Chimeric peptides that degrade cxcr4 and uses thereof

Abstract

The application provides a chimeric peptide for degrading CXCR4 and an application, and belongs to the technical field of biological medicine. The chimeric peptide is one of K-6, K-8, K-17, K-4 and K-9. The application research finds that the chimeric peptide can not only effectively degrade CXCR4, but also can significantly inhibit the migration and invasion of tumor cells and significantly inhibit the proliferation ability of tumor cells, and the chimeric peptide provides more choices for anti-tumor or anti-tumor metastasis drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a chimeric peptide that degrades CXCR4 and its application. Background Technology

[0002] Prostate cancer is the second most common cancer among men worldwide, posing a significant threat to public health. While early-stage prostate cancer can be effectively treated with procedures such as surgery, radiotherapy, or chemotherapy, effective treatments are lacking and prognosis is extremely poor once the disease progresses to intermediate or advanced stages, namely locally advanced (e.g., infiltrating adjacent tissues) or distant metastases (e.g., bone metastases). Studies show that the 5-year survival rate for locally advanced prostate cancer (PCa) is close to 100%, but once distant metastases occur, the 5-year survival rate drops sharply to below 30%. Therefore, effective treatments are urgently needed for patients with intermediate or advanced PCa, aiming to treat the primary tumor while simultaneously inhibiting distant metastasis.

[0003] CXCR4 is a transmembrane protein that transmits intracellular signals by binding to CXCL12. CXCR4 is highly expressed in prostate cancer and plays a crucial role in tumor metastasis. Disrupting the CXCL12-CXCR4 axis can effectively inhibit tumor metastasis. Currently licensed CXCR4-targeted therapy AMD3100 inhibits CXCR4 through single-molecule receptor interactions; these methods, by blocking the binding of CXCL12 to CXCR4, inevitably suffer from off-target effects. Therefore, providing a CXCR4-targeted therapy that effectively prevents off-target effects and enhances its anti-tumor metastasis capabilities remains a pressing issue. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a chimeric peptide that degrades CXCR4 and its application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a chimeric peptide that degrades CXCR4, wherein the chimeric peptide is one of K-6, K-8, K-17, K-4 and K-9;

[0007] The amino acid sequence of K-6 is KFERQRFFESH;

[0008] The amino acid sequence of K-8 is KFERQKPVSLSYR;

[0009] The amino acid sequence of K-17 is KFERQRFFESHAPAKPVSLSYR;

[0010] The amino acid sequence of K-4 is KFERQYRR-2Nal-G;

[0011] The amino acid sequence of K-9 is KFERQGGP. D YR D VGR- D 2Nal-P.

[0012] This invention provides the application of the above-mentioned chimeric peptide in the preparation of CXCR4 degradation products.

[0013] Preferably, the chimeric peptide targets and degrades CXCR4 in tumor cells.

[0014] Preferably, the tumor is prostate cancer.

[0015] This invention provides the application of the above-mentioned chimeric peptide in the preparation of tumor-inhibiting drugs.

[0016] This invention provides the use of the above-mentioned chimeric peptide in the preparation of drugs that inhibit tumor migration and / or invasion.

[0017] The present invention provides a drug for treating prostate cancer, the drug comprising one or more of the above-mentioned K-6, K-8, K-17, K-4 and K-9.

[0018] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0019] Preferably, the drug further includes a lysosomal autophagy agonist and / or inhibitor.

[0020] Preferably, the active pharmaceutical ingredient is one or more of K-6, K-8, K-17, K-4 and K-9.

[0021] Beneficial effects

[0022] This invention provides a chimeric peptide that degrades CXCR4 and its applications. The research of this invention found that the chimeric peptide can not only effectively degrade CXCR4, but also significantly inhibit the migration and invasion of tumor cells and significantly inhibit the proliferation of tumor cells. The chimeric peptide of this invention provides a new option for anti-tumor or anti-tumor metastasis drugs. Attached Figure Description

[0023] Figure 1 HPLC and MS results for K-6;

[0024] Figure 2 HPLC and MS results for K-8;

[0025] Figure 3 HPLC and MS results for K-17;

[0026] Figure 4 HPLC and MS results for K-4;

[0027] Figure 5 HPLC and MS results for K-9;

[0028] Figure 6 The chemical structures of the chimeric peptides K-6, K-8, K-17, K-4 and K-9 prepared in Examples 1-5 are shown in the diagrams.

[0029] Figure 7 The image shows the Western blot results of the degradation capacity of Control, peptides K-6, K-8, K-17, K-4 and K-9 on CXCR4.

[0030] Figure 8 The effects of Control, K-6, K-8, K-17, K-4, and K-9 on RM-1 cell migration and invasion were presented.

[0031] Figure 9 The results show the effects of Control, K-6, K-8, K-17, K-4, and K-9 on the lateral migration, longitudinal migration, and invasion rate of RM-1 cells. Specifically, A represents the effect of Control, K-6, K-8, K-17, K-4, and K-9 on the lateral migration rate of RM-1 cells; B represents the effect of Control, K-6, K-8, K-17, K-4, and K-9 on the longitudinal migration rate of RM-1 cells; and C represents the effect of Control, K-6, K-8, K-17, K-4, and K-9 on the invasion rate of RM-1 cells. A p < 0.05 indicates a significant difference compared to the Control group.

[0032] Figure 10 The results show the effects of different concentrations of Control, K-6, K-8, K-17, K-4 and K-9 on the viability of RM-1 cells. Among them, p < 0.05 indicates a significant difference compared with Control.

[0033] Figure 11 To investigate the mechanism of CXCR4 degradation by chimeric peptides, lane 1 was the control group, lane 2 was the K-9 group, lane 3 was the K-9+QX77 group, and lane 4 was the K-9+VER155008 group. Detailed Implementation

[0034] This invention provides a chimeric peptide of CXCR4, wherein the chimeric peptide is one of K-6, K-8, K-17, K-4 and K-9;

[0035] The amino acid sequence of K-6 is KFERQRFFESH;

[0036] The amino acid sequence of K-8 is KFERQKPVSLSYR;

[0037] The amino acid sequence of K-17 is KFERQRFFESHAPAKPVSLSYR;

[0038] The amino acid sequence of K-4 is KFERQYRR-2Nal-G;

[0039] The amino acid sequence of K-9 is KFERQGGPDYRDVGR-D2Nal-P.

[0040] In this invention, the chimeric peptides are prepared by the Fmoc solid-phase peptide synthesis method. The K-6, K-8, K-17, K-4 and K-9 prepared by the Fmoc solid-phase peptide synthesis method are all detected by HPLC and have a purity of more than 95%.

[0041] The chimeric peptide of CXCR4 of the present invention is a lysosome-targeting chimaera (LYTAC), which can guide the CXCR4 protein to lysosomes for efficient degradation.

[0042] This invention has found that the amino acid sequence of chimeric peptides has a significant impact on their activity in degrading CXCR4 in tumors and in inhibiting tumor growth and metastasis. Not every chimeric peptide can achieve the same effect of degrading CXCR4 in tumors and in inhibiting tumor growth and metastasis.

[0043] This invention provides the application of the above-mentioned chimeric peptide in the preparation of CXCR4 degradation products.

[0044] In this invention, the chimeric peptide significantly targets and degrades CXCR4 in tumor cells. The tumor is preferably prostate cancer, such as RM-1 cells. The product can be a reagent or a drug.

[0045] This invention provides the application of the above-mentioned chimeric peptide in the preparation of tumor-inhibiting drugs, wherein the tumor is preferably prostate cancer, such as RM-1 cells.

[0046] This invention provides the use of the above-mentioned chimeric peptide in the preparation of drugs that inhibit tumor migration and / or invasion, wherein the tumor is preferably prostate cancer, such as RM-1 cells.

[0047] The present invention provides a drug for treating prostate cancer, the drug comprising one or more of the above-mentioned K-6, K-8, K-17, K-4 and K-9.

[0048] In this invention, the medicament further includes pharmaceutically acceptable excipients, such as one or more of flavoring agents, excipients, preservatives, solvents, or antioxidants. In the medicament, one or more of K-6, K-8, K-17, K-4, and K-9 constitute 50-100% of the medicament by mass. The medicament can use one or more of K-6, K-8, K-17, K-4, and K-9 as the sole active ingredient for degrading CXCR4, anti-tumor activity, or anti-tumor metastasis, or it can be used in combination with anti-tumor or anti-tumor metastasis drugs to jointly treat tumors or inhibit tumor metastasis. As a preferred embodiment, the active ingredient of the medicament is one or more of K-6, K-8, K-17, K-4, and K-9, more preferably K-9. The active ingredient, also known as the effective ingredient, refers to the component in the medicament that directly exerts the effect or function of degrading CXCR4, anti-tumor activity, or anti-tumor metastasis.

[0049] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0050] 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.

[0051] In the following embodiments, the QX77 was purchased from MCE with part number HY-112483; the VER155008 was purchased from MCE with part number HY-10941.

[0052] Example 1

[0053] A polypeptide K-6, the amino acid sequence of which is KFERQRFFESH (SEQ ID NO.5).

[0054] Synthesis of peptide K-6: Synthesized using the standard Fmoc solid-phase peptide synthesis method (SPPS). In simple terms, 0.5 mol of dichloromethane resin was dissolved in 15 mL of dichloromethane (DCM) and added to a solid-phase tube. After swelling for 10 min, the solution was extruded using a bulb syringe. 1 mol of Fmoc-protected histidine (Fmoc-His-OH) was dissolved in 15 mL of N,N-dimethylformamide (DMF), and 330 μL of N,N-diisopropylethylamine (DIPEA) was added to adjust the pH. The reaction was carried out at room temperature for 2 h. Subsequently, after extruding the solvent using a bulb syringe, DCM and DMF were added sequentially, followed by washing five times each for 1 min. The Fmoc protecting group was cleaved using a DMF solution containing 20% ​​piperidine for 30 min, followed by extrusion of the solvent and washing five times with DMF for 1 min each time. The second amino acid, 1 mol Fmoc-protected serine (Fmoc-Ser-OH), was dissolved in 15 mL of DMF along with 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. This process was repeated for subsequent amino acids: 1 mol Fmoc-protected glutamic acid, phenylalanine, arginine, glutamine, arginine, glutamic acid, phenylalanine, and lysine, each dissolved in 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) in 15 mL of DMF. After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. Finally, the peptide was cleaved for 45 min using a cleavage buffer of 95% trifluoroacetic acid (TFA), 2.5% trimethylsilane (TIS), and 2.5% water. The cleavage buffer was collected and evaporated to dryness using a rotary evaporator. Anhydrous diethyl ether was then added to precipitate the KFERQRFFESH peptide.

[0055] The chimeric peptide K-6 was detected by HPLC and MS, respectively. The results are shown in the figure. Figure 1 .

[0056] Figure 1 The results showed that the above preparation method successfully prepared polypeptide K-6 with a purity of 95%.

[0057] After sequencing, the amino acid sequence of peptide K-6 is shown in SEQ ID NO.5.

[0058] Example 2

[0059] A chimeric peptide K-8 that degrades CXCR4, wherein the amino acid sequence of K-8 is KFERQKPVSLSYR (SEQ ID NO.1).

[0060] Synthesis of the chimeric peptide K-8 degraded from CXCR4: Synthesized using the standard Fmoc solid-phase peptide synthesis method (SPPS). In simple terms, 0.5 mol of dichloromethane resin was dissolved in 15 mL of dichloromethane (DCM) and added to a solid-phase tube. After swelling for 10 min, the solution was extruded using a bulb syringe. 1 mol of Fmoc-protected arginine (Fmoc-Arg-OH) was dissolved in 15 mL of N,N-dimethylformamide (DMF), and 330 μL of N,N-diisopropylethylamine (DIPEA) was added to adjust the pH. The reaction was carried out at room temperature for 2 h. Subsequently, after extruding the solvent using a bulb syringe, DCM and DMF were added sequentially for washing five times, 1 min each time. The Fmoc protecting group was cleaved using a DMF solution containing 20% ​​piperidine for 30 min, after which the solvent was extruded and washed five times with DMF for 1 min each time. The second amino acid, 1 mol Fmoc-protected tyrosine (Fmoc-Tyr-OH), was dissolved in 15 mL of DMF along with 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. This process was repeated for subsequent amino acids: 1 mol Fmoc-protected serine, leucine, serine, valine, proline, lysine, glutamine, arginine, glutamic acid, phenylalanine, and lysine were dissolved in 15 mL of DMF along with 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. Finally, the peptide was cleaved for 45 min using a cleavage buffer of 95% trifluoroacetic acid (TFA), 2.5% trimethylsilane (TIS), and 2.5% water. The cleavage buffer was collected and evaporated to dryness using a rotary evaporator. Anhydrous diethyl ether was then added to precipitate the KFERQKPVSLSYR peptide.

[0061] The chimeric peptide K-8 was detected by HPLC and MS, respectively. The results are shown in the figure. Figure 2 .

[0062] Figure 2 The results showed that the chimeric peptide K-8 was successfully prepared using the above preparation method, with a purity of 95%.

[0063] After sequencing, the amino acid sequence of the chimeric peptide K-8 is shown in SEQ ID NO.1.

[0064] Example 3

[0065] A chimeric peptide K-17 that degrades CXCR4, wherein the amino acid sequence of K-17 is KFERQRFFESHAPAKPVSLSYR (SEQ ID NO.2).

[0066] Synthesis of the CXCR4-degraded chimeric peptide K-17: Synthesized using the standard Fmoc solid-phase peptide synthesis method (SPPS). In simple terms, 0.5 mol of dichloromethane resin was dissolved in 15 mL of dichloromethane (DCM) and added to a solid-phase tube. After swelling for 10 min, the solution was extruded using a bulb syringe. 1 mol of Fmoc-protected arginine (Fmoc-Arg-OH) was dissolved in 15 mL of N,N-dimethylformamide (DMF), and 330 μL of N,N-diisopropylethylamine (DIPEA) was added to adjust the pH. The reaction was carried out at room temperature for 2 h. Subsequently, after extruding the solvent using a bulb syringe, DCM and DMF were added sequentially for washing five times, 1 min each time. The Fmoc protecting group was cleaved using a DMF solution containing 20% ​​piperidine for 30 min, after which the solvent was extruded and washed five times with DMF for 1 min each time. The second amino acid, 1 mol of Fmoc-protected tyrosine (Fmoc-Tyr-OH), was dissolved in 15 mL of DMF along with 1 mol of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. This process was repeated for subsequent amino acids: 1 mol of Fmoc-protected serine, leucine, serine, valine, proline, lysine, alanine, proline, alanine, histidine, serine, glutamic acid, phenylalanine, arginine, glutamine, arginine, glutamic acid, phenylalanine, and lysine were dissolved in 15 mL of DMF along with 1 mol of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. Finally, the peptide was cleaved for 45 min using a cleavage buffer of 95% trifluoroacetic acid (TFA), 2.5% trimethylsilane (TIS), and 2.5% water. The cleavage buffer was collected and evaporated to dryness using a rotary evaporator. Subsequently, anhydrous diethyl ether was added to precipitate the KFERQRFFESHAPAKPVSLSYR peptide.

[0067] The chimeric peptide K-17 was detected by HPLC and MS, respectively. The results are shown in the figure. Figure 3 .

[0068] Figure 3 The results showed that the chimeric peptide K-17 was successfully prepared using the above preparation method, with a purity of 95%.

[0069] After sequencing, the amino acid sequence of the chimeric peptide K-17 is shown in SEQ ID NO.2.

[0070] Example 4

[0071] A chimeric peptide K-4 that degrades CXCR4, wherein the amino acid sequence of K-4 is KFERQYRR-2Nal-G (SEQ ID NO. 3-2Nal-G).

[0072] Synthesis of the chimeric peptide K-4 degraded from CXCR4: Synthesized using the standard Fmoc solid-phase peptide synthesis method (SPPS). In simple terms, 0.5 mol of dichloromethane resin was dissolved in 15 mL of dichloromethane (DCM) and added to a solid-phase tube. After swelling for 10 min, the solution was extruded using a bulb syringe. 1 mol of Fmoc-protected 2-naphthylglycine (Fmoc-2Nal-G-OH) was dissolved in 15 mL of N,N-dimethylformamide (DMF), and 330 μL of N,N-diisopropylethylamine (DIPEA) was added to adjust the pH. The reaction was carried out at room temperature for 2 h. Subsequently, after extruding the solvent using a bulb syringe, DCM and DMF were added sequentially for washing five times, 1 min each time. The Fmoc protecting group was cleaved using a DMF solution containing 20% ​​piperidine for 30 min, after which the solvent was extruded and washed five times with DMF for 1 min each time. The second amino acid, 1 mol Fmoc-protected arginine (Fmoc-Arg-OH), was dissolved in 15 mL of DMF along with 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. This process was repeated for subsequent amino acids: 1 mol Fmoc-protected arginine, tyrosine, glutamine, arginine, glutamic acid, phenylalanine, and lysine, each dissolved in 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) in 15 mL of DMF. After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. Finally, the peptide was cleaved for 45 min using a cleavage buffer of 95% trifluoroacetic acid (TFA), 2.5% trimethylsilane (TIS), and 2.5% water. The cleavage buffer was collected and evaporated to dryness using a rotary evaporator. Anhydrous diethyl ether was then added to precipitate the KFERQYRR-2Nal-G peptide.

[0073] The chimeric peptide K-4 was detected by HPLC and MS, respectively. The results are shown in the figure. Figure 4 .

[0074] Figure 4 The results showed that the chimeric peptide K-4 was successfully prepared using the above preparation method, with a purity of 95%.

[0075] After sequencing, the amino acid sequence of the chimeric peptide K-4 is KFERQYRR-2Nal-G.

[0076] Example 5

[0077] A chimeric peptide K-9 that degrades CXCR4, wherein the amino acid sequence of K-9 is KFERQGGP D YR D VGR- D 2Nal-P (SEQ ID NO.4- D YR DVGR- D 2Nal-P), where D Y、 D V and D 2Nal-P represent D-tyrosine, D-valine, and D-2-naphthylproline, respectively.

[0078] Synthesis of chimeric peptide K-9: Synthesized using the standard Fmoc solid-phase peptide synthesis method (SPPS). In simple terms, 0.5 mol of dichloromethane resin was dissolved in 15 mL of dichloromethane (DCM) and added to a solid-phase tube. After swelling for 10 min, it was extruded using a suction bulb. 1 mol of Fmoc-protected D-2-naphthylproline (Fmoc- D 2Nal-P-OH was dissolved in 15 mL of N,N-dimethylformamide (DMF), and 330 μL of N,N-diisopropylethylamine (DIPEA) was added to adjust the pH. The reaction was carried out at room temperature for 2 h. Subsequently, the solvent was squeezed out using a bulb syringe, and the mixture was washed five times each with DCM and DMF, 1 min each time. The Fmoc protecting group was cleaved with a DMF solution containing 20% ​​piperidine for 30 min, and then the solvent was squeezed out and washed five times with DMF, 1 min each time. The second amino acid, 1 mol Fmoc-protected arginine (Fmoc-Arg-OH), was dissolved in 15 mL of DMF along with 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU). After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. Subsequent amino acids were added sequentially: 1 mol Fmoc-protected glycine, D-valine, arginine, D-tyrosine, proline, glycine, glycine, glutamine, arginine, glutamic acid, phenylalanine, and lysine, each dissolved in 1 mol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) along with 1 mol of DMF. After adjusting the pH with DIPEA, the solution was added to a solid-phase tube and reacted for 2 h. Finally, the peptide was cleaved for 45 min using a cleavage buffer of 95% trifluoroacetic acid (TFA), 2.5% trimethylsilane (TIS), and 2.5% water. The cleavage buffer was collected and evaporated to dryness using a rotary evaporator. Anhydrous diethyl ether was then added to precipitate KFERQGGP. D YR D VGR- D 2Nal-P polypeptide.

[0079] The chimeric peptide K-9 was detected by HPLC and MS, respectively. The results are shown in the figure. Figure 5 .

[0080] Figure 5 The results showed that the chimeric peptide K-9 was successfully prepared using the above preparation method, with a purity of 95%.

[0081] After sequencing, the amino acid sequence of the chimeric peptide K-9 is KFERQGGP. D YR D VGR- D 2Nal-P.

[0082] The chemical structures of the chimeric peptides K-6, K-8, K-17, K-4, and K-9 prepared in Examples 1-5 are shown in the figure below. Figure 6 .

[0083] Example 6

[0084] Examples 1-5 show the functional verification of the prepared chimeric peptides K-6, K-8, K-17, K-4, and K-9.

[0085] (1) Effects of peptides K-6, K-8, K-17, K-4 and K-9 on the degradation ability of CXCR4

[0086] 2×10 5 RM-1 mouse prostate cancer cells were seeded per well in 6-well plates and cultured at 37°C and 5% CO2 for 12 h. Then, peptides K-6, K-8, K-17, K-4 and K-9 were added to a final concentration of 100 μM and incubated at 37°C and 5% CO2 for 18 h. Total protein was extracted and subjected to Western blotting to verify the degradation ability of different peptides on CXCR4.

[0087] like Figure 7 As shown, K-6, K-8, K-17, K-4 and K-9 prepared in this invention can all degrade CXCR4 protein in RM-1 cells, with K-9 showing the best degradation effect.

[0088] (2) Study on the effects of peptides K-6, K-8, K-17, K-4 and K-9 on the migration and invasion ability of prostate cancer cells

[0089] Cell scratch assay: RM-1 cells at a density of 200,000 cells / well were seeded into 6-well plates. Once the cells reached 80% confluence, uniformly sized, cell-free regions were physically scratched at the bottom of each well. After washing away suspended cell debris, complete culture media containing PBS (Control), K-6, K-8, K-17, K-4, and K-9 (the final concentrations of K-6, K-8, K-17, K-4, and K-9 in the complete culture media were all 100 μM) were added. The plates were incubated at 37°C and 5% CO2 for 24 h. Images of each group were taken under a microscope at 0 h and 24 h, and quantitative analysis was performed using ImageJ. The area of ​​the cell-free region was calculated using ImageJ software. The lateral migration rate was obtained by dividing the area at 24 h by the area at 0 h.

[0090] Cell migration assay: RM-1 cells at a density of 50,000 cells / well were seeded into transwell chambers of 24-well plates. PBS (Control), K-6, K-8, K-17, K-4, and K-9 were added (the final concentrations of K-6, K-8, K-17, K-4, and K-9 were all 100 μM), and the cells were incubated for 18 h. Following this, crystal violet staining was performed for 30 min, and the cells were washed multiple times with light PBS buffer. The chambers were then inverted and the number of cells that migrated through the chambers was observed under a microscope. Quantitative analysis was performed using ImageJ. The number of cells per field of view was obtained using ImageJ, with the Control group as 100%. The percentage of cells in the remaining groups divided by the number of cells in the Control group was used to determine the longitudinal migration rate.

[0091] Cell invasion assay: RM-1 cells at a density of 50,000 cells / well were seeded into transwell chambers containing matrix gel in 24-well plates. PBS (Control), K-6, K-8, K-17, K-4, and K-9 were added (the final concentrations of K-6, K-8, K-17, K-4, and K-9 were all 100 μM), and the cells were incubated for 18 h. Following this, crystal violet staining was performed for 30 min, and the cells were washed multiple times with light PBS buffer. The chambers were then inverted and the number of cells that had penetrated the chambers was observed under a microscope. Quantitative analysis was performed using ImageJ. The cell count per field of view was obtained using ImageJ, with the Control group representing 100%. The percentage of cells in the remaining groups divided by the Control group cell count was the invasion rate.

[0092] like Figure 8 and Figure 9 As shown, except for K-6, peptides K-8, K-17, K-4 and K-9 can significantly inhibit the migration and invasion of RM-1 cells, with K-9 showing the best inhibitory effect, consistent with the trend of CXCR4 degradation level.

[0093] (2) Study on the effects of peptides K-6, K-8, K-17, K-4 and K-9 on the inhibitory ability of prostate cancer cells

[0094] RM-1 prostate cancer cells at 3000 cells / well were seeded into 96-well plates and cultured for 12 h at 37°C and 5% CO2. Then, different final concentrations (0, 10, 25, 50, 100, and 200 μM) of peptides K-6, K-8, K-17, K-4, and K-9 were added and incubated for 24 h at 37°C and 5% CO2. The control group was incubated for 24 h at 37°C and 5% CO2 with the same concentration of PBS. Cytotoxicity was detected using the CCPK-8 assay.

[0095] like Figure 10As shown, K-6, K-8, K-17, K-4 and K-9 can all significantly inhibit prostate cancer cells. K-9 can significantly inhibit the proliferation of prostate cancer cells in a concentration-dependent manner.

[0096] (3) Study on the mechanism of CXCR4 degradation by chimeric peptides

[0097] This embodiment investigates the mechanism of CXCR4 degradation through K-9 treatment.

[0098] RM-1 cells at a density of 200,000 cells / well were seeded into 6-well plates and cultured at 37°C and 5% CO2 for 12 hours. The cells were then divided into four groups: Control group, K-9 group, K-9+QX77 group, and K-9+VER155008 group, with three replicates per group. In the K-9 group, RM-1 cells were cultured for another 4 hours, followed by incubation with 100 μM K-9 at 37°C and 5% CO2 for 18 hours. In the K-9+QX77 group, RM-1 cells were treated with 10 μM lysosomal autophagy agonist QX77 for 24 hours, followed by incubation with 100 μM K-9 at 37°C and 5% CO2 for 18 hours. The K-9+VER155008 group involved treating RM-1 cells with the lysosomal autophagy inhibitor VER155008 at a final concentration of 0.1 μM for 4 h, followed by incubation with K-9 at a final concentration of 100 μM at 37℃ and 5% CO2 for 18 h. The control group consisted of RM-1 cells cultured without any drug treatment for 22 h. Total protein was extracted from each group after treatment, and Western blotting was performed to detect CXCR4 expression levels and verify the mechanism of CXCR4 degradation.

[0099] like Figure 11 As shown, after pretreatment with the lysosomal autophagy agonist QX77, the CXCR4 degradation capacity of the K-9+QX77 group increased compared to the K-9 group; while after pretreatment with the lysosomal autophagy inhibitor VER155008, the CXCR4 degradation capacity of the K-9+VER155008 group decreased compared to the K-9 group. This indicates that the CXCR4-degrading chimeric peptides of the present invention (such as K-9) degrade CXCR4 via the lysosomal autophagy pathway.

[0100] In summary, the chimeric peptide that degrades CXCR4 of the present invention can degrade CXCR4 through lysosomal autophagy, thereby effectively inhibiting tumor migration and invasion, and even effectively inhibiting tumor proliferation.

[0101] 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. A chimeric peptide degrading CXCR4, characterized in that, The amino acid sequence of the chimeric peptide is KFERQRFFESH.

2. The use of the chimeric peptide according to claim 1 in the preparation of prostate cancer drugs.

3. Use according to claim 2, characterized in that, The drug is used to target and degrade CXCR4 on tumor cell membranes.

4. Use according to claim 2, characterized in that, The drug is used to degrade the CXCR4 protein in prostate cancer cells.

5. Use according to claim 2, characterized in that, The drug is used to inhibit the migration and invasion of prostate cancer cells.

6. The application according to claim 2, characterized in that, The drug is used to inhibit the proliferation of prostate cancer cells.

7. A drug for treating prostate cancer, characterized in that, The drug comprises the chimeric peptide that degrades CXCR4 as described in claim 1.

8. The medicament according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The drug according to claim 7, characterized in that, The drug also includes lysosomal autophagy agonists and / or inhibitors.

10. The medicament according to claim 9, characterized in that, The final concentration of the autophagy agonist and / or inhibitor is not less than 0.1 μM.

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