A peptide that inhibits CK2 in the NCI-H460 cell line

Abstract

The invention discloses a polypeptide that inhibits CK2 in NCI-H460 cell line, belongs to the field of biopharmaceutical technology, includes an amino acid sequence of an inhibitory polypeptide as shown in SEQ ID NO.1; and also includes a method for preparing a polypeptide that inhibits CK2 in NCI-H460 cell line, including the following steps: S1. removing the Fmoc protecting group after pretreatment of Fmoc-Wang amide-MBHA Resin resin; S2. coupling Fmoc-Cys(R1)-OH to the deprotected resin and methylating the terminal Arg carboxyl hydroxyl group; S3. repeating the above S1-S2 from C-terminus to N-terminus to connect the corresponding amino acids to obtain a polypeptide-resin complex; S4. preparing an N-terminal Pro phosphate derivative and coupling it with the polypeptide-resin complex; S5. cleaving the polypeptide-resin complex obtained by solid phase synthesis to release the synthesized inhibitory polypeptide. The problem of abnormal proliferation and spread of tumor cells is solved, and the catalytic activity of CK2 is effectively antagonized.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a polypeptide which inhibits CK2 in an NCI-H460 cell line. Background Art

[0002] Today, lung cancer remains the second most common cancer and the leading cause of cancer-related death worldwide. Over the past decade, advances in research have led to major breakthroughs in the understanding, diagnosis, and management of this disease, which is now accepted as a heterogeneous entity that can be divided into two major histological types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC comprises approximately 80-85% of lung cancer cases, and is divided into three major histological subtypes based on tissue origin: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (LCLC). The histological diagnosis of NSCLC is crucial to achieve accurate treatment, as the effectiveness of some therapeutic drugs varies depending on tumor histology.

[0003] In the field of lung cancer research, the emergence of new treatment methods and technologies has brought hope for improving patient prognosis. In recent years, research on key molecular targets in non-small cell lung cancer (NSCLC) has been particularly active. Casein kinase 2 (CK2), as a multifunctional serine / threonine protein kinase, plays an important role in the development of various cancers, especially in regulating cell cycle progression, inhibiting cell apoptosis, and promoting tumor angiogenesis. It shows abnormal activity, which makes it a very potential anti-tumor drug target.

[0004] Casein kinase 2 (CK2) is a serine / threonine protein kinase widely present in eukaryotic cells. Studies have shown that it is overexpressed in many types of cancer and is closely related to malignant behaviors such as tumor cell proliferation, survival, invasion and metastasis. In the NCI-H460 cell line (derived from human lung squamous cell carcinoma), CK2 may also have similar abnormal expression and enhanced activity. Given the key role of CK2 in tumor cells, it is considered an attractive anti-tumor drug target. By inhibiting the activity of CK2, it is possible to prevent the abnormal proliferation and spread of cancer cells, thereby opening up new avenues for cancer treatment.

[0005] Peptide inhibitors tend to have higher specificity and lower side effects. Compared with small molecule inhibitors, peptides can better simulate the three-dimensional spatial conformation of protein interactions, accurately identify and bind to the active site of CK2, and thus achieve effective inhibition. The NCI-H460 cell line is a commonly used in vitro experimental model for studying the pathogenesis and drug screening of lung cancer and other related diseases. Verifying the effect of CK2 inhibitors in this cell line can preliminarily evaluate the potential role of the peptide inhibitor in the treatment of lung cancer and accumulate scientific evidence for further in vivo experiments and clinical applications.

[0006] Therefore, developing a peptide that can effectively inhibit CK2 activity in the NCI-H460 cell line can not only provide a deep understanding of the specific mechanism of CK2 in the occurrence and development of lung cancer, but also has the potential to develop new anti-tumor drugs and improve the treatment status of lung cancer patients. Summary of the invention

[0007] To this end, the present invention provides a polypeptide that inhibits CK2 in the NCI-H460 cell line to solve the above problems.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] According to a first aspect of the present invention, a polypeptide that inhibits CK2 in the NCI-H460 cell line is provided, and the amino acid sequence of the inhibitory polypeptide is shown in SEQ ID NO.1.

[0010] Furthermore, the Pro at the N-terminus of the polypeptide is phosphate-modified, and the hydroxyl group in the carboxyl group in the Arg structure at the C-terminus is replaced by a methyl group.

[0011] According to a second aspect of the present invention, there is provided a method for preparing a polypeptide that inhibits CK2 in an NCI-H460 cell line, comprising the following steps:

[0012] S1. Pre-treat Fmoc-Wang amide-MBHA Resin to remove Fmoc protecting group;

[0013] S2. coupling Fmoc-Cys(R1)-OH to the deprotected resin and methylating the terminal Arg carboxyl hydroxyl group;

[0014] S3. Repeat the above S1-S2 from the C-terminus to the N-terminus to connect the corresponding amino acids to obtain a polypeptide-resin complex;

[0015] S4. Preparation of N-terminal Pro phosphate derivatives and coupling with peptide-resin complexes;

[0016] S5. Cleavage of the peptide-resin complex obtained by solid phase synthesis to release the synthesized inhibitory peptide.

[0017] Furthermore, the specific steps of S1 include:

[0018] (1) Resin pretreatment: Use DCM and NMP to swell the Fmoc-Wang amide-MBHA Resin;

[0019] (2) Fmoc deprotection: The swollen resin was mixed with a piperidine / NMP solution containing 0.2 M 6-Cl-HOBT, stirred in a constant temperature water bath at 50-60°C for 15-30 min, cooled and filtered to remove the solution, and then mixed with a new piperidine / NMP solution containing 0.2 M 6-Cl-HOBT, placed in a constant temperature water bath, stirred at 50-60°C for 20-40 min, and cooled to obtain a resin with the initial Fmoc protecting group removed.

[0020] Furthermore, the specific steps of S2 are:

[0021] (1) using a mixed solution of NMP containing Fmoc-Cys(R1)-OH, HBTU, HOBT and DIPEA as a promoting liquid, mixing it with the resin from which the Fmoc protecting group has been removed, performing a third water bath reaction, filtering and removing the reaction solution after cooling, and obtaining a resin containing the Fmoc-Cys(R1) residue after washing;

[0022] (2) Mix the resin and CH3I in a molar ratio of 1:(5-10), then add C6H 15 N, and then place the mixture in an inert gas and react at 0-4°C for 12-24h. After the reaction is completed, wash the resin with appropriate amounts of DMF, DCM and methanol to remove unreacted reagents.

[0023] Furthermore, the specific steps of S4 are:

[0024] (1) Under anhydrous and oxygen-free conditions, Fmoc-Pro-OH is taken, DCC and C2H7O4P are added, and then the reaction system is fully stirred at room temperature under nitrogen protection to generate Fmoc-Pro-PO(OEt)2;

[0025] (2) Purify Fmoc-Pro-PO(OEt)2 and use the purified Fmoc-Pro-PO(OEt)2 as the N-terminal amino acid unit and HBTU / HOBT / DIPEA as coupling reagents to perform an amide bond coupling reaction with the prepared polypeptide-resin complex, so that Fmoc-Pro-PO(OEt)2 forms an amide bond with the C-terminal residue on the complex.

[0026] Furthermore, the specific steps of S5 are:

[0027] The coupled peptide-resin complex was mixed with the cleavage agent Reagent K, shaken at 0°C for 30 minutes, and then the reaction was continued for 3 hours. After filtration, the filtrate was mixed with C4H8O2, and the precipitate was collected to obtain the crude product of the inhibitory peptide.

[0028] Furthermore, after obtaining the crude product of the inhibitory polypeptide in S5, liquid chromatography purification is also included, and the chromatographic conditions include: C18 reverse phase column; mobile phase A: 0.10% formic acid aqueous solution and methanol by volume percentage; mobile phase B: 0.1% TFA / acetonitrile by volume percentage; mobile phase gradient: mobile phase B20-80%, 30min; flow rate is 500mL / min, detection wavelength is 220nm, main peak is collected in segments, and segmented purified liquids with any single impurity not exceeding 0.5% are combined, and unqualified segments are combined and concentrated and then purified repeatedly until the purified liquid is qualified.

[0029] Further, the tumor is selected from large cell carcinoma.

[0030] According to the third aspect of the present invention, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition include an inhibitory polypeptide or a salt of an inhibitory polypeptide prepared by the preparation method according to any one of claims 3 to 9 and a pharmaceutically acceptable carrier.

[0031] The present invention has the following advantages:

[0032] The present invention proposes a polypeptide that can inhibit CK2 in the NCI-H460 cell line, which is unique in that it can specifically act on the overactive CK2 in the NCI-H460 cell line. The polypeptide of the present application can efficiently bind tightly to the active site of CK2, thereby effectively antagonizing the catalytic activity of CK2. In this way, the polypeptide of the present application can systematically interfere with multiple oncogenic signaling pathways driven by CK2, thereby producing the effects of inhibiting tumor cell proliferation, migration and promoting cell apoptosis. This invention provides a new candidate drug molecule for the treatment of lung cancer, which is expected to improve existing treatment options and improve patient prognosis by targeting CK2 activity. DETAILED DESCRIPTION

[0033] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The present invention provides a polypeptide that inhibits CK2 in the NCI-H460 cell line, comprising the amino acid sequence of the inhibitory polypeptide as shown in SEQ ID NO.1;

[0035] Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Lys-Leu-Pro-Ile-Lys-Gly-Ala-Glu-His-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg.

[0036] The Pro at the N-terminus of the polypeptide is phosphate-modified, and the hydroxyl group in the carboxyl group in the Arg structure at the C-terminus is replaced by a methyl group.

[0037] The present invention provides a method for preparing a polypeptide that inhibits CK2 in an NCI-H460 cell line, comprising the following steps:

[0038] S1. Pre-treat Fmoc-Wang amide-MBHA Resin to remove Fmoc protecting group;

[0039] S2. coupling Fmoc-Cys(R1)-OH to the deprotected resin and methylating the terminal Arg carboxyl hydroxyl group;

[0040] S3. Repeat the above S1-S2 from the C-terminus to the N-terminus to connect the corresponding amino acids to obtain a polypeptide-resin complex;

[0041] S4. Preparation of N-terminal Pro phosphate derivatives and coupling with peptide-resin complexes;

[0042] S5. Cleavage of the peptide-resin complex obtained by solid phase synthesis to release the synthesized inhibitory peptide.

[0043] Among them, the specific steps of S1 include:

[0044] (1) Resin pretreatment: Use DCM and NMP to swell the Fmoc-Wang amide-MBHA Resin;

[0045] (2) Fmoc deprotection: The swollen resin was mixed with a piperidine / NMP solution containing 0.2 M 6-Cl-HOBT, stirred in a constant temperature water bath at 50-60°C for 15-30 min, cooled and filtered to remove the solution, and then mixed with a new piperidine / NMP solution containing 0.2 M 6-Cl-HOBT, placed in a constant temperature water bath, stirred at 50-60°C for 20-40 min, and cooled to obtain a resin with the initial Fmoc protecting group removed.

[0046] Among them, the specific steps of S2 are:

[0047] (1) using a mixed solution of NMP containing Fmoc-Cys(R1)-OH, HBTU, HOBT and DIPEA as a promoting liquid, mixing it with the resin from which the Fmoc protecting group has been removed, performing a third water bath reaction, filtering and removing the reaction solution after cooling, and obtaining a resin containing the Fmoc-Cys(R1) residue after washing;

[0048] (2) The resin obtained in (1) was mixed with CH3I in a molar ratio of 1:8, and then CH 15 N mixed, C6H 15 The molar ratio of N to CH3I was 1:1. The mixture was placed in nitrogen and reacted at 4°C for 24 h. After the reaction was completed, the resin was washed with appropriate amounts of DMF, DCM and methanol to remove unreacted reagents.

[0049] Among them, the specific steps of S4 are:

[0050] (1) Under anhydrous and oxygen-free conditions, Fmoc-Pro-OH is taken, DCC is added as a dehydrating agent and C2H7O4P is added as a phosphating agent, and then the reaction system is fully stirred at room temperature under nitrogen protection to generate Fmoc-Pro-PO(OEt)2;

[0051] (2) Purify Fmoc-Pro-PO(OEt)2 and use the purified Fmoc-Pro-PO(OEt)2 as the N-terminal amino acid unit and HBTU / HOBT / DIPEA as coupling reagents to perform an amide bond coupling reaction with the prepared polypeptide-resin complex, so that Fmoc-Pro-PO(OEt)2 forms an amide bond with the C-terminal residue on the complex.

[0052] Among them, the specific steps of S5 are:

[0053] The coupled peptide-resin complex was mixed with the cleavage agent Reagent K, shaken at 0°C for 30 minutes, and then the reaction was continued for 3 hours. After filtration, the filtrate was mixed with C4H8O2, and the precipitate was collected to obtain the crude product of the inhibitory peptide.

[0054] Among them, after obtaining the crude product of the inhibitory polypeptide in S5, it also includes liquid chromatography purification, and the chromatographic conditions include: C18 reverse phase column; mobile phase A: 0.10% formic acid aqueous solution and methanol by volume, mobile phase B: 0.1% TFA / acetonitrile by volume; mobile phase gradient: mobile phase B20-80%, 30min; flow rate is 500mL / min, detection wavelength is 220nm, the main peak is collected in segments, and the segmented purified liquid with any single impurity not exceeding 0.5% is combined, and the unqualified segments are combined and concentrated and then purified repeatedly until the purified liquid is qualified.

[0055] Specifically, the tumor is selected from large cell carcinoma.

[0056] The present invention provides a pharmaceutical composition, wherein the active ingredients of the pharmaceutical composition include the inhibitory polypeptide or the inhibitory polypeptide salt prepared by the above preparation method and a pharmaceutically acceptable carrier.

[0057] Embodiment 1:

[0058] Preparation of a peptide that inhibits CK2 in NCI-H460 cell line;

[0059] S1. Pre-treat Fmoc-Wang amide-MBHA Resin to remove Fmoc protecting group;

[0060] S2. coupling Fmoc-Cys(R1)-OH to the deprotected resin and methylating the terminal Arg carboxyl hydroxyl group;

[0061] S3. Repeat the above S1-S2 from the C-terminus to the N-terminus to connect the corresponding amino acids to obtain a polypeptide-resin complex;

[0062] S4. Preparation of N-terminal Pro phosphate derivatives and coupling with peptide-resin complexes;

[0063] S5. Cleavage of the peptide-resin complex obtained by solid phase synthesis to release the synthesized inhibitory peptide.

[0064] The specific steps of S1 are:

[0065] (1) Resin pretreatment: Take 10 mL of DCM to swell the Fmoc-Wang amide-MBHA Resin for 30 min, remove DCM by filtration, and then swell with 10 mL of NMP for 30 min, rinse and set aside;

[0066] (2) Fmoc deprotection: The swollen resin was mixed with 8 mL of a 30% piperidine / NMPP (v / v) solution containing 0.2 M 6-Cl-HOBT, stirred in a 50° C. constant temperature water bath for 30 min, cooled and filtered to remove the solution, then mixed with a new 30% piperidine / NMP solution containing 0.2 M 6-Cl-HOBT for 5 min, placed in a constant temperature water bath, stirred at 60° C. for 40 min, and cooled to obtain a resin with the initial Fmoc protecting group removed.

[0067] The specific steps of S2 are:

[0068] (1) 15 mL of a mixed solution of 0.05 mmol Fmoc-Cys(R1)-OH, 0.05 mmol HBTU, 0.05 mmol HOBT and 0.05 mmol DIPEA in NMP was taken as a promoter solution, and then the promoter solution was mixed with the resin from which the Fmoc protecting group was removed, and a third water bath reaction was carried out, and the mixture was stirred at 60° C. for 60 min. After cooling, the reaction solution was filtered to remove the reaction solution, and the resin containing the Fmoc-Cys(R1) residue was obtained after washing;

[0069] (2) The resin and CH3I were mixed in a molar ratio of 1:8, and then CH 15 N mixed, C6H 15 The molar ratio of N to CH3I was 1:1. The obtained mixture was placed in nitrogen and reacted at 4°C for 24 hours. After the reaction was completed, the resin was washed with appropriate amounts of DMF, DCM and methanol to remove unreacted reagents.

[0070] The specific steps of S4 are:

[0071] (1) Under anhydrous and oxygen-free conditions, Fmoc-Pro-OH is added with DCC as a dehydrating agent and C2H7O4P as a phosphating agent, and then the reaction system is fully stirred at room temperature under nitrogen protection to generate Fmoc-Pro-PO(OEt)2.

[0072] (2) Purify Fmoc-Pro-PO(OEt)2 and use the purified Fmoc-Pro-PO(OEt)2 as the N-terminal amino acid unit and HBTU / HOBT / DIPEA as coupling reagents to perform an amide bond coupling reaction with the prepared polypeptide-resin complex, so that Fmoc-Pro-PO(OEt)2 forms an amide bond with the C-terminal residue on the complex.

[0073] The specific steps of S5 are:

[0074] The coupled peptide-resin complex was mixed with 12 mL of cleavage agent Reagent K, shaken at 0°C for 30 min, and then reacted for 3 h. After filtration, the filtrate was mixed with C4H8O2 in a volume ratio of 2:1, and the precipitate was collected to obtain a crude inhibitory peptide.

[0075] Peptide purification:

[0076] The crude polypeptide was dissolved in 50% acetonitrile / water and purified by preparative liquid chromatography. The chromatographic conditions were: C18 reverse phase column (300mm×25mm, 10μm); mobile phase A: 0.10% formic acid aqueous solution and methanol (v / v), mobile phase B: 0.1% TFA / acetonitrile (v / v); mobile phase gradient: 20-80% mobile phase, 30min; flow rate 500mL / min detection wavelength 220nm. The main peak was collected in sections, and the purified solutions with any single impurity not exceeding 0.5% were combined. The unqualified sections were combined and concentrated, and then purified repeatedly until the purified solution was qualified. The collected solution was lyophilized to obtain 50mg of pure product.

[0077] Example 2: Evaluation of the targeting affinity of inhibitory peptides for CK2 in the NCI-H460 cell line

[0078] 1. Cell culture and treatment: NCI-H460 was originally from the American type culture specimen (ATCC, Manassas, Virginia, USA). The cell line was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 50 μg / mL gentamicin. The cells were maintained at 37°C and 5% CO2 to maintain good growth. The cells were digested with trypsin at a concentration of 0.05% trypsin + 0.02% EDTA, a single cell suspension was prepared, and dead cells were excluded by trypan blue staining to ensure that only living cells were used for the experiment.

[0079] 2. Quantification of intracellular CK2 protein: The total protein content of NCI-H460 cells was extracted using RIPA lysis buffer, and the content was 9.72 mg / g wet gel. The expression level of CK2 protein in NCI-H460 cells was quantitatively detected using ELISA method using specific anti-CK2 antibody.

[0080] 3. Incubation of inhibitory peptides with cells: Prepare cell culture medium (add 10% (v / v) fetal bovine serum and 50 μg / mL gentamicin) containing different concentration gradients with initial concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μM of the compound and the control Scr peptide, respectively, to ensure that the final concentration of DMSO has no obvious toxicity to the cells.

[0081] The cells were inoculated in a flat-bottom 96-well plate (6000 cells / mL) with multiple replicates for each concentration. After 24 hours or after the cells adhered to the wall, the culture medium containing different concentrations of inhibitory peptides was replaced and incubated at 37°C and 5% CO2 for 24 hours to ensure that the inhibitory peptides were fully in contact with the intracellular CK2 and reached adsorption equilibrium.

[0082] 4. Cell lysis and inhibitory peptide concentration determination:

[0083] (1) After the incubation, carefully collect the cells and lyse them to release the intracellular inhibitory peptides. Lyse the cells in the same way as in step 2 to ensure that CK2 and its bound inhibitory peptides are fully released. Then, remove the cell debris and precipitate by centrifugation and collect the supernatant.

[0084] (2) The concentration of the remaining inhibitory peptide in the supernatant of each concentration group was measured at a wavelength of 280 nm using a NanoDrop 2000 ultra-micro spectrophotometer, and untreated blank cells (without inhibitory peptide added) were recorded as a control.

[0085] 5. Data analysis: The inhibitory peptide binding data of each concentration group were statistically analyzed. The nonlinear fitting tool in the Origin software was used to fit the Langmuir isotherm adsorption equation to calculate the maximum static adsorption value Qmax (nmol / cell number or nmol / total protein amount) and equilibrium dissociation constant Kd (μmol / L) of the example compound and the control Scr peptide in NCI-H460 cells.

[0086] Table 1 Static adsorption maximum and equilibrium dissociation constant of Example compounds and control Scr

[0087] Group Qmax(nmol / g) Kd(umol / L) Scr small peptide 15.48±0.62 5.32±0.43 Example 1 97.43±0.57 <![CDATA[0.05±0.02 ** ]]>

[0088] Note: * P≤0.05 and ** P≤0.01 is the Student's t test result relative to the Scr peptide control group.

[0089] in conclusion:

[0090] 1. The maximum static adsorption value Qmax (nmol / cell number or nmol / total protein amount) of the polypeptide prepared in this example in NCI-H460 cells is significantly higher than the Qmax value of the Scr small peptide in the control group. The Qmax value of the polypeptide prepared in this example is 97.43±0.57nmol / g, while the Qmax value of the Scr small peptide in the control group is 15.48±0.62nmol / g.

[0091] 2. The equilibrium dissociation constant Kd (μmol / L) of the polypeptide prepared in this example is significantly lower than the Kd value of the Scr peptide in the control group. The Kd value of the example compound is 0.05±0.02μmol / L, while the Kd value of the Scr peptide in the control group is 5.32±0.43μmol / L.

[0092] Taking into account the Qmax and Kd values, the polypeptide prepared in this example has stronger binding ability and higher affinity, and has significant targeting affinity and specificity.

[0093] Example 3: Evaluation of in vitro antitumor activity

[0094] 1. Cell inoculation and inhibitory polypeptide incubation: NCI-H460 cells in the logarithmic growth phase were inoculated in a 96-well culture plate at a density of 6000 cells / mL and cultured for 24 hours to allow them to adhere to the wall.

[0095] 2. Cell viability detection: Prepare inhibitory peptide solutions with different concentration gradients, with initial concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μM, and dilute with low serum medium to ensure no cytotoxicity. Add the prepared inhibitory peptide solution to NCI-H460 cells cultured for 24 hours, with at least three replicates for each concentration, and set up a control group without inhibitory peptide. Continue incubation for 48 hours to allow the inhibitory peptide to fully interact with intracellular CK2.

[0096] After the incubation, 20 μl of CCK-8 solution was added to each well, and the incubation was continued for 2 h. The OD value of each well was detected at a wavelength of 450 nm using an enzyme reader, and the data was recorded.

[0097] 3. Data analysis: GraphPad Prism 7.0 software was used to calculate the half inhibitory concentration (IC 50 ), reflecting the degree of inhibition of cell growth by the inhibitory polypeptide.

[0098] Table 2: In vitro cytotoxicity of inhibitory peptides

[0099]

[0100] in conclusion:

[0101] 1. The inhibitory peptide had a significant inhibitory effect on the growth of NCI-H460 cells at different concentrations. As the concentration of the inhibitory peptide increased, the survival rate of the cells decreased, indicating that the inhibitory peptide had anti-tumor activity.

[0102] 2. The inhibitory effects of different concentrations of inhibitory peptides on the growth of NCI-H460 cells vary. At lower concentrations, the inhibitory effect of the inhibitory peptide is weaker, while at higher concentrations, the inhibitory effect is stronger. This difference is related to the concentration of the inhibitory peptide, and a high concentration of the inhibitory peptide can produce a stronger inhibitory effect on cells.

[0103] 3. According to IC 50 IC 50 The lower the value, the stronger the inhibitory effect of the inhibitory peptide on cell growth. 50 The values ​​were between 9.46 and 20.91 μM, indicating that the inhibitory peptide had significant anti-tumor activity.

[0104] Example 4: In vivo anti-tumor activity evaluation

[0105] NCI-H460 cells were inoculated subcutaneously in the right axilla of balb / c nude mice to establish a human lung cancer xenograft model. When the tumor grew to about 50 mm 3 Afterwards, the animals were randomly divided into blank control group and treatment group. The treatment group was intravenously injected with inhibitory peptide, the dosage was set at 5 mg / kg, and the administration volume was 0.5 ml / kg; the blank control group was intravenously injected with an equal volume of normal saline as a control. The drug was administered once every other day. After 14 days of administration, the mice were killed, the tumor was surgically removed and weighed, and the relative tumor volume V (mm 3 ).

[0106] Table 3 In vivo tumor volume (mm 3 )

[0107] Note: * P≤0.05 and ** P ≤ 0.01 is the Student's t test result relative to the blank control.

[0108] in conclusion:

[0109] 1. Inhibitory effect of inhibitory polypeptide on tumor growth: The tumor volume of the drug-treated group (i.e., inhibitory polypeptide group) was significantly smaller than that of the blank control group (i.e., saline control group) from the 1st day to the 13th day after administration, indicating that the inhibitory polypeptide has a significant anti-tumor effect on the human lung cancer xenograft model.

[0110] 2. Dynamic changes of inhibitory effect over time: As can be seen from the table, the tumor volume of the drug-treated group showed a gradual increase from the 1st day to the 13th day after administration, but the growth rate was significantly slower than that of the blank control group, which indicates that the inhibitory polypeptides provided by the present invention can effectively inhibit the growth and proliferation of lung cancer cells in nude mice and have good in vivo anti-tumor activity.

[0111] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A polypeptide that inhibits CK2 in the NCI-H460 cell line, characterized in that: The amino acid sequence of the inhibitory polypeptide is shown in SEQ ID NO.1; The Pro at the N-terminus of the polypeptide is phosphate-modified, and the hydroxyl group in the carboxyl group in the Arg structure at the C-terminus is replaced by a methyl group.

2. A method for preparing a polypeptide that inhibits CK2 in the NCI-H460 cell line, for preparing the polypeptide that inhibits CK2 in the NCI-H460 cell line as claimed in claim 1, characterized in that: The following steps are involved: S1. Pre-treat Fmoc-Wang amide-MBHA Resin to remove Fmoc protecting group; S2. coupling Fmoc-Cys(R1)-OH to the deprotected resin and methylating the terminal Arg carboxyl hydroxyl group; S3. Repeat the above S1-S2 from the C-terminus to the N-terminus to connect the corresponding amino acids to obtain a polypeptide-resin complex; S4. Preparation of N-terminal Pro phosphate derivatives and coupling with peptide-resin complexes; S5. Cleavage of the peptide-resin complex obtained by solid phase synthesis to release the synthesized inhibitory peptide.

3. The preparation method according to claim 2, characterized in that: The specific steps of S1 include: (1) Resin pretreatment: Use DCM and NMP to swell the Fmoc-Wang amide-MBHA Resin; (2) Fmoc deprotection: The swollen resin was mixed with a piperidine / NMP solution containing 0.2 M 6-Cl-HOBT, stirred in a constant temperature water bath at 50-60°C for 15-30 min, cooled and filtered to remove the solution, and then mixed with a new piperidine / NMP solution containing 0.2 M 6-Cl-HOBT, placed in a constant temperature water bath, stirred at 50-60°C for 20-40 min, and cooled to obtain a resin with the initial Fmoc protecting group removed.

4. The preparation method according to claim 2, characterized in that: The specific steps of S2 are: (1) using a mixed solution of NMP containing Fmoc-Cys(R1)-OH, HBTU, HOBT and DIPEA as a promoting liquid, mixing it with the resin from which the Fmoc protecting group has been removed, performing a third water bath reaction, filtering and removing the reaction solution after cooling, and obtaining a resin containing the Fmoc-Cys(R1) residue after washing; (2) Mix the resin and CH3I in a molar ratio of 1:(5-10), then add C6H 15 N, and then place the mixture in an inert gas and react at 0-4°C for 12-24h. After the reaction is completed, wash the resin with appropriate amounts of DMF, DCM and methanol to remove unreacted reagents.

5. The preparation method according to claim 2, characterized in that: The specific steps of S4 are: (1) Under anhydrous and oxygen-free conditions, Fmoc-Pro-OH is taken, DCC and C2H7O4P are added, and then the reaction system is fully stirred at room temperature under nitrogen protection to generate Fmoc-Pro-PO(OEt)2; (2) Purify Fmoc-Pro-PO(OEt)2 and use the purified Fmoc-Pro-PO(OEt)2 as the N-terminal amino acid unit and HBTU / HOBT / DIPEA as coupling reagents to perform an amide bond coupling reaction with the prepared polypeptide-resin complex, so that Fmoc-Pro-PO(OEt)2 forms an amide bond with the C-terminal residue on the complex.

6. The preparation method according to claim 2, characterized in that: The specific steps of S5 are: The coupled peptide-resin complex was mixed with the cleavage agent Reagent K, shaken at 0°C for 30 minutes, and then the reaction was continued for 3 hours. After filtration, the filtrate was mixed with C4H8O2, and the precipitate was collected to obtain the crude product of the inhibitory peptide.

7. The preparation method according to claim 2, characterized in that: After the crude product of the inhibitory polypeptide is obtained in S5, liquid chromatography purification is also included, and the chromatographic conditions include: C18 reverse phase column; mobile phase A: 0.10% by volume formic acid aqueous solution and methanol; mobile phase B: 0.1% by volume TFA / acetonitrile; mobile phase gradient: mobile phase B20-80%, 30min; flow rate is 500mL / min, detection wavelength is 220nm, main peak is collected in segments, and segmented purified liquids with any single impurity not exceeding 0.5% are combined, and unqualified segments are combined and concentrated and then purified repeatedly until the purified liquid is qualified.

8. Use of the polypeptide having an inhibitory effect on CK2 prepared by the preparation method according to claim 2 in the preparation of anti-tumor drugs, characterized in that: The tumor is selected from large cell carcinoma.

9. A pharmaceutical composition, characterized in that The active ingredients of the pharmaceutical composition include the inhibitory polypeptide or the inhibitory polypeptide salt prepared by the preparation method according to any one of claims 2 to 8 and a pharmaceutically acceptable carrier.

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