011] Preferably, the flexible connector is GSGS.

[0012] Preferably, the amino acid sequence of the drug is shown in SEQ ID NO.1~4; SEQ ID NO.1: TVFTSWEEYLGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO.2: VFTSWEEYLDGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO.3: HICRKLKIGYGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO.4: TSWEEYLDWVGSGSALAPYIPYGRKKRRQRRR.

[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 application also provides the use of the drug in the preparation of a product for treating lipid metabolism disorder.

[0016] Preferably, the lipid metabolism disorder is hypercholesterolemia or atherosclerosis.

[0017] The present application provides a PROTAC drug targeting PCSK9, and a preparation method and application thereof, the drug comprising the following structures: a PCSK9 binding peptide, an E3 ubiquitin ligase ligand, a cell penetrating peptide and a flexible linker. The PCSK9 binding peptide of the present application can specifically bind to the active site of PCSK9; the E3 ubiquitin ligase ligand is used to recruit the VHL E3 ubiquitin ligase complex; the cell penetrating peptide promotes the transmembrane delivery of the degrader; and the flexible linker adopts a GSGS linker to maintain the spatial conformation of the functional domains. The drug provided by the present application can be used for preparing a product for treating lipid metabolism disorders such as hypercholesterolemia and atherosclerosis. Furthermore, the drug of the present application can be used in combination with statins to synergistically enhance the lipid-lowering effect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Structure of the PROTAC drug for PCSK9.

[0019] Figure 2 Degradation efficiency of Cadd4 on PCSK9.

[0020] Figure 3 Inhibitory effect of MG132 on Cadd4.

[0021] Figure 4 Effect of Cadd4 on LDLR.

[0022] Figure 5 In vivo distribution of Cadd4 after intraperitoneal injection.

[0023] Figure 6 Effect of Cadd4 on lipid metabolism in vivo.

[0024] Figure 7 Lipid-lowering effect of Cadd4 on ApoE - / - .

[0025] Figure 8 Sustained lipid-lowering effect of Cadd4.

[0026] Figure 9 Effect of Cadd4 on liver function in mice.

[0027] Figure 10 Effect of Cadd4 in human liver tissue. DETAILED DESCRIPTION

[0028] The application provides a PROTAC drug targeting PCSK9, which comprises a PCSK9 binding peptide, an E3 ubiquitin ligase ligand, a cell penetrating peptide and a flexible linker.

[0029] In the application, the amino acid sequence of the PCSK9 binding peptide is TVFTSWEEYL (SEQ ID NO. 5), VFTSWEEYLD (SEQ ID NO. 6), HICRKLKIGY (SEQ ID NO. 7) or TSWEEYLDWV (SEQ ID NO. 8).

[0030] In the application, the E3 ubiquitin ligase ligand is ALAPYIP (SEQ ID NO. 9).

[0031] In the application, the cell penetrating peptide is YGRKKRRQRRR (SEQ ID NO. 10).

[0032] In the application, the flexible linker is GSGS (SEQ ID NO. 11).

[0033] In the application, the amino acid sequence of the drug is shown in SEQ ID NO. 1-4. SEQ ID NO. 1: TVFTSWEEYLGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO. 2: VFTSWEEYLDGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO. 3: HICRKLKIGYGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO. 4: TSWEEYLDWVGSGSALAPYIPYGRKKRRQRRR.

[0034] In the application, the drug further comprises a fluorescent label, and the fluorescent label is Rhodamine.

[0035] In the application, the drug is prepared by solid phase synthesis.

[0036] The application further provides application of the drug in preparation of a product for treating lipid metabolism disorder diseases.

[0037] In the application, the lipid metabolism disorder disease is hypercholesterolemia or atherosclerosis.

[0038] The technical solutions provided by the application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the application.

[0039] Example 1 PROTAC drug design targeting PCSK9

[0040] In order to obtain a binding peptide capable of efficiently inducing ubiquitination and subsequent degradation of a target protein, a target protein sequence targeting PCSK9 is designed based on computer-aided drug design (CADD) technology, which can specifically bind to the active site of PCSK9; an E3 ubiquitin ligase ligand is used to recruit a VHL E3 ubiquitin ligase complex; a cell-penetrating peptide promotes the transmembrane delivery of the degrader; a flexible linker is a GSGS linker, which maintains the spatial conformation of the functional domains. In order to facilitate the observation of in vitro and in vivo experimental verification of the PROTAC efficiency, a fluorescent tag is added. The specific design results and sequences are shown in Table 1: Table 1

[0041] The preparation method of the drug comprises the following steps: (1) Take 2-CTC resin, swell in DCM for 1 hour, and wash with DMF for 3 times; (2) Take 1 eq of the first protected amino acid, 1.5 eq of DIEA, and DMF as the solvent, and react with the resin for 2 hours; (3) Dry the resin, wash with DMF for 3 times, add methanol+DIEA to cap for 1 hour; (4) Wash the resin, and remove the Fmoc with 20% piperidine in DMF, and react for 10 min x 2 times; (5) Wash the resin, and react 3 eq of the second amino acid+3 eq of HOBT+3 eq of DIC in DMF as the solvent for 1.5 hours; (6) Repeat steps (4)-(5) until the last amino acid residue at the N-terminus is coupled, and the Fmoc at the N-terminus is removed; (7) Add 3 eq of Rhodamine+3 eq of HBTU+3 eq of DIEA, and react in DMF as the solvent for 4 hours; (8) Wash the resin and dry the resin; (9) Cut the resin and the side chain protection group of the polypeptide with 95% TFA+2% Tis+2% EDT+1% H2O, and react for 2 hours; (10) Filter the resin, wash the filtrate with ice ethyl ether, and centrifuge to obtain the precipitate, which is the crude product; (11) Purify by liquid chromatography and freeze-dry.

[0042] Example 2 Verification of PROTAC targeting PCSK9

[0043] 1. CADD-driven rational design and verification: Based on the PCSK9 crystal structure (PDB: 4NMX) Figure 1 Peptide-protein molecular docking experiments were conducted using the HPEPDOCK server. Docking simulations were performed with default parameters, generating multiple conformations for each peptide. Initial screening of these conformations was conducted based on predicted binding affinity, spatial complementarity, and the interaction ability with key residues at the PCSK9 binding interface. To improve the rigor of the screening, a diverse set of conformations for each peptide was generated using the MODPEP tool before docking. The MDock scoring function was used for evaluation, integrating van der Waals forces, electrostatic interactions, and geometric fit indices. Finally, the target sequences were selected based on two indices: the lowest predicted binding energy and the highest structural stability. In addition to the overall scoring indicators, residue-level interaction patterns were also emphasized, especially hydrogen bond formation and hydrophobic interactions, which are crucial for stable peptide-protein binding. Based on the molecular docking calculation results, the peptide Cadd4, with the highest predicted binding affinity, was selected. LX-2 cells were treated with Cadd4 for 8 hours, and cell proteins were extracted. Western blotting was used to detect the PCSK9 level in the lysate. The results showed that Cadd4 significantly improved the PCSK9 degradation efficiency (DC50≈20μm). Figure 2 ).

[0044] 2. Dual mechanism of action: 1) Simultaneously binds to PCSK9 and E3 ligase to form a ternary complex, which degrades PCSK9 via the ubiquitin-proteasome pathway (verified by MG132 inhibitors). Figure 3 The specific protocol is as follows: LX-2 cells were treated with 20 μm of Cadd4 and 5 μm of MG123 for 8 h, respectively. Cell proteins were extracted and subjected to Western blotting experiments to detect PCSK9. The results showed that MG132 could effectively block Cadd4-induced PCSK9 degradation, confirming that this degradation process is proteasome-dependent. Furthermore, immunofluorescence experiments confirmed a clear co-localization and binding phenomenon between Cadd4 and intracellular PCSK9.

[0045] 2) Relieve the inhibition of low-density lipoprotein receptor (LDLR) by PCSK9 ( Figure 4 The specific protocol is as follows: Mice with hypercholesterolemia induced by a high-fat diet (HFD) were intraperitoneally injected with 20 mg / kg Cadd4. Four hours later, the livers of these mice were removed for Western blotting and immunofluorescence staining. The results showed that Cadd4 reduced PCSK9 expression in the liver while upregulating LDLR expression.

[0046] 3. Liver-targeting properties: It accumulates in the liver 4 hours after intraperitoneal injection ( Figure 5), to achieve organ-specific distribution. The specific steps are as follows: to evaluate the distribution of Cadd4 in C57BL / 6 mice, 20 mg / kg of Cadd4 labeled with rhodamine was injected intraperitoneally, and the organs of the animals were taken out at different time points, and the drug distribution was detected by InSyTe FLECT / CT imaging system. The results show that Cadd4 is mainly enriched in the liver, and reaches the peak concentration 4 hours after injection, and decreases significantly after 8 hours Figure 5 ).

[0047] Example 3 Application of PROTAC targeting PCSK9

[0048] 1. High efficiency of lowering cholesterol: In a self-fed high-fat diet (HFD) mouse model, the serum total cholesterol (TC) was reduced by 25%, and the low-density lipoprotein cholesterol (LDL-C) was reduced by 29% in 7 days; the same effect was verified in ApoE-deficient mice Figure 6 、 Figure 7 ). The specific steps are as follows: after the HFD mice were injected intraperitoneally with 20 mg / kg of Cadd4 for 3 times on the first day, the third day and the fifth day, the whole blood samples were collected from the retro-orbital plexus and the serum was separated. The total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) detection kit was used to quantitatively detect the TC and LDL-C concentrations in the serum. The same method was used to detect the TC and LDL-C levels in the purchased ApoE-deficient mice. The results show that the metabolic changes of the high-fat diet mice are significantly reversed.

[0049] 2. Long-acting: the effect can last at least 7 days after a single dose Figure 8 ). The specific steps are as follows: after the HFD mice were injected intraperitoneally with 20 mg / kg of Cadd4 on the first day, the whole blood samples were collected from the retro-orbital plexus and the serum was separated to detect the TC level.

[0050] 3. Safety: no significant effect on liver function (ALT / AST), body weight or food intake Figure 9 ).

[0051] No genetic toxicity risk (the effect is at the protein level rather than the DNA level). The specific steps are as follows: after the HFD mice were injected intraperitoneally with 20 mg / kg of Cadd4 for 3 times on the first day, the third day and the fifth day, the whole blood samples were collected from the retro-orbital plexus and the serum was separated to detect the ALT and AST levels. The results show that Cadd4 treatment has no significant effect on the body weight, food intake or liver function indicators (ALT and AST levels) of the mice, indicating that it has excellent safety characteristics.

[0052] Cross-species applicability: verify the PCSK9 degradation and LDLR up-regulation ability in human liver tissue Figure 10). The specific steps are as follows: the protein is extracted after the surgically removed human liver sample is soaked in a Cadd4 solution of 20 mg / kg for 4 hours, and the expression of PCSK9 and LDLR is detected by immunoblotting. The results show that the polypeptide can effectively degrade PCSK9 and significantly up-regulate the expression of LDLR.

[0053] It can be known from the above examples that the present application provides a PROTAC drug targeting PCSK9 and a preparation method and application thereof, the drug comprising the following structures: a PCSK9 binding peptide, an E3 ubiquitin ligase ligand, a cell penetrating peptide and a flexible linker. The drug provided by the present application can be used for preparing a product for treating lipid metabolism disorder diseases such as hypercholesterolemia and atherosclerosis. Furthermore, the drug of the present application can be used in combination with statins to synergistically enhance the lipid-lowering effect.

[0054] The above only describes the preferred embodiments of the present application, and it should be pointed out that, for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A PROTAC drug targeting PCSK9, characterized in that, The drug comprises a PCSK9 binding peptide, an E3 ubiquitin ligase ligand, a cell penetrating peptide and a flexible linker.

2. The medicament according to claim 1, characterized in that, The amino acid sequence of the PCSK9 binding peptide is TVFTSWEEYL, VFTSWEEYLD, HICRKLKIGY or TSWEEYLDWV.

3. The medicament according to claim 1, characterized in that, The E3 ubiquitin ligase ligand is ALAPYIP.

4. The medicament according to claim 1, characterized in that, The cell penetrating peptide is YGRKKRRQRRR.

5. The medicament according to claim 1, characterized in that, The flexible linker is GSGS.

6. The medicament according to claim 1, characterized in that, The amino acid sequence of the drug is shown in SEQ ID NO. 1~4. SEQ ID NO. 1: TVFTSWEEYLGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO. 2: VFTSWEEYLDGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO. 3: HICRKLKIGYGSGSALAPYIPYGRKKRRQRRR; SEQ ID NO. 4: TSWEEYLDWVGSGSALAPYIPYGRKKRRQRRR.

7. The medicament according to any one of claims 1 to 6, characterized in that, The drug further comprises a fluorescent label, and the fluorescent label is Rhodamine.

8. The medicament according to claim 7, characterized in that, The drug is prepared by solid phase synthesis.

9. Use of the drug of any one of claims 1~8 in the preparation of a product for treating lipid metabolism disorder diseases.

10. Use according to claim 9, characterized in that, The lipid metabolism disorder diseases are hypercholesterolemia or atherosclerosis.

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