A beta-catenin targeted degradation cyclic peptide modified by d-amino acid and n-methylation and a preparation method and application thereof

Abstract

This invention discloses a β-catenin-targeting degradation cyclic peptide modified with D-amino acids and N-methylation, its preparation method, and its application, belonging to the field of peptide drug technology. The amino acid sequence of the cyclic peptide is shown in SEQ ID NO.1, wherein the cysteine ​​residues at positions 9 and 21 form a cyclic structure through disulfide bonds, the serine residue at position 2 and the phenylalanine residue at position 7 are D-configured amino acids, and the threonine residue at position 3 is N-methylated. After modification, this cyclic peptide significantly improves serum stability, resistance to enzymatic degradation, and cell membrane permeability, while maintaining a high affinity for β-catenin. It can effectively induce ubiquitination-dependent proteasome degradation of β-catenin and inhibit the expression of its downstream target genes Cyclin D1 and c-Myc. The invention also provides the application of this cyclic peptide in the preparation of drugs for treating diseases related to abnormal accumulation of β-catenin.

Description

Technical Field

[0001] This invention relates to the field of peptide drug technology, specifically to a β-catenin-targeted degradation cyclic peptide modified with D-amino acid and N-methylation, its preparation method, and its application. Background Technology

[0002] β-catenin is a core effector protein in the Wnt / β-catenin signaling pathway, playing a crucial regulatory role in cell proliferation, differentiation, migration, and stem cell maintenance. In normal cells, β-catenin is continuously phosphorylated by the disruption complex composed of APC, AXIN, CK1, and GSK3β, and degraded by the ubiquitin-proteasome system, thus maintaining a low intracellular level. When APC undergoes inactivating mutations, the disruption complex is impaired, leading to abnormally stable and abundant accumulation of β-catenin. After translocating into the nucleus, it continuously activates the TCF / LEF transcriptional complex, inducing abnormal expression of target genes such as c-Myc, Cyclin D1, and Axin2, ultimately resulting in colorectal adenoma formation and malignant progression. Therefore, β-catenin has become one of the most important therapeutic targets for familial adenomatous polyposis (FAP) and APC-mutant colorectal cancer.

[0003] Because β-catenin lacks a deep binding pocket suitable for small molecule binding, it is a typical protein-protein interaction (PPI) target and has long been considered a "difficult-to-drug" target protein. In recent years, intervention strategies targeting β-catenin have mainly included different technical routes such as small molecule inhibitors, PROTAC degraders, peptide degraders, and cyclic peptide degraders.

[0004] Table 1 Comparison of existing β-catenin targeting strategies

[0005] Compared to traditional linear peptides, cyclic peptides, due to their closed-loop molecular backbone, effectively reduce conformational freedom and entropy loss during binding, thus typically exhibiting higher target protein binding affinity, better protease tolerance, and longer in vivo duration of action. For typical protein-protein interaction targets like β-catenin, cyclic peptides can better mimic the natural protein binding interface, making them an important direction in the development of β-catenin-targeted drugs in recent years. Furthermore, by combining cyclic peptides with functional modules such as cell-penetrating peptides (CPPs) and E3 ubiquitin ligands, novel peptide degraders with both cell delivery and target protein degradation capabilities can be constructed, providing new therapeutic strategies for diseases such as APC-mutant colorectal cancer and familial adenomatous polyposis.

[0006] Currently, a large number of studies have been conducted both domestically and internationally on β-catenin targeted degradation and peptide drug optimization. For example, CN111298106A discloses a polypeptide conjugate capable of simultaneously binding to β-catenin and VHL ligase, providing a new technical route for targeted degradation of β-catenin by recruiting VHL to mediate β-catenin ubiquitination; CN118496322A discloses a polypeptide degrader combining a cell-penetrating peptide, a β-catenin-targeting cyclic peptide, and a VHL ligand, achieving intracellular targeted degradation of β-catenin; CN121800938A discloses a polypeptide degradation system based on a cell-penetrating peptide coupled with a VHL ligand, further validating the feasibility of CPP promoting intracellular delivery and E3 ligase recruitment; CN119751593A discloses a VHL polypeptide ligand containing a disulfide bond structure, providing a new connection method for constructing cyclic polypeptide degraders; in addition, CN118638189A improves the protease tolerance and cell penetration ability of polypeptides by constructing a bicyclic polypeptide restrictive molecular conformation, providing a new design idea for polypeptide stabilization.

[0007] Table 2 Comparison of existing β-catenin targeted degradation and peptide optimization technologies

[0008] As shown in Table 2, existing technologies mainly focus on the construction of β-catenin degradation systems, improvement of cell delivery efficiency, optimization of cyclization methods, and improvement of peptide stability, solving technical problems at different levels and laying the foundation for the development of peptide degradative agents. However, these technologies pay more attention to the combined design of functional modules or general stabilization modifications, and lack in-depth research on the spatial conformation optimization of β-catenin-targeted degradation cyclic peptides themselves.

[0009] For β-catenin-targeted degradation of cyclic peptides, it is necessary not only to maintain a high affinity for β-catenin but also to preserve the peptide's ability to transmembrane and the effective recruitment of E3 ligase ligands, thereby promoting the formation of a stable ternary complex and achieving sustained degradation. Therefore, conformational changes or backbone modifications of any amino acid residue in the cyclic peptide can cause a rearrangement of the overall spatial conformation, thus affecting β-catenin recognition, cellular uptake efficiency, and degradation activity. While existing modification techniques for improving peptide stability, such as D-amino acid substitution and N-methylation, have been applied to some linear peptides or other bioactive peptides, they are primarily general stabilization strategies and cannot directly derive modification schemes suitable for β-catenin-targeted degradation of cyclic peptides. Furthermore, there are no reports of precise conformational optimization design targeting key sites.

[0010] Therefore, how to achieve a synergistic enhancement of anti-protease degradation ability, spatial conformational stability and sustained degradation activity by precisely regulating the conformation of key sites of cyclic peptides while maintaining β-catenin recognition ability, cell membrane penetration ability and VHL ligase recruitment ability remains a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0011] The present invention aims to overcome the shortcomings of the prior art and provide a β-catenin-targeting cyclic peptide with high stability, high cell permeability and high degradation activity modified by a combination of D-amino acids and N-methylation, as well as its preparation method and its application in the treatment of related diseases.

[0012] First, the present invention provides a β-catenin-targeted degradation cyclic peptide modified with D-amino acid and N-methylation, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] The cysteine ​​residues at positions 9 and 21 form a cyclized structure via a disulfide bond (-SS-). This cyclization significantly enhances the conformational stability and resistance to protease degradation of the cyclic peptide.

[0014] The core cyclic peptide recognition sequence of the cyclic peptide is derived from the β-catenin binding fragment of the APC protein. By introducing D-Ser, D-Phe, and N-Me-Thr combined modifications at specific sites that contribute little to the β-catenin binding interface but have a key impact on backbone flexibility and enzyme cleavage sensitivity, the local conformational rigidity is enhanced, backbone flexibility is reduced, and enzyme cleavage tolerance is improved.

[0015] Specifically, the cyclic peptide contains the following non-natural amino acid combinations for modification. The modification sites were screened based on the structural features of the APC-β-catenin interaction interface, selecting residues that contribute little to the binding interface but have a key impact on the flexibility of the polypeptide backbone and the sensitivity to enzymatic cleavage for site-specific modification: D-amino acids: The second position is D-serine, and the seventh position is D-phenylalanine. The introduction of D-amino acids can effectively resist the hydrolysis of endogenous proteases and prolong the in vivo half-life of the peptide.

[0016] N-methylation modification: The 3rd position is N-methylated threonine. N-methylation can reduce the flexibility of peptide bonds, enhance their affinity for binding to targets, and further improve metabolic stability.

[0017] The cyclic peptide, through combined modification with D-serine, D-phenylalanine, and N-methylthreonine, and disulfide bond cyclization, achieves local conformational constraint, main chain flexibility regulation, and protease recognition site shielding. Compared with traditional natural L-amino acid peptides or single-modified peptides, the combined modification of this invention not only maintains the high affinity of the cyclic peptide for β-catenin but also outperforms single modifications or other non-specific site combined modifications in overall performance. This site-specific combined modification method achieves a synergistic optimization effect between improved stability, enhanced cell permeability, and functional persistence, thereby significantly enhancing the conformational stability, enzyme tolerance, and cell membrane permeability of the peptide while maintaining high specific binding capacity.

[0018] Preferably, in some preferred embodiments, a cell-penetrating fragment (such as the polyarginine sequence SEQ ID NO.2: RRRRRRRRR) can be selectively introduced at the N-terminus, and a flexible linker (such as aminocaproic acid) and an E3 ubiquitin ligand ligand (such as the VHL ligand SEQ ID NO.3: ALAPYIP) can be linked at the C-terminus to construct a peptide degradation system based on PROTAC or a molecular glue mechanism, thereby achieving selective ubiquitination and degradation of β-catenin.

[0019] Secondly, this invention provides the application of the above-mentioned β-catenin-targeting degradation cyclic peptide in the preparation of drugs for inhibiting diseases related to abnormal accumulation of β-catenin.

[0020] Preferably, the disease associated with abnormal accumulation of β-catenin is APC-mutant colorectal cancer or familial adenomatous polyposis (FAP).

[0021] The mechanism of action of the drug is as follows: through the specific binding of the polypeptide to β-catenin, it induces ubiquitination-dependent proteasome degradation of β-catenin, thereby effectively inhibiting the activity of the Wnt / β-catenin signaling pathway and downregulating the expression of downstream target proteins of β-catenin, Cyclin D1 and c-Myc.

[0022] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0023] Furthermore, the present invention also provides a pharmaceutical composition comprising the above-described β-catenin-targeted degradation cyclic peptide and pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutically acceptable excipients include, but are not limited to: stabilizers selected from one or more of mannitol, sucrose, trehalose, dextran 40, lactose, or sorbitol; buffers selected from one or more of histidine, citrate, acetate, or phosphate; surfactants, such as polysorbate 80; preservatives, such as phenol or benzyl alcohol; antioxidants, such as glutathione or methionine; and polymer carriers or liposome materials for preparing long-acting or targeted formulations, such as polylactic acid-glycolic acid copolymer, polylactic acid, or distearate phosphatidylethanolamine. The above excipients can be selected and combined according to dosage form requirements (e.g., lyophilized powder for injection, injection solution, or liposome formulation) to maintain the stability, safety, and efficacy of the cyclic peptide drug.

[0025] Finally, the present invention also provides a method for preparing the above-mentioned β-catenin-targeted degradation cyclic peptide, comprising the following steps: (1) β-catenin-targeted degradation peptides were synthesized using a solid-phase peptide synthesis method; (2) The β-catenin-targeted degradation peptide obtained in step (1) is subjected to an oxidation reaction to form a disulfide bond between two cysteine ​​residues, thereby achieving cyclization; (3) The cyclized product was purified to obtain the β-catenin-targeted degradation cyclic peptide.

[0026] Compared with the prior art, the present invention has the following advantages: Compared with existing technologies, this invention, through D-amino acid substitution and N-methylation modification, significantly improves the stability, cell permeability, and functional persistence of cyclic peptides while maintaining β-catenin binding capacity, specifically in the following aspects: 1. After incubation in a 50% fetal bovine serum system for 24 h, the modified cyclic peptide retained approximately 38% of its intact cyclic peptide, while the unmodified cyclic peptide retained only about 10%, representing an improvement in integrity of approximately 3.8 times. At 8 h, the residual rate of the modified cyclic peptide was approximately 68%, significantly higher than the 40% of the unmodified cyclic peptide (an improvement of approximately 70%). These results indicate that D-amino acid and N-methylation modification significantly enhanced the cyclic peptide's resistance to protease degradation and markedly improved serum stability.

[0027] 2. In the washout experiment, 48 h after drug withdrawal, the expression level of β-catenin in the modified cyclic peptide group remained at approximately 0.68, while that in the unmodified cyclic peptide group had recovered to approximately 0.90, close to the baseline level; in contrast, the modified cyclic peptide still additionally inhibited β-catenin by approximately 24% at 48 h. The results indicate that this modification strategy can significantly prolong the duration of cyclic peptide function in cells.

[0028] 3. In SW480 and DLD-1 cells, the mean intracellular fluorescence intensity of the modified cyclic peptide increased from 1.00 to 1.62 and 1.70, respectively, which were approximately 62% and 70% higher than that of the unmodified cyclic peptide (P<0.05), indicating that D-amino acid and N-methylation modification can significantly enhance the cellular uptake capacity of cyclic peptides.

[0029] 4. In SW480 cells, the modified cyclic peptide reduced β-catenin expression to 0.18, a further decrease of approximately 45.5% compared to the unmodified cyclic peptide (0.33); in DLD-1 cells, it decreased from 0.39 to 0.24, a reduction of approximately 38.5% (P<0.05). This indicates that the modified cyclic peptide has a stronger β-catenin degradation capacity in the APC mutant background.

[0030] 5. Under 8 μM treatment, Cyclin D1 levels in SW480 and DLD-1 cells decreased from 1.00 to 0.24 and 0.26, respectively, representing reductions of approximately 74% and 74%; c-Myc levels decreased from 1.00 to 0.21 and 0.24, respectively, representing reductions of approximately 79% and 76%. This indicates that the modified cyclic peptide can effectively inhibit the activity of the Wnt / β-catenin signaling pathway.

[0031] 6. In a nude mouse xenograft model, the tumor volume of the modified cyclic peptide treatment group decreased by approximately 28%, the tumor weight decreased significantly, and the expression of β-catenin protein in the tumor tissue decreased by approximately 34%, demonstrating a stronger tumor growth inhibitory effect than the unmodified cyclic peptide, suggesting that its anti-tumor effect is closely related to the inhibition of the β-catenin signaling pathway.

[0032] In summary, this invention significantly improves the stability, cellular uptake, and functional persistence of cyclic peptides through a combination of D-amino acid and N-methylation modifications. It can also efficiently promote β-catenin degradation and inhibit the Wnt / β-catenin signaling pathway, showing promising application prospects in the treatment of APC-mutant colorectal cancer and related diseases. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the chemical structure of the modified cyclic peptide prepared in Example 1 of the present invention, wherein the green box represents the D-amino acid modification site, the yellow box represents the N-methylation modification site, and the blue box represents the disulfide bond cyclization structure.

[0034] Figure 2 The above are the analytical characterization results of the modified cyclic peptide prepared in Example 1 of the present invention, wherein A is the LC-MS mass spectrometry analysis chromatogram and B is the RP-HPLC purity analysis chromatogram.

[0035] Figure 3 The results of serum stability experiments of modified and unmodified cyclic peptides in Example 2 of this invention are shown. In this example, A is the curve showing the change in the proportion of intact cyclic peptides at different time points (0-24 h); B is the RP-HPLC chromatogram of the combined modified and unmodified cyclic peptides at 0 h and 8 h.

[0036] Figure 4 This is a comparison of the effects of modified and unmodified cyclic peptides on β-catenin protein expression in the Washout experiment in Example 2 of the present invention. In this figure, A is the result of Western blot detection; B is the quantitative analysis curve of the relative expression level of β-catenin protein over time.

[0037] Figure 5 The image shows the uptake of modified and unmodified cyclic peptides in cells in Example 3 of this invention. A is a fluorescence microscopy image of SW480 cells; B is a fluorescence microscopy image of DLD-1 cells; and C is a statistical analysis of the relative fluorescence intensity within cells.

[0038] Figure 6 The following are the Western blot analysis results of the effects of modified cyclic peptides, unmodified cyclic peptides, and the small molecule inhibitor MSAB on β-catenin protein expression in Example 4 of the present invention. Among them, A is the Western blot detection result of SW480 cells; B is the Western blot detection result of DLD-1 cells; and C is the quantitative analysis diagram of the relative expression level of β-catenin protein.

[0039] Figure 7The following is a Western blot analysis result of the concentration-dependent downregulation of β-catenin protein expression by the modified cyclic peptide in Example 5 of the present invention. In this figure, A is the Western blot detection result of SW480 cells; B is the Western blot detection result of DLD-1 cells; and C is a quantitative analysis graph of the relative expression level of β-catenin protein after treatment with different concentrations of modified cyclic peptide.

[0040] Figure 8 The following is a Western blot analysis result of the time-dependent downregulation of β-catenin protein expression by the modified cyclic peptide in Example 6 of the present invention. In this figure, A is the Western blot detection result of SW480 cells; B is the Western blot detection result of DLD-1 cells; and C is a quantitative analysis graph of the relative expression level of β-catenin protein by the modified cyclic peptide at different treatment times.

[0041] Figure 9 The following are the Western blot analysis results of the concentration-dependent regulation of the expression of downstream target proteins Cyclin D1 and c-Myc in the Wnt / β-catenin signaling pathway by modified cyclic peptides in Example 7 of this invention. Among them, A is the Western blot detection result of SW480 cells; B is the Western blot detection result of DLD-1 cells; C is the quantitative analysis diagram of the relative expression level of Cyclin D1 protein; and D is the quantitative analysis diagram of the relative expression level of c-Myc protein.

[0042] Figure 10 The following are Western blot analysis results of the time-dependent regulation of the expression of downstream target proteins Cyclin D1 and c-Myc of the Wnt / β-catenin signaling pathway by modified cyclic peptides in Example 8 of this invention. Among them, A is the Western blot detection result of SW480 cells; B is the Western blot detection result of DLD-1 cells; C is the quantitative analysis diagram of the relative expression level of Cyclin D1 protein; and D is the quantitative analysis diagram of the relative expression level of c-Myc protein.

[0043] Figure 11 The images show the results of in vitro tumor and tumor volume analysis of nude mice in different treatment groups in Example 9 of the present invention. A shows photographs of in vitro tumors of nude mice in each group; B shows a statistical analysis chart of tumor volume in each group.

[0044] Figure 12 The results of immunohistochemical staining analysis of β-catenin in Example 10 of the present invention are shown. In this figure, A is the immunohistochemical staining map of β-catenin in each group of tumor tissues; B is the statistical analysis map of β-catenin positive expression.

[0045] Figure 13 The results of Western blot analysis of β-catenin protein in tumor tissue in Example 10 of the present invention are shown. In this figure, A is the Western blot detection result of β-catenin protein; B is a quantitative analysis diagram of the relative expression level of β-catenin protein. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0047] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0048] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0049] Example 1 Solid-phase synthesis of combinatorially modified cyclic peptides A cell-penetrating fragment (RRRRRRRRR) was introduced at the N-terminus of the core cyclic peptide (SEQ ID NO.1), and a flexible linker (6-aminocaproic acid, Ahx) and an E3 ubiquitin ligase ligand (ALAPYIP) were linked at the C-terminus to construct a combinatorial modified cyclic peptide with cell-penetrating ability and targeted protein degradation function. Its chemical structure is shown in [reference needed]. Figure 1 The preparation method is as follows: (1) β-catenin-targeted degradation peptides were synthesized using a solid-phase peptide synthesis method; Rink Amide MBHA resin (0.56 mmol / g degree of substitution) was used as the solid-phase support, and N,N-dimethylformamide (DMF) was used as the reaction solvent. Each amino acid coupling step was activated with 4 molar equivalents of Fmoc-protected amino acids, 4 molar equivalents of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), 4 molar equivalents of 1-hydroxybenzotriazole (HOBt), and 8 molar equivalents of N,N-diisopropylethylamine (DIEA), and the reaction was carried out at room temperature with shaking for 45 min. After each coupling step, the Fmoc protecting groups were removed using a 20% piperidine / DMF solution for 10 min each time, for a total of 2 times. The resin was then thoroughly washed with DMF.

[0050] Following the target sequence, amino acid coupling was performed sequentially, with Fmoc-D-Ser(OtBu)-OH at position 2, Fmoc-N-Me-Thr(tBu)-OH at position 3, and Fmoc-D-Gly-OH at position 6, to achieve D-amino acid and N-methylation modification, thereby improving the serum stability and protease tolerance of the target peptide. After the core peptide sequence was synthesized, a nonamericarin (RRRRRRRRR) cell-penetrating peptide was coupled to the N-terminus, and a 6-aminocaproic acid (Ahx) flexible linker and an E3 ubiquitin ligase ligand sequence (ALAPYIP) were sequentially coupled to the C-terminus to obtain the target linearly combined modified peptide. After synthesis, the peptide was cleaved at room temperature for 2 h using a lysis buffer (trifluoroacetic acid / triisopropylsilane / ultrapure water = 95:2.5:2.5, v / v / v) to cleave the peptide from the resin and simultaneously remove the side-chain protecting groups. After filtering the lysis buffer to remove the resin, the filtrate was added dropwise to pre-cooled anhydrous ether to precipitate the crude peptide. The precipitate was collected by centrifugation, washed three times with anhydrous ether, and vacuum dried to obtain crude β-catenin-targeted degradation peptide.

[0051] (2) The β-catenin-targeted degradation peptide obtained in step (1) is subjected to an oxidation reaction, causing two cysteine ​​residues to form disulfide bonds, thereby achieving cyclization: The β-catenin-targeted degradation peptide obtained in step (1) was dissolved in PBS buffer (pH 7.4) containing 20% ​​(v / v) dimethyl sulfoxide (DMSO) to a final peptide concentration of 0.2–0.5 mg / mL. The solution was magnetically stirred at room temperature for 12 h to form intramolecular disulfide bonds under air oxidation conditions, thus achieving a cyclization reaction. After the reaction was completed, the crude β-catenin-targeted degradation cyclic peptide was obtained by freeze-drying.

[0052] (3) The cyclized product was purified to obtain the β-catenin-targeted degradation cyclic peptide.

[0053] The crude β-catenin-targeted degradation cyclic peptide obtained in step (2) was purified by C18 reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic column was a C18 preparative column; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid (TFA), and mobile phase B was an acetonitrile solution containing 0.1% TFA; the gradient elution program was 5%–60% B (30 min); the flow rate was 1.0 mL / min; and the detection wavelength was 214 nm. The target peak was collected and freeze-dried to obtain the β-catenin-targeted degradation cyclic peptide.

[0054] The molecular weight of the purified target cyclic peptide was determined using liquid chromatography-mass spectrometry (LC-MS).

[0055] The liquid chromatography conditions were as follows: the column was a C18 analytical column (2.1 mm × 100 mm, 1.7 μm); mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid; the gradient elution program was 0–2 min, 5% B; 2–15 min, 5%–60% B; 15–18 min, 60%–95% B; 18–20 min, maintaining 95% B; the flow rate was 0.30 mL / min; the column temperature was 35℃; and the injection volume was 5 μL.

[0056] Mass spectrometry was performed using an electrospray ionization (ESI) source in positive ion mode with a capillary voltage of 4.5 kV, a cone voltage of 40 V, an ion source temperature of 120 °C, a desolventizing temperature of 350 °C, a desolventizing gas flow rate of 800 L / h, and a scanning mass range of m / z 300–6000. Multicharged ion peaks of the target peptide were acquired, and deconvolution analysis was performed using software to calculate the molecular weight of the target peptide to confirm its structure.

[0057] MS characterization results (see) Figure 2 As shown in Figure A, the ionic peak of the target polypeptide after cyclization and combinatorial modification mainly appears at m / z = 4286.80 Da ([M+H)). + The result is consistent with the theoretical molecular weight of 4290.82 Da, indicating that the target polypeptide was successfully synthesized and has the correct structure. HPLC analysis results (see...) Figure 2 Table B shows that the cyclic peptide exhibits a single major chromatographic peak at 7.128 min. The peak integral results are shown in Table 3. The area of ​​the main peak of the target cyclic peptide accounts for 95.734% of the total peak area, indicating that the obtained product has high chemical purity and good uniformity, which can meet the requirements of subsequent in vitro cell function evaluation, β-catenin targeted degradation experiment and animal experiment research.

[0058] Table 3. HPLC peak integration results of the target cyclic peptide

[0059] Comparative Example 1: Preparation of Unmodified Cyclic Peptides The unmodified cyclic peptide was prepared according to the method of Example 1. The difference from Example 1 is that the second position is L-serine, the seventh position is L-phenylalanine, and the third position is L-threonine without N-methylation modification.

[0060] Example 2: Effects of D-amino acid and N-methylation modification on serum stability and functional retention of cyclic peptides 1. Serum stability test The unmodified cyclic peptides prepared in Comparative Example 1 (all L-amino acids, without N-methylation modification, but retaining the same disulfide bond cyclization structure) and the modified cyclic peptides prepared in Example 1 were prepared as 1 mM stock solutions. 20 μL of each stock solution was added to 980 μL of PBS buffer (pH 7.4) containing 50% (v / v) fetal bovine serum to a final volume of 1 mL and a final concentration of 20 μM, and incubated at 37 °C. Samples were taken at 0, 2, 4, 8, 12, and 24 h. After each sampling, an equal volume of ice-cold acetonitrile was added to terminate the reaction, and the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was used for analysis. The peptide content was determined by reversed-phase high-performance liquid chromatography (RP-HPLC), and the proportion of the main peak area at each time point to the initial (0 h) main peak area was calculated as the proportion of the remaining intact peptide. The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase A was 0.1% TFA aqueous solution, mobile phase B was 0.1% TFA acetonitrile solution, gradient elution of 5%–60% B (30 min), flow rate 1.0 mL / min, and detection wavelength 214 nm.

[0061] The peak area of ​​the intact peptide at 0 h was set to 100%, and the proportion of intact peptide residue at each time point was calculated. The results are as follows: Figure 3 As shown, the residual rates of the modified cyclic peptide at 2, 4, 8, 12, and 24 h were approximately 90%, 81%, 68%, 55%, and 38%, respectively; while those of the unmodified cyclic peptide were approximately 78%, 61%, 40%, 25%, and 10%, respectively. Notably, at 8 h, the residual rate of the modified cyclic peptide was approximately 68%, significantly higher than the 40% of the unmodified cyclic peptide; at 24 h, the modified cyclic peptide still retained approximately 38% of the complete peptide, while the unmodified cyclic peptide retained only about 10%. These results indicate that D-amino acid substitution and N-methylation modification significantly slowed the degradation rate of cyclic peptides in serum and significantly improved serum stability.

[0062] 2. Washout Experiment SW480 cells were seeded in 6-well plates and cultured at 37°C and 5% CO2 in DMEM medium containing 10% (v / v) fetal bovine serum to approximately 70% confluence. Unmodified cyclic peptides prepared in Comparative Example 1 and modified cyclic peptides prepared in Example 1 were added to a final concentration of 4 μM (diluted from 1 mM stock solution and added to the medium), and incubation continued for 24 h. After treatment, the cells were washed three times with PBS buffer and then cultured in fresh medium free of peptides.

[0063] Cell samples were collected at 0, 12, 24, and 48 h. Total protein was extracted using RIPA lysis buffer, and the supernatant was collected after centrifugation. Protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using anti-β-catenin primary antibody, with GAPDH used as an internal control.

[0064] The results are as follows Figure 4 As shown, grayscale analysis was performed using ImageJ software, with the untreated group normalized to 1. After 24 h of treatment, the relative expression levels of β-catenin in the unmodified cyclic peptide group and the modified cyclic peptide group decreased to approximately 0.50 and 0.40, respectively. 12 h after drug withdrawal, the unmodified cyclic peptide group recovered to approximately 0.60, while the modified cyclic peptide group only recovered to approximately 0.46. After 24 h of drug withdrawal, the two groups recovered to approximately 0.75 and 0.55, respectively. After 48 h of drug withdrawal, the unmodified cyclic peptide group further recovered to approximately 0.90, approaching the baseline expression level, while the modified cyclic peptide group only recovered to approximately 0.68, still significantly lower than the unmodified cyclic peptide group.

[0065] Example 3: Effects of D-amino acid and N-methylation modifications on cell permeability To evaluate the cellular uptake capacity of the modified cyclic peptides of this invention, the unmodified cyclic peptides prepared in Comparative Example 1 and the modified cyclic peptides prepared in Example 1 were labeled with FITC. The peptides were fluorescently labeled using the FITC-NHS method. After labeling, free FITC was removed by dialysis or HPLC purification, and a 1 mM stock solution was prepared. SW480 and DLD-1 cells were seeded in confocal culture dishes and cultured in DMEM medium containing 10% (v / v) fetal bovine serum at 37°C and 5% CO2 until approximately 70% confluence. FITC-labeled unmodified and modified cyclic peptides were added to a final concentration of 5 μM (diluted from the 1 mM stock solution and added to the medium), and incubated for another 6 h. After incubation, the cells were washed three times with PBS buffer to remove unbound peptides. Fluorescence imaging was performed using a confocal laser scanning microscope. All samples were subjected to the same excitation and acquisition parameters (FITC excitation wavelength 488 nm, emission wavelength 500–550 nm). After image acquisition, the mean intracellular fluorescence intensity was quantitatively analyzed using ImageJ software. The experiment was repeated three times, with multiple fields of view taken for statistical analysis each time.

[0066] The results are as follows Figure 5As shown, the unmodified cyclic peptide group exhibited intracellular fluorescence signals in both SW480 and DLD-1 cells, while the modified cyclic peptide group showed significantly enhanced fluorescence signals. Quantitative analysis using ImageJ software revealed that in SW480 cells, the relative fluorescence intensity of the modified cyclic peptide group increased from 1.00±0.08 to 1.62±0.18; and in DLD-1 cells, it increased from 1.00±0.09 to 1.70±0.11, with statistically significant differences. P <0.05). The results indicate that D-amino acid and N-methylation modifications can effectively enhance the cellular uptake capacity of cyclic peptides and improve their cellular permeability.

[0067] Example 4: Effect of modified cyclic peptides on β-catenin protein levels SW480 and DLD-1 cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 in DMEM medium containing 10% (v / v) fetal bovine serum until the cells reached approximately 70% confluence. The cells were then treated with unmodified cyclic peptides (final concentration 4 μM) prepared in Comparative Example 1, modified cyclic peptides (final concentration 4 μM) prepared in Example 1, and the reported β-catenin degradation inhibitor MSAB (final concentration 10 μM), respectively. A solvent control group (DMSO, final concentration ≤0.1%) was also included. Treatment time for each group was 24 h. After treatment, cells were washed twice with PBS, and total protein was extracted using RIPA lysis buffer. The supernatant was collected after centrifugation, and protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using anti-β-catenin primary antibody, with GAPDH used as an internal control.

[0068] The results are as follows Figure 6 As shown, after grayscale analysis and normalization using GAPDH as an internal reference, the relative expression levels of β-catenin in SW480 cells were 1.00±0.01, 0.82±0.02, 0.33±0.02, and 0.18±0.01 in the DMSO group, MSAB group, unmodified cyclic peptide group, and modified cyclic peptide group, respectively; and in DLD-1 cells, they were 1.00±0.02, 0.70±0.02, 0.39±0.01, and 0.24±0.01, respectively. Compared with the unmodified cyclic peptide, the modified cyclic peptide further reduced β-catenin protein expression by approximately 45.5% and 38.5% in SW480 and DLD-1 cells, respectively, with statistically significant differences. P <0.05). The above results indicate that the cyclic peptide modified with D-amino acids and N-methylation can more effectively downregulate the expression level of β-catenin protein in APC mutant colorectal cancer cells.

[0069] Example 5: Concentration-dependent analysis of β-catenin degradation induced by modified cyclic peptides To further evaluate the dose-dependent effect of the modified cyclic peptide of this invention on β-catenin protein expression, different concentration treatment experiments were conducted in SW480 and DLD-1 cells. Cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 in DMEM medium containing 10% (v / v) fetal bovine serum until the cells reached approximately 70% confluence. The modified cyclic peptide prepared in Example 1 was then added to final concentrations of 0, 2, 4, 6, and 8 μM, respectively, and treated for 48 h. Culture conditions were maintained constant throughout the treatment. After treatment, cells were washed twice with PBS, and total protein was extracted using RIPA lysis buffer. The supernatant was collected by centrifugation, and protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using anti-β-catenin primary antibody, with GAPDH used as an internal control.

[0070] The results are as follows Figure 7 As shown, after grayscale analysis and normalization using GAPDH as an internal reference, with the increase of modified cyclic peptide concentration, the relative expression level of β-catenin in SW480 cells gradually decreased from 0.90±0.01 to 0.20±0.01, and in DLD-1 cells from 0.94±0.01 to 0.17±0.01, both showing a significant dose-dependent decreasing trend. This indicates that the modified cyclic peptide can continuously enhance the downregulation effect on β-catenin protein with increasing concentration.

[0071] Example 6: Time-dependent analysis of β-catenin degradation induced by modified cyclic peptides To further evaluate the time-dependent effect of the modified cyclic peptide of this invention on β-catenin protein expression, a time-gradient experiment was performed in SW480 and DLD-1 cells. Cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 in DMEM medium containing 10% (v / v) fetal bovine serum until the cells reached approximately 70% confluence. The modified cyclic peptide prepared in Example 1 was added to a final concentration of 4 μM, and the cells were cultured continuously. Cell samples were collected at 0, 6, 12, 24, and 48 h. After treatment at each time point, the cells were washed twice with PBS, and total protein was extracted using RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using anti-β-catenin primary antibody, with GAPDH used as an internal control.

[0072] The results are as follows Figure 8As shown, after grayscale analysis and normalization using GAPDH as an internal control, with the extension of the modified cyclic peptide treatment time, the relative expression level of β-catenin in SW480 cells gradually decreased from 0.91±0.01 to 0.18±0.01, and in DLD-1 cells from 0.87±0.01 to 0.22±0.01, both showing a significant time-dependent decreasing trend. Combined with the aforementioned concentration gradient experiment results, this indicates that the modified cyclic peptide of this invention can continuously downregulate β-catenin protein expression in a concentration- and time-dependent manner.

[0073] Example 7: Concentration-dependent regulation of Wnt downstream target proteins Cyclin D1 and c-Myc expression by modified cyclic peptides To further verify the effect of the modified cyclic peptide of this invention on the expression of downstream target proteins of the Wnt / β-catenin signaling pathway, a concentration gradient experiment was performed in SW480 and DLD-1 cells. Cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 in DMEM medium containing 10% (v / v) fetal bovine serum until the cells reached approximately 70% confluence. The modified cyclic peptide prepared in Example 1 was added to final concentrations of 0, 1, 2, 4, and 8 μM, respectively, and treated for 48 h. After treatment, the cells were washed twice with PBS, and total protein was extracted using RIPA lysis buffer. The supernatant was collected after centrifugation, and protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using anti-Cyclin D1 and anti-c-Myc primary antibodies, respectively, with GAPDH used as an internal control.

[0074] The results are as follows Figure 9 As shown, after cells were treated with 0, 2, 4, 6, and 8 μM modified cyclic peptides for 48 h, the expression of Cyclin D1 and c-Myc proteins gradually decreased with increasing modified cyclic peptide concentration, exhibiting a significant concentration-dependent effect. The relative expression levels of Cyclin D1 in SW480 and DLD-1 cells decreased from 1.00 in the control group to 0.24 and 0.26 at 8 μM, respectively; c-Myc protein expression decreased from 1.00 to 0.21 and 0.24, respectively, representing a reduction of approximately 75% or more compared to the control group. These results indicate that the modified cyclic peptides of this invention can inhibit the activity of the Wnt / β-catenin signaling pathway by promoting β-catenin degradation and further downregulating the expression of its downstream target proteins Cyclin D1 and c-Myc.

[0075] Example 8: Time-dependent regulation of Wnt downstream target proteins Cyclin D1 and c-Myc expression by modified cyclic peptides To further verify the time-dependent effect of the modified cyclic peptide of this invention on the expression of downstream target proteins in the Wnt / β-catenin signaling pathway, a time-gradient experiment was performed in SW480 and DLD-1 cells. Cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 in DMEM medium containing 10% (v / v) fetal bovine serum until approximately 70% confluence. The modified cyclic peptide prepared in Example 1 was added to a final concentration of 4 μM, and the cells were continuously cultured. Cell samples were collected at 0, 6, 12, 24, and 48 h. After treatment at each time point, cells were washed twice with PBS, and total protein was extracted using RIPA lysis buffer. The supernatant was collected after centrifugation, and protein concentration was determined using the BCA method. Equal amounts of protein from each group were subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using anti-Cyclin D1 and anti-c-Myc primary antibodies, with β-actin used as an internal control.

[0076] The results are as follows Figure 10 As shown, after treatment with the modified cyclic peptide for 0, 6, 12, 24, and 48 h, the expression of Cyclin D1 and c-Myc proteins gradually decreased with increasing treatment time, exhibiting a significant time-dependent effect. Specifically, the relative expression levels of Cyclin D1 in SW480 and DLD-1 cells decreased from 1.00 in the control group to 0.19 and 0.21 at 48 h, respectively; c-Myc protein levels decreased to 0.16 and 0.21, respectively, representing a reduction of approximately 80% compared to the control group. The expression of both proteins had already significantly decreased at 24 h, reaching its lowest level at 48 h. These results indicate that the modified cyclic peptide of this invention can continuously promote the degradation of β-catenin with increasing treatment time and further downregulate the expression of its downstream target proteins Cyclin D1 and c-Myc.

[0077] Example 9: Antitumor effect of modified cyclic peptide in nude mouse xenograft model and its influence on β-catenin expression To verify the antitumor activity of the modified cyclic peptide of this invention in vivo and its regulatory effect on the β-catenin signaling pathway, an SW480 cell subcutaneous xenograft model in nude mice was established for animal experiments. 4-6 week old BALB / c nude mice were selected, and SW480 cells were distributed at a rate of 5 × 10⁶ cells per mouse. 6Cells were subcutaneously inoculated into the right axilla of nude mice. When the tumor volume grew to approximately 80–100 mm³, the mice were randomly divided into an unmodified peptide group and a modified cyclic peptide group, with four mice in each group. The modified cyclic peptide group was treated with the modified cyclic peptide prepared in Example 1, while the unmodified peptide group was treated with an equal dose of the unmodified peptide. Intraperitoneal injection was administered at the prescribed dose every other day for four consecutive weeks. During the experiment, the long and short diameters of the tumors in the nude mice were measured periodically, and the tumor volume was calculated using the formula V = 1 / 2 × L × W². Tumor growth was continuously recorded. After the experiment, the nude mice were sacrificed, the tumor tissue was dissected and weighed, and images of the tumor were taken. Total protein was extracted from some tumor tissue using RIPA lysis buffer, and the expression level of β-catenin protein was detected by Western blot. Other tumor tissue was paraffin-embedded and immunohistochemically stained to detect the expression of β-catenin in the tumor tissue.

[0078] The results are as follows Figure 11 As shown, compared with the unmodified peptide group, the growth of xenografts in nude mice was significantly inhibited in the modified cyclic peptide group. At week 4, the average tumor volume in the modified cyclic peptide group was approximately 0.73 cm³, which was about 28% lower than that in the unmodified peptide group (approximately 1.02 cm³). The volume and mass of the tumors dissected at the endpoint were also significantly reduced, indicating that the modified cyclic peptide has good in vivo antitumor activity.

[0079] Immunohistochemical results ( Figure 12 The results showed that the positive expression of β-catenin in tumor tissues was significantly reduced in the modified cyclic peptide group, with a relative expression level decreasing by approximately 34% compared to the unmodified peptide group (P<0.05). Western blot results ( Figure 13 Further investigation confirmed that the relative expression level of β-catenin protein in the modified cyclic peptide group was approximately 0.37, which was about 34% lower than that in the unmodified peptide group (approximately 0.56) (P<0.05).

[0080] The above results indicate that the modified cyclic peptide can significantly inhibit the growth of xenografts in nude mice and reduce the expression of β-catenin protein in tumor tissues. Its in vivo antitumor effect may be related to the inhibition of the Wnt / β-catenin signaling pathway.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A β-catenin-targeted degradation cyclic peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The β-catenin-targeted degradation cyclic peptide according to claim 1, characterized in that, The serine at position 2 and the phenylalanine at position 7 in the cyclic peptide are D-configuration amino acids.

3. The β-catenin-targeted degradation cyclic peptide according to claim 1, characterized in that, The threonine at position 3 in the cyclic peptide is N-methylated.

4. The β-catenin-targeted degradation cyclic peptide according to claim 1, characterized in that, The cysteine ​​residues at positions 9 and 21 in the cyclic peptide form a cyclic structure via disulfide bonds.

5. The β-catenin-targeted degradation cyclic peptide according to claim 1, characterized in that, The cyclic peptide has a cell-penetrating sequence attached to its N-terminus, and the amino acid sequence is shown in SEQ ID NO.

2.

6. The β-catenin-targeted degradation cyclic peptide according to claim 1, characterized in that, The cyclic peptide has an E3 ubiquitin ligase ligand attached to its C-terminus.

7. The β-catenin-targeted degradation cyclic peptide according to claim 6, characterized in that, The E3 ubiquitin ligand is a VHL ligand, and its amino acid sequence is shown in SEQ ID NO.

3.

8. The use of the β-catenin-targeting degradation cyclic peptide according to any one of claims 1-7 in the preparation of a medicament for treating diseases related to abnormal accumulation of β-catenin.

9. The application according to claim 8, characterized in that, The diseases associated with abnormal accumulation of β-catenin are APC-mutant colorectal cancer or familial adenomatous polyposis.

10. A method for preparing the β-catenin-targeted degradation cyclic peptide according to any one of claims 1-7, characterized in that, Includes the following steps: (1) The β-catenin-targeted degradation peptide was synthesized using a solid-phase peptide synthesis method; (2) The β-catenin-targeted degradation peptide is oxidized to form a disulfide bond between two cysteine ​​residues, thereby achieving cyclization; (3) The cyclized product was purified to obtain the β-catenin-targeted degradation cyclic peptide.

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