Polypeptide for treating or preventing cancer invasion and cancer metastasis and application thereof

Abstract

The invention discloses a polypeptide. The polypeptide refers to a peptide fragment containing a co-crystallization site of non-muscle myosin (Myosin IIA) and S100A4 and an allosteric peptide of the peptide fragment. The polypeptide contains amino acids from the 1894th site to the 1924th site of non-muscle myosin IIA, and the nitrogen terminal of the polypeptide is acylated by using C10-C16 acid; the polypeptide disclosed by the invention has an action mechanism for competitively inhibiting the physiological activity of S100A4, stays at a primary part by controlling the development of tumor cells, and has an excellent inhibiting effect on invasion and metastasis of cancers.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more specifically, to a polypeptide for treating or preventing cancer invasion and cancer metastasis and its uses. Background Art

[0002] Cancer, also known as malignant tumor, is an important worldwide health problem, and its mortality rate is second only to cardiovascular and cerebrovascular diseases. The main reason is the uncontrolled growth and metastasis of tumor cells, which then leads to the failure of the main organ functions and even death. The most common cancers in men are prostate cancer, colorectal cancer, lung cancer and bladder cancer; the most common cancers in women are breast cancer, colorectal cancer and lung cancer.

[0003] One of the main causes of cancer death is the metastasis of cancer cells, which is directly related to the cancer development process, anti-cancer treatment resistance and poor patient survival. Tumor metastasis is a complex process involving multiple pathogenic steps. In recent years, the research on the potential molecular mechanisms of cancer metastasis has attracted particular attention from cancer researchers. Many metastasis-related proteins have been found to participate in the related processes. S100A4, a calcium-binding protein, is overexpressed in the occurrence and migration of some cancer cells and is one of the cancer cell metastasis-related proteins. The high expression of S100A4 is closely related to various metastatic cancers.

[0004] The S100A4 gene is located on human chromosome 1q21. It is a dimer structure and is a Ca2+-binding protein with an EF double helix domain. At the molecular level, S100A4 is considered a Ca 2+ activation switch. Ca 2+ loading enables it to bind to target proteins, including non-muscle myosin (Myosin IIA), Annexin A2, tumor suppressor gene p53, advanced glycation end products (RAGE), intracellular molecular target liprin-β1, and intracellular molecular target Ezrin. The interaction between S100A4 and Myosin IIA can prevent oligomerization and inhibit the assembly of Myosin IIA filaments, promoting filament disassembly. The research results show that S100A4 can regulate cell polarization during directed movement through its interaction with Myosin IIA. Thereby leading to enhanced cell motility and invasiveness and playing a positive role in cell adhesion in metastatic tumor cells.

[0005] In a variety of malignant tumors, such as breast cancer, lung cancer, ovarian cancer, urological malignancies, etc., S100A4 has overexpression and is accompanied by cancer metastasis. Therefore, inhibiting S100A4 can effectively inhibit the development and metastasis of tumor cells.

[0006] Before the present invention, there have not been many drug studies targeting S100A4 for cancer intervention. After the Wnt signaling pathway is activated, β-catenin accumulates in the cell and enters the nucleus to transcribe the S100A4 gene. S100A4 transcriptional inhibitors discovered based on inhibiting the Wnt / β-catenin signaling pathway are the only type of inhibitors, including sulindac, calcimycin, and niclosamide. Commonly used cancer treatment drugs in clinical practice are very limited in terms of selectivity and effectiveness.

[0007] The main difference between the present invention and existing treatment methods is that, different from traditional cancer radiotherapy and chemotherapy that eliminate tumors, it controls the development of tumor cells and makes them stay at the primary site, becoming a controllable non-fatal chronic disease, which can greatly extend the life expectancy and quality of life of patients. In addition, such polypeptides can be used as supplementary drugs for transcriptional inhibitors to double-block the role of S100A4 in cancer metastasis and development, so that cancer can be better controlled.

[0008] Before the present invention, no drugs with the treatment mechanism of inhibiting cancer metastasis have been marketed, and no reports or patents have been found on the mechanism of action based on the binding target protein Myosin IIA of the cancer metastasis-related protein S100A4. By analyzing the co-crystallization site of non-muscle myosin (Myosin IIA) and S100A4, a peptide segment containing the co-crystallization site is intercepted and the effective site is mutated to obtain its allosteric peptide, which can competitively bind to S100A4 and effectively inhibit the biological activities of non-muscle myosin (Myosin IIA), Annexin A2, tumor suppressor gene p53, advanced glycation end product (RAGE), intracellular molecular target liprin-β1, and intracellular molecular target Ezrin with S100A4 in vivo, thereby significantly reducing the growth and metastasis of cancer cells. Summary of the Invention

[0009] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is the defect that existing cancer treatments cannot effectively inhibit the invasion and metastasis of cancer cells.

[0010] The object of the present invention is to provide a use of a novel polypeptide targeting the cancer metastasis-related protein S100A4 in drugs for treating and preventing cancer metastasis, so as to provide more drug options for treating and preventing cancer metastasis. The object of the present invention is achieved by the following technical solutions: A use of a novel polypeptide targeting the cancer metastasis-related protein S100A4 in anti-cancer drugs for treating and preventing the growth and metastasis of cancer cells is provided. The aim is to analyze the co-crystallization site of non-muscle myosin (Myosin IIA) and S100A4, intercept the peptide segment containing the co-crystallization site and mutate one or several amino acid sites in its amino acid sequence to obtain an allosteric peptide, improve its activity, increase its affinity for the cancer metastasis-related protein S100A4, thereby competitively inhibiting the growth and metastasis of cancer cells, and providing more drug options for cancer treatment.

[0011] In the first aspect of the present invention, a polypeptide is provided, which refers to a peptide segment containing the co-crystallization site of non-muscle myosin (Myosin IIA) and S100A4 and its allosteric peptide;

[0012] Further, the polypeptide contains the amino acids at positions 1894-1924 of non-muscle myosin (Myosin IIA);

[0013] Further, the polypeptide is acylated at its N-terminus with a C10-C16 acid.

[0014] Further, the polypeptide sequence is R`RKLQREX1EDATETADAMNREVX2SLKNKLRR;

[0015] wherein, X1 is selected from one or more of L, I, Y, F, V; X2 is selected from one or more of S, T, E, Q, N, D;

[0016] wherein, R` is selected from one or more of caproyl, lauroyl, myristoyl, palmitoyl;

[0017] Further, the polypeptide is:

[0018] M1: Myristoyl-RKLQRELEDATETADAMNREVSSLKNKLRR; or

[0019] M2: Myristoyl-RKLQRELEDATETADAMNREVESLKNKLRR; or

[0020] M3: Myristoyl-RKLQREFEDATETADAMNREVSSLKNKLRR; or

[0021] M4: Myristoyl-RKLQREFEDATETADAMNREVESLKNKLRR; or

[0022] M5: Lauroyl—RKLQREFEDATETADAMNREVESLKNKLRR; or

[0023] M6: Myristoyl—RKLQREFEDATETADAMNREVESLKNKLRR; or

[0024] M7: Palmitoyl—RKLQREFEDATETADAMNREVESLKNKLRR; or

[0025] M8: RKLQRELEDATETADAMNREVESLKNKLRR; or

[0026] M9: RKLQREFEDATETADAMNREVSSLKNKLRR; or

[0027] M10: RKLQREFEDATETADAMNREVESLKNKLRR;

[0028] Further, for the polypeptide, X1 is Phe (F);

[0029] Further, for the polypeptide, X2 is Glu (E);

[0030] Further, for the polypeptide, R' is myristoyl;

[0031] Further, the polypeptide is: Myristoyl—RKLQREFEDATETADAMNREVESLKNKLRR;

[0032] Further, the polypeptide is prepared by microwave solid-phase synthesis method:

[0033] Using 2-chlorotrityl chloride resin with a loading level of 1.4 mmol / g as the carrier, HATU / HOAt as the condensing agent, adopting Fmoc protection strategy, 20% piperidine / DMF as the deprotecting agent, and HFIP / DCM = 1:4 as the cleavage reagent, the target peptide is synthesized by gradually lengthening the peptide chain;

[0034] In the second aspect of the present invention, there is provided the use of the polypeptide described in the first aspect above in the preparation of a drug for treating and / or preventing cancer and cancer metastasis;

[0035] The cancer metastasis is achieved by the binding of the cancer metastasis-related protein S100A4 on cancer cells to non-muscle myosin (Myosin IIA), Annexin A2, tumor suppressor gene p53, advanced glycation end products (RAGE), intracellular molecular target liprin-β1, and intracellular molecular target Ezrin, realizing the growth, invasion, and metastasis of cancer cells; inhibiting the process of cancer development, and making it stay at the primary site by controlling the development of cancer;

[0036] Further, the cancer includes breast cancer, osteosarcoma, lung cancer, prostate cancer, colorectal cancer, brain cancer, gastric cancer, bladder cancer, pancreatic cancer, melanoma, kidney cancer, thyroid cancer, liver cancer;

[0037] In the third aspect of the present invention, the present invention provides a pharmaceutical composition for treating and / or preventing cancer and cancer metastasis, the pharmaceutical composition comprising the polypeptide described in the first aspect of the present invention above; and a pharmaceutically acceptable carrier;

[0038] Further, the pharmaceutical composition may further include a S100A4 transcription inhibitor found by inhibiting the Wnt / β-catenin signaling pathway;

[0039] The pharmaceutical composition exerts its effect by competitively inhibiting the binding of the cancer metastasis-related protein S100A4 to non-muscle myosin (Myosin IIA), Annexin A2, tumor suppressor gene p53, advanced glycation end products (RAGE), intracellular molecular target liprin-β1, and intracellular molecular target Ezrin, inhibiting the process of cancer development, and making it stay at the primary site by controlling the development of cancer, inhibiting the invasion and metastasis of cancer;

[0040] The control of cancer development specifically refers to the growth and metastasis of cancer cells during the development of cancer diseases;

[0041] Further, the pharmaceutical composition can be prepared into different dosage forms, and the dosage forms are selected from any one of tablets, pills, capsules, granules, microencapsulated tablets, suspensions, dripping pills, oral liquids, injections, aerosols, suppositories or subcutaneous dosage forms;

[0042] Compared with the prior art, the present invention for the first time proposes to use the binding target protein Myosin IIA of the cancer metastasis-related protein S100A4 as a basis, intercept peptide segments by analyzing the co-crystallization sites of non-muscle myosin (Myosin IIA) and S100A4, and mutate its effective sites to increase the allosteric peptide with the affinity for S100A4, which has the activity of competitively binding with S100A4 and effectively inhibiting the biological activities of S100A4 in vivo. Compared with current therapeutic drugs, such novel polypeptides have a mechanism of competitively inhibiting the physiological activities of S100A4, and by controlling the development of tumor cells, making them stay at the primary site, and the treatment theory of becoming a controllable non-fatal chronic disease has not been reported before. Description of the Drawings

[0043] Figure 1 It is the HPLC detection chart of the M1 polypeptide in Example 1 of the present invention;

[0044] Figure 2 It is the mass spectrometry detection chart of the M1 polypeptide in Example 1 of the present invention;

[0045] Figure 3 It is the HPLC detection chart of the M8 polypeptide in Example 1 of the present invention;

[0046] Figure 4 It is the mass spectrometry detection chart of the M8 polypeptide in Example 1 of the present invention;

[0047] Figure 5 It is the column chart of the invasion of A375 cells by the polypeptides M1 - M10 and S100A4 in Example 4 of the present invention;

[0048] Figure 6 It is the column chart of the migration of A375 cells by the polypeptides M1 - M10 and S100A4 in Example 4 of the present invention;

[0049] Figure 7 It is the column chart of the invasion of A375 cells by the polypeptides M1 - M10 in Example 4 of the present invention;

[0050] Figure 8 It is the column chart of the migration of A375 cells by the polypeptides M1 - M10 in Example 4 of the present invention;

[0051] Figure 9 It is the plasma concentration-time curve of M1 in rats in Example 5 of the present invention;

[0052] Figure 10 It is the plasma concentration-time curve of M5 in rats in Example 5 of the present invention. Detailed Embodiments

[0053] All features disclosed in this specification, or steps in all methods or processes disclosed, can be combined in any way, except for mutually exclusive features and / or steps.

[0054] Any feature disclosed in this specification, unless specifically stated, can be replaced by other equivalent or similar-purpose alternative features. That is, unless specifically stated, each feature is just an example in a series of equivalent or similar features.

[0055] Example 1: Taking M1 and M8 as examples, prepare polypeptides and detect them.

[0056] Prepared by microwave solid-phase synthesis method, using 2-chlorotrityl chloride resin with a loading level of 1.4 mmol / g as the carrier, HATU / HOAt as the condensing agent, adopting the Fmoc protection strategy, 20% piperidine / DMF as the deprotecting agent, and HFIP / DCM = 1:4 as the cleavage reagent, and synthesize the target peptide by gradually lengthening the peptide chain according to the sequence order of M1 and M8.

[0057] M1: Myristoyl—RKLQRELEDATETADAMNREVSSLKNKLRR;

[0058] M8: RKLQRELEDATETADAMNREVESLKNKLRR;

[0059] Detect M1 and M8 prepared by HPLC and mass spectrometry. The results are as Figures 1 to 4 shown. It can be known from Figures 1 to 2 that the structure of the prepared M1 polypeptide is correct and the purity is greater than 90%; it can be known from Figures 3 to 4 that the structure of the prepared M8 polypeptide is correct and the purity is greater than 90%.

[0060] Prepare other M1 - M10 polypeptides and detect them by similar operations as above. The obtained polypeptides have correct structures and meet the purity requirements.

[0061] Example 2: Fluorescence polarization (FP) assay for polypeptide affinity test

[0062] Configure the reaction system: 384-well plate, with 21 μL of reaction system in each well, containing 7 μL of 2 μM S100A4, 7 μL of 500 nM fluorescently labeled protein Myosin IIA1894 - 1937, and 7 μL of gradient-diluted test polypeptides M1 - M10. Set up one well containing only the fluorescently labeled protein and PBS buffer and one well without the test polypeptide as quality control wells. Oscillate for 10 min and read the fluorescence polarization values.

[0063] The detection results are shown in Table 1. The polypeptides M1 - M10 of this application bind to S100A4, and the fluorescence polarization IC read during the detection50 The numerical value is less than 8.7 μM, indicating that the polypeptides M1 - M10 of the present application have good affinity with S100A4. Especially for M4 - M6, the IC 50 value is lower than that of other polypeptides, showing more excellent affinity.

[0064] Table 1

[0065]

[0066] Example 3: MTT assay for the inhibitory effect of polypeptides on different cancer cells

[0067] Prepare the reaction system: Taking the A375 cell line as an example, suspend the cell solution containing 3×10⁵ cells in 6 mL of complete medium, then take 100 μL of the suspension per well and spread it on a 96 - well plate, with 6 parallel control groups. Incubate in a CO₂ incubator for 12 h. After the cells are completely adherent, aspirate the cell supernatant, add the medium with different concentrations of the polypeptides M1 - M10 to be tested, and incubate in a CO₂ incubator for 24 h. Add 20 μL / well of the pre - diluted thiazolyl blue solution in the dark, and then continue to incubate for 4 h. Add 150 μL / well of DMSO, measure the absorbance at 490 nm after shaking for 15 min, draw a curve, and obtain the half - maximal inhibitory concentration (IC 50 ) of cancer cells.

[0068] Using the above similar operations, detect the inhibitory activities of the peptides M1 - M10 of the present application on HepG - 2 cells, HCT - 116 cells, and 231 cells;

[0069] The results are shown in Table 2 below. The half - maximal inhibitory concentrations (IC 50 ) of the polypeptides M1 - M10 of the present application on A375 cells, HepG - 2 cells, HCT - 116 cells, and 231 cells are in the range of 4 - 30 μM, all showing excellent inhibitory activities; especially for M4, the IC 50 value on each type of cell is lower than 6 μM, much lower than that of other polypeptides, showing more excellent cell inhibitory activity.

[0070] Table 2

[0071]

[0072] Example 4: Invasion and migration inhibition assay of malignant melanoma A375 cells:

[0073] (1) Using melanoma cells (A375), Matrigel matrix gel, and DMEM complete medium, a blank control group with only DMEM complete medium added was set up, a positive control group with only S100A4 added was set up, a positive control group with TGF-β added was set up, a control group with Myosin IIA and S100A4 proteins added as a control, and an experimental group with the test polypeptides M1 - M10 and S100A4 protein added was set up. The total volume of the above test system was 100 μL, and DMEM basal medium was used. 600 μL of DMEM complete medium was added below. After culturing in an incubator for 24 hours, the migrated and invaded cells were observed, photographed, and counted under a microscope.

[0074] The results are as Figures 5 to 6 shown. Compared with the blank control group, the positive control group, the protein control group, the number of invaded and migrated malignant melanoma A375 cells in the group containing the polypeptides M1 - M10 of the present application and S100A4 is much lower than that of the control group, showing an excellent effect of inhibiting cancer cell invasion and migration.

[0075] (2) Using melanoma cells (A375), Matrigel matrix gel, and DMEM complete medium, a blank control group with only DMEM complete medium added was set up, a positive control group with only S100A4 added was set up, a positive control group with TGF-β added was set up, a control group with only Myosin IIA added as a control, and an experimental group with only the test polypeptide added was set up. The total volume of the above test system was 100 μL, and DMEM basal medium was used. 600 μL of DMEM complete medium was added below. After culturing in an incubator for 24 hours, the migrated and invaded cells were observed, photographed, and counted under a microscope.

[0076] The results are as Figures 7 to 8 shown. Compared with the blank control group, the positive control group, the protein control group, the number of invaded and migrated malignant melanoma A375 cells in the group containing the polypeptides M1 - M10 of the present application is much lower than that of the control group, showing an excellent effect of inhibiting cancer cell invasion and migration.

[0077] Example 5: Pharmacokinetic Test of Polypeptide in Mice

[0078] Drug preparation: Accurately weigh about 100 mg, measure 1 mL of DMSO and dissolve it by ultrasound, measure 0.1 mL of the stock solution, and mix it with 0.9 mL of pure water in parallel for 6 portions.

[0079] Intraperitoneal administration: Twelve healthy rats weighing 250±10 g were randomly divided into 2 groups, with an equal number of males and females. The rats were fasted for 12 h before administration and allowed free access to water. 1 mL of the drug solution was injected intraperitoneally into each rat, so that the dosage for each group was 0.4 mg / g. Blood was collected from the orbital sinus of the rats before administration and at 5 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h, and 24 h after administration, placed in heparinized biochemical test tubes, and plasma was separated and collected by centrifugation and stored at -20°C.

[0080] Take 100 μL of the internal standard and place it in a 2 mL conical plastic centrifuge tube. Add 50 μL of the plasma to be tested and 100 μL of acetonitrile, vortex for 60 s, centrifuge at 10000 rpm for 10 min, and inject 20 μL of the supernatant for analysis.

[0081] Chromatographic conditions:

[0082] High performance liquid chromatograph:

[0083] Chromatographic column: thermo acclaim C18 250 mm * 4.6 mm, 5 μm

[0084] Mobile phase: Mobile phase A: 0.1% trifluoroacetic acid in water

[0085] Mobile phase B: Acetonitrile

[0086] Mobile phase A: Mobile phase B = 30:70

[0087] Flow rate: 1.0 mL / min Wavelength: 276 nm Column temperature: 30°C Injection volume: 20 μL;

[0088] The results are as Figures 9 to 10 shown in Table 3 and the important pharmacokinetic parameters of the polypeptide in rats;

[0089] Table 3

[0090]

[0091] Pharmacokinetic experiments showed that the half-life of the fatty acid acylated polypeptide inhibitors was effectively prolonged, up to more than 11 hours. As shown in the figure, the half-life of M1 was 10.483 h and that of M5 was 7.265 h, both higher than that of the unmodified polypeptide inhibitors. For example, the half-life of M8 was only 3.873 h. In addition, the time to peak plasma concentration of the fatty acid acylated polypeptide inhibitors was also prolonged compared with that of the unacylated inhibitors, from 1 h to 2 h. The relatively better pharmacokinetic parameters provided a good basis for the distribution and efficacy of the drug in vivo. The relevant metabolic parameters met the basic requirements for drug development.

[0092] The present invention is not limited to the foregoing specific embodiments. The present invention extends to any new feature or any new combination disclosed in this specification, as well as to any new method or process step or any new combination disclosed.

Claims

1. A polypeptide, which refers to a peptide segment containing the co-crystallization site of non-muscle myosin (Myosin IIA) and S100A4 and its allosteric peptide; characterized in that, The polypeptide contains amino acids at positions 1894-1924 of non-muscle myosin (Myosin IIA), and the polypeptide is acylated at its N-terminus with C10-C16 acids.

2. The polypeptide according to claim 1, wherein The polypeptide sequence is R`RKLQREX1EDATETADAMNREVX2SLKNKLRR; wherein, X1 is selected from one or more of L, I, Y, F, V; X2 is selected from one or more of S, T, E, Q, N, D; wherein, R` is selected from one or more of myristoyl, lauroyl, myristoyl, palmitoyl.

3. The polypeptide according to claim 2, wherein The X1 is Phe (F).

4. The polypeptide according to claim 2, wherein The X2 is Glu (E).

5. The polypeptide according to claim 2, wherein The R` is myristoyl.

6. The polypeptide according to claim 2, characterized in that The polypeptide is: M1: Myristoyl-RKLQRELEDATETADAMNREVSSLKNKLRR; or M2: Myristoyl-RKLQRELEDATETADAMNREVESLKNKLRR; or M3: Myristoyl-RKLQREFEDATETADAMNREVSSLKNKLRR; or M4: Myristoyl-RKLQREFEDATETADAMNREVESLKNKLRR; or M5: Lauroyl-RKLQREFEDATETADAMNREVESLKNKLRR; or M6: Myristoyl-RKLQREFEDATETADAMNREVESLKNKLRR; or M7: Palmitoyl-RKLQREFEDATETADAMNREVESLKNKLRR; or M8: RKLQRELEDATETADAMNREVESLKNKLRR; or M9: RKLQREFEDATETADAMNREVSSLKNKLRR; or M10: RKLQREFEDATETADAMNREVESLKNKLRR.

7. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating and / or preventing cancer and cancer metastasis, characterized in that, The cancers include breast cancer, osteosarcoma, lung cancer, prostate cancer, colorectal cancer, brain cancer, gastric cancer, bladder cancer, pancreatic cancer, melanoma, kidney cancer, thyroid cancer, liver cancer.

8. A pharmaceutical composition for treating and / or preventing cancer and cancer metastasis, characterized in that, The pharmaceutical composition contains the polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

9. The use according to claim 7 or the pharmaceutical composition according to claim 8, characterized in that The cancer metastasis is that the cancer metastasis-related protein S100A4 on cancer cells binds to non-muscle myosin (Myosin IIA), Annexin A2, tumor suppressor gene p53, advanced glycation end products (RAGE), intracellular molecular target liprin-β1, and intracellular molecular target Ezrin, to achieve the growth, invasion, and metastasis of cancer cells; inhibit the process of cancer development, and stay at the primary site by controlling the development of cancer.

10. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition can be prepared into different dosage forms, and the dosage forms are selected from any one of tablets, pills, capsules, granules, microcapsule tablets, suspensions, dripping pills, oral liquids, injections, aerosols, suppositories or subcutaneous dosage forms.

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