A protac-wlp polypeptide and uses thereof

Abstract

This invention discloses a PROTAC-WLP polypeptide and its applications. Its amino acid sequence, from the N-terminus to the C-terminus, consists of a VHL ligand pentapeptide, an AHX linker, a WLP sequence, and a TAT transmembrane peptide sequence linked sequentially. The amino acid sequence of the VHL ligand pentapeptide is shown in SEQ ID NO: 01, with a hydroxyl modification at the 4th carbon atom of the proline residue. The amino acid sequence of the WLP sequence is shown in SEQ ID NO: 02, and the amino acid sequence of the TAT transmembrane peptide sequence is shown in SEQ ID NO: 03. The PROTAC-WLP polypeptide and its pharmaceutically acceptable salt provided by this invention, by organically combining the WLP targeting sequence with the TAT transmembrane sequence and the VHL E3 ligase ligand in a PROTAC manner, can specifically target the CARM1 protein and induce its degradation via the ubiquitin-proteasome pathway. This achieves a shift from traditional enzyme activity inhibition to protein-level clearance, exhibiting higher catalytic efficiency and potential advantages in overcoming drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a PROTAC-WLP polypeptide and its applications. Background Technology

[0002] Cancer is one of the major diseases that seriously threaten human health, with persistently high incidence and mortality rates, and in recent years showing a continuous upward trend and a younger age of onset. Therefore, in-depth research into the molecular mechanisms of cancer development and progression, identifying key regulatory factors, and designing and developing safe, effective, and highly specific inhibitors based on these factors are of significant academic and social value for cancer prevention and treatment.

[0003] CARM1 (protein arginine methyltransferase 4, PRMT4), also known as coactivator-associated arginine methyltransferase 1, is an important epigenetic regulator. This enzyme primarily catalyzes methylation of arginine residues in histones and other protein substrates, thereby participating in various biological processes such as gene transcription regulation and cell signal transduction. Current research indicates that CARM1 is aberrantly expressed or exhibits enhanced activity in various malignant tumors, and is closely related to tumor occurrence, development, invasion, and prognosis, making it a potentially significant target for anti-tumor drugs.

[0004] Proteolysis-targeting chimeras (PROTACs) are a novel class of bifunctional molecules composed of an E3 ubiquitin ligase ligand, a target protein (POI) ligand, and a chemical linker connecting the two. These molecules can simultaneously bind to both the target protein and the E3 ligase, forming a ternary complex that induces polyubiquitination of the target protein, which is then recognized and degraded by the 26S proteasome, thus achieving selective clearance of the target protein at the protein level. Compared to traditional enzyme activity inhibitors, PROTAC technology offers potential advantages such as a catalytic mode of action and the ability to effectively overcome drug resistance, making it a research hotspot in the field of targeted drug development.

[0005] In recent years, researchers have reported several small-molecule inhibitors of CARM1, which have shown certain anti-tumor effects in in vitro and in vivo experiments. The applicant has also previously designed and synthesized peptides targeting CARM1 and verified their anti-tumor cell activity in various cancer cell lines. However, apart from the applicant's work, no other peptide inhibitors capable of independently and effectively inhibiting CARM1 enzyme activity have been publicly reported in the existing technology, especially peptide molecules that utilize the PROTAC strategy to achieve CARM1 protein degradation, which remains a gap in the field.

[0006] Despite some progress made by existing CARM1 inhibitors, they still have problems such as relatively simple mechanisms of action, limited cell penetration, insufficient in vivo stability, and potential off-target risks, making it difficult to fully meet the needs of clinical treatment.

[0007] Therefore, developing novel CARM1 peptide PROTAC inhibitors with good cell permeability, target specificity, and efficient degradation ability is of great significance for further improving the therapeutic effect of CARM1-related tumors. Summary of the Invention

[0008] The purpose of this invention is to provide a PROTAC-WLP polypeptide.

[0009] Another object of the present invention is to provide the application of the above-mentioned PROTAC-WLP polypeptide.

[0010] The technical solution of the present invention is as follows:

[0011] A PROTAC-WLP polypeptide has an amino acid sequence consisting of, from the N-terminus to the C-terminus, a VHL ligand pentapeptide, an AHX linker, a WLP sequence, and a TAT transmembrane peptide sequence linked sequentially.

[0012] The amino acid sequence of the VHL ligand pentapeptide is shown in SEQ ID NO: 01, and the 4th carbon atom of the proline residue has a hydroxyl modification.

[0013] The amino acid sequence of the WLP sequence is shown in SEQ ID NO: 02.

[0014] The amino acid sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO: 03.

[0015] The use of the above-mentioned PROTAC-WLP peptide or its bioacceptable salt in the preparation of CARM1 degrading agents.

[0016] A CARM1 degrading agent, the raw materials of which include the above-mentioned PROTAC-WLP peptide or its biocompatible salt.

[0017] In a preferred embodiment of the present invention, the raw material is the above-mentioned PROTAC-WLP polypeptide or a chemically acceptable salt thereof.

[0018] In a preferred embodiment of the invention, the raw materials further include pharmaceutically acceptable excipients.

[0019] The use of the above-mentioned PROTAC-WLP peptide in the preparation of breast cancer treatment compositions.

[0020] The use of the above-mentioned CARM1 degrading agent in the preparation of breast cancer treatment compositions.

[0021] A breast cancer treatment composition comprising the above-mentioned PROTAC-WLP peptide and / or the above-mentioned CARM1 degrading agent.

[0022] The beneficial effects of this invention are:

[0023] 1. The PROTAC-WLP polypeptide and its pharmaceutically usable salt provided by the present invention can specifically target CARM1 protein and induce its degradation mediated by the ubiquitin-proteasome pathway by organically combining the WLP targeting sequence with the TAT membrane-penetrating sequence and VHL E3 ligase ligand in a PROTAC manner. This achieves a shift from traditional enzyme activity inhibition to protein-level clearance, and has higher catalytic efficiency and potential advantages in overcoming drug resistance in terms of mechanism of action.

[0024] 2. The PROTAC-WLP polypeptide of the present invention can effectively inhibit the proliferation activity of cancer cells in in vitro cancer cell level experiments, showing good anti-tumor cell effects.

[0025] 3. In in vivo animal model experiments, the PROTAC-WLP peptide of the present invention can effectively inhibit tumor growth, while having no significant adverse effects on general physiological indicators such as the weight of experimental animals, indicating that it has good safety.

[0026] 4. The PROTAC-WLP polypeptide of the present invention is modified with a TAT membrane-penetrating sequence, which significantly enhances cell permeability and facilitates drug delivery; its pharmaceutically acceptable salt form further improves stability and formulation suitability, and can be formulated into suitable pharmaceutical compositions with pharmaceutically acceptable excipients, suitable for the prevention or treatment of breast cancer.

[0027] 5. This invention provides a novel peptide inhibitor regimen for the treatment of CARM1-related tumors, which has positive significance in terms of the feasibility of industrial preparation and the potential for clinical application. Attached Figure Description

[0028] Figure 1 This is a graph showing the results of Western blot analysis of the degradation of CARM1 protein by PROTAC-WLP-TAT in Example 2 of this invention.

[0029] Figure 2 This demonstrates the inhibitory effect of PROTAC-WLP-TAT on MCF7 subcutaneous xenografts in Example 3 of the present invention.

[0030] Figure 3 This demonstrates the effect of PROTAC-WLP-TAT on mouse weight changes in Example 3 of the present invention.

[0031] Figure 4This demonstrates the effects of PROTAC-WLP-TAT on changes in the heart, liver, spleen, lungs, and kidneys of mice in Example 3 of this invention.

[0032] Figure 5 This demonstrates the inhibitory effect of the combination of PROTAC-WLP-TAT and anti-CTLA4 antibody on 4T1 subcutaneous xenografts in Example 4 of the present invention.

[0033] Figure 6 This demonstrates the effect of the combined use of PROTAC-WLP-TAT and anti-CTLA4 antibody on changes in mouse body weight in Example 4 of this invention. Detailed Implementation

[0034] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0035] Example 1

[0036] Different breast cancer cell lines, MCF7, MDA-MB-231, and 4T1, were seeded in 96-well plates at a density of 20%–30%. After 24 h, aqueous solutions containing different concentrations of PROTAC-WLP-TAT (i.e., the PROTAC-WLP polypeptide of this invention: IYP(OH)AL(SEQ ID NO: 01){AHX}FSLNWRPPCLF(SEQ ID NO: 02)YGRKKRRQRRR(SEQ ID NO: 03), synthesized by GenScript) were added. The test drug (PROTAC-WLP-TAT) was administered at 12 concentration gradients: 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and 0 μM (blank group, H2O added only). The control group consisted of a short peptide of TAT containing the membrane-penetrating peptide, with the amino acid sequence SQQTPRFNPIM (SEQ ID NO: 04). Each concentration gradient included three replicates. Cell culture medium was changed before drug administration. One week after drug administration, cytotoxicity was assessed colorimetrically using the CellTiter 96® AQueous Single Solution Cell Proliferation Detection Kit. CellTiter 96® AQueous single-solution reagent contains 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (ethylthiophene, PES). 20 μL of CellTiter 96® AQueous reagent was added to every 100 μL of culture medium and incubated at 37 °C in a 5% CO2 incubator for 1 h. The reaction was terminated by adding 25 μL of 10% SDS. Absorbance data at 490 nm were recorded using a Thermo Multiskan MK3 microplate reader. Data were processed, and the IC50 of the drug was analyzed using GraphPad Prism 6 software. 50 Values. The test results are shown in Table 1.

[0037] Table 1. Cell proliferation inhibition data

[0038] PROTAC-WLP-TAT 3.146 33.77 21.28 Blank control - - -

[0039] As shown in Table 1, the blank control group had no inhibitory effect on cells, while PROTAC-WLP-TAT had a significant inhibitory effect on the proliferation of breast cancer cells. It can be used in the preparation of drugs for the prevention and / or treatment of diseases related to abnormal CARM1 activity, especially in the preparation of drugs for the treatment of cancer.

[0040] Example 2

[0041] In MCF7 breast cancer cells, PROTAC-WLP-TAT was administered at concentration gradients of 0 mM, 0.01 mM, 0.1 mM, 1 mM, 10 mM, and 20 mM. After 48 h, cells were collected and lysed for Western blotting analysis. The results are as follows: Figure 1 As shown.

[0042] according to Figure 1 It is known that PROTAC-WLP-TAT can effectively degrade CARM1 protein. However, in addition to CARM1, PROTAC-WLP-TAT can also degrade PRMT5 and PRMT6 proteins in the PRMT family, which are closely related to the occurrence and development of various cancers.

[0043] Example 3

[0044] On the skin of nude mice, according to 5x10 6 MCF7 breast cancer cells were subcutaneously inoculated into the bilateral axillae of nude mice at a density equal to the tumor density. One week after tumor formation, the mice were randomly divided into two groups of four mice each. Each group of mice received an intraperitoneal injection of the drug every two days, and tumor size and mouse weight were measured and recorded. The drugs administered to each group were physiological saline and PROTAC-WLP-TAT 50 mg / kg. Two weeks after treatment, the tumors were dissected, weighed, and recorded. The results are as follows: Figure 2 , Figure 3 and Figure 4 As shown.

[0045] according to Figure 2 , Figure 3 and Figure 4 It can be seen that PROTAC-WLP-TAT significantly inhibits the growth of MCF7 subcutaneous xenografts, while having no significant effect on mouse body weight or organs such as the heart, liver, spleen, lungs, and kidneys, indicating that it has good safety.

[0046] Example 4

[0047] In BALB / c mice, at a dose of 1x10 64T1 breast cancer cells were subcutaneously inoculated into the bilateral axillae of mice at a density that allowed for tumor formation. One week after tumor formation, the mice were randomly divided into four groups of four mice each. Each group of mice received an intraperitoneal injection of the drug once daily, and tumor size and mouse weight were measured and recorded. The drug administration methods for each group were as follows: saline, PROTAC-WLP-TAT 30 mg / kg, anti-CTLA-4 antibody 10 mg / kg, and PROTAC-WLP-TAT + anti-CTLA-4 antibody, respectively. After 10 days of treatment, the tumors were dissected, weighed, and recorded. Results are as follows: Figure 5 and Figure 6 As shown.

[0048] according to Figure 5 and Figure 6 It can be seen that the combination of PROTAC-WLP-TAT and anti-CTLA4 antibody significantly inhibits the growth of 4T1 subcutaneous xenografts, while having no significant effect on mouse body weight, indicating that it has good safety.

[0049] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A PROTAC-WLP polypeptide, characterized in that: Its amino acid sequence, from the N-terminus to the C-terminus, consists of a VHL ligand pentapeptide, an AHX linker, a WLP sequence, and a TAT transmembrane peptide sequence linked sequentially. The amino acid sequence of the VHL ligand pentapeptide is shown in SEQ ID NO: 01, and the 4th carbon atom of the proline residue has a hydroxyl modification. The amino acid sequence of the WLP sequence is shown in SEQ ID NO:

02. The amino acid sequence of the TAT membrane-penetrating peptide is shown in SEQ ID NO:

03.

2. Use of the PROTAC-WLP polypeptide of claim 1 or a chemically acceptable salt thereof in the preparation of a CARM1 degrading agent.

3. A CARM1 degrading agent, characterized in that: Its raw materials include the PROTAC-WLP polypeptide of claim 1 or its technically acceptable salt.

4. The CARM1 degrading agent as described in claim 3, characterized in that: The raw material is the PROTAC-WLP polypeptide of claim 1 or its chemically acceptable salt.

5. A CARM1 degrading agent as described in claim 3 or 4, characterized in that: Its raw materials also include pharmaceutically acceptable excipients.

6. Use of the PROTAC-WLP polypeptide of claim 1 in the preparation of a breast cancer treatment composition.

7. Use of the CARM1 degrading agent according to any one of claims 3 to 5 in the preparation of breast cancer treatment compositions.

8. A breast cancer treatment composition, characterized in that: Its raw materials include the PROTAC-WLP polypeptide of claim 1 and / or the CARM1 degrading agent of any one of claims 3 to 5.

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