GB2621640A - Active polypeptide for inhibiting growth of liver cancer cells, and preparation method therefor and use thereof - Google Patents
Active polypeptide for inhibiting growth of liver cancer cells, and preparation method therefor and use thereof Download PDFInfo
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- GB2621640A GB2621640A GB2215958.6A GB202215958A GB2621640A GB 2621640 A GB2621640 A GB 2621640A GB 202215958 A GB202215958 A GB 202215958A GB 2621640 A GB2621640 A GB 2621640A
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- active polypeptide
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to an active polypeptide for inhibiting the growth of liver cancer cells, and a preparation method therefor and the use thereof. The amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK. The active polypeptide can target tumor cells, inhibit DNA synthesis of liver cancer cells, inhibit the proliferation and growth thereof, accelerate the repair of liver tissues and recover the liver function. The synthesized active polypeptide can be used for preparing and developing a drug for preventing or treating hepatitis, cirrhosis and hepatic fibrosis.
Description
ACTIVE POLYPEPTIDE FOR INHIBITING GROWTH OF LIVER
CANCER CELLS AS WELL AS PREPARATION METHOD AND USE
THEREOF
TECHNICAL FIELD
The present invention relates to the technical field of biomedicine, and particularly relates to an active polypeptide for inhibiting growth of liver cancer cells as well as a preparation method and use thereof.
BACKGROUND
In recent years, hepatitis has always been one of important global public health problems. According to latest data from the World Health Organization, nearly 2 billion of human beings are infected with hepatitis viruses in the world, in which about 500 million people are infected with chronic hepatitis, leading to about 1 million people dead every year. Liver-related diseases, including various hepatitis, liver cirrhosis, and liver fibrosis, are major lethal diseases affecting human health. Due to the hepatitis, the liver cirrhosis, and the liver fibrosis, necrosis of a large amount of liver cells will be caused, leading to liver failure and even a life risk.
As a novel protein drug, active polypeptides have aroused strong interest among scholars and industries. Due to the unique composition of amino acids and the structural amphiphilicity, the active polypeptide can bind to macromolecules such as nucleic acids and proteins in nuclei, and negatively charged components on surfaces of viruses or bacteria to destroy cell membrane structures or intracellular macromolecules, so as to destroy normal functions of cells and to cause cell death. In recent years, a lot of progress has been made in the treatment of liver diseases with polypeptide drugs, but so far, there is still a lack of effective therapeutic strategies and effective drugs for the treatment of liver diseases.
Therefore, an urgent problem to be solved by those skilled in the art is to provide an active polypeptide capable of inhibiting the synthesis of DNA of cancer cells, especially inhibiting the regeneration of liver cancer cells.
SUMMARY
In order to overcome the defects of the prior art, an objective of the present invention is to provide an active polypeptide for inhibiting growth of liver cancer cells as well as a preparation method and use thereof. The active polypeptide of the present invention can target the liver organ, inhibit proliferation and growth of liver cancer cells, accelerate repair of liver tissues, and restore functions of the liver.
In order to resolve the above technical problems, the present invention provides the following technical solutions: In a first aspect, the present invention provides an active polypeptide for inhibiting growth of liver cancer cells. The active polypeptide has an amino acid sequence of VNNSSLIGLGYTQTLKPGIK.
In a second aspect, the present invention provides a method for preparing the active polypeptide as described in the first aspect. The method includes the following steps: (I) carrying out a synthesis reaction with chlorotrityl chloride resin as a starting raw material and a 9-fluorenylmethoxycarbonyl protected amino acid as a monomer for connecting the chlorotrityl chloride resin with the amino acid to synthesize a polypeptide solution; (2) after the synthesis reaction is completed, adding a cutting solution to cut the polypeptide solution from the chlorotrityl chloride resin; and (3) precipitating the polypeptide solution with ether and/or blow-drying the polypeptide solution with nitrogen, and then performing purification by preparative HPLC to obtain the active polypeptide.
Further, the synthesis reaction is carried out in a synthesis direction of sequentially performing condensation from a C terminal to an N terminal according to the amino acid sequence of the active polypeptide.
Further, the cutting solution includes, by mass percentage, 95% of TEA, I% of water, 2% of EDT, and 2% of TIS.
In a third aspect, the present invention provides use of the active polypeptide as described in the first aspect in preparation of a drug for preventing or treating hepatitis, liver cirrhosis, and liver fibrosis.
As described above, the drug of the present invention can prevent or treat hepatitis, liver cirrhosis, and liver fibrosis. Usually, the active polypeptide of the present invention can be formulated as an active ingredient in a nontoxic, inert, and pharmaceutically acceptable carrier medium. The formulated drug can be administered by conventional routes, including but not limited to oral, intramuscuhu; intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
When formulated in a dosage form for oral administration, the drug contains a safe and effective amount of the active polypeptide of the present invention and a pharmaceutically acceptable carrier and/or adjuvant. The drug for oral administration can be formulated into a tablet, powder, a granule, a capsule, and other common dosage forms. An excipient used may be at least one of starch, lactose, sucrose, mannose, and hydroxymethyl cellulose. A disintegrant may he at least one of cross-linked polyvinyl pyrrolidonc and sodium carboxymethyl starch. A binder may be at least one of gelatin and polyethylene glycol. In addition to the above-mentioned dosage forms, the drug for oral administration can also be formulated into an emulsion, a syrup, and the like.
The drug of the present invention can also be formulated into an injection with water for injection, normal saline, and glucose water by a conventional method.
In addition, the active polypeptide of the present invention can also be used in combination with other drugs for treating hepatitis, liver cirrhosis, and liver fibrosis.
Compared with the prior art, the present invention has the following beneficial effects: The active polypeptide provided in the present invention can target the liver organ, significantly inhibit synthesis of DNA of tumor cells, inhibit proliferation and growth of liver cancer cells, accelerate repair of liver tissues, and restore functions of the liver. The additional aspects and advantages of the present invention will be set forth in part in the description below, parts of which will become apparent from the description below, or will be understood by the practice of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings described herein are intended to provide further understanding of the present invention and constitute a part of this application, but do not constitute the improper limitation to the present invention, hi the accompanying drawings: FIG. 1 is a high-performance liquid chromatogram of an active polypeptide provided in the present invention; FIG. 2 is a mass spectrogram of the active polypeptide provided in the present invention; and FIG. 3 is a diagram showing an inhibition effect of the active polypeptide provided in the present invention on growth of SMMC-7721, LO-2, Chang liver, HepG-2, and Huh-7 cells.
DETAILED DESCRIPTION
In order to understand the technical contents of the present invention more fully, the present invention is further introduced and described below with reference to the accompanying drawings and specific embodiments. Apparently, the embodiments described are merely a part, rather than all of the embodiments of the present invention, and all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.
Those skilled in the art will easily understand the features, beneficial effects, and advantages of the present invention by reading the contents disclosed in the specification.
Unless otherwise indicated, all percentages, fractions, and ratios are calculated based on total weights of compositions of the present invention. The term "weight content" herein may be represented by the symbol "%".
The terms "include", "comprise", "comprising", "contain", "have", and other variants herein are intended to encompass non-exclusive inclusion, and are not distinguished therebetween. The term "comprise" indicates that other steps and ingredients may be added without affecting final results. The term "comprise" also includes the terms "consist of" and "basically consist of". The composition and method/process of the present invention may comprise, consist of, or basically consist of essential elements and limitations described herein as well as any additional or optional ingredients, components, steps or limitations described herein.
In the solutions of the present invention, DCM refers to dichloromethane; 11BTU refers to benzotriazole-N,N,N',Nt-tetramethylurea hexafluorophosphate; DIEA refers to N,N-diisopropyl ethylamine; DMF refers to N,N-dimethylformamide; TFA refers to trifluoroacetic acid; EDT refers to 1,2-ethanedithiol; Fmoc refers to 9-11uorenylmethoxycarbonyl; and T1S refers to triisopropylsilane.
Example 1
In this example, a method for preparing an active polypeptide is provided. The method includes the following steps: (1) 3 g of chlorotrityl chloride resin (with a substitution degree of 1.03 mmol/g) was put into a reaction tube, and 45 mL of DCM was added for shaking for 30 mm.
(2) Connection with a first amino acid A solvent was removed by suction filtration with a sand core, 10 mmol of a Fmoc-Gly amino acid was added, DMF was added for dissolution, and 30 Immo' of DIEA was added for shaking for 60 mm.
(3) Deprotection The DMF was removed, 45 mL of a 20% piperidine DMF solution was added for reaction for 5 mm, the DMF was removed, and a 20% piperidine DMF solution was added again for reaction for 15 min (4) Washing mL of DMF was added for washing twice, 30 mL of DCM was added for washing twice, and 30 mL of DMF was added for washing twice.
(5) Condensation 12 mmol of a protected amino acid and 13 Ennio] of EIBTU were added to the reaction tube, 15 mL of DMF was added for dissolution, and 13 mum] of DIEA was immediately added for reaction for 30 min. (6) Washing mL of DMF was added for washing once, 30 mL of DCM was added for washing twice, and 30 mL of DMF was added for washing twice.
(7) Steps 3 to 6 were repeated for sequentially connecting amino acids in a sequence from right to left until completion.
(8) Cleaning of the resin After the resin is connected with all the amino acids in the sequence, draining was performed, 30 mL of DMF was added for washing twice, 30 mL of methanol was added for washing twice, 30 mL of DMF was added for washing twice, and 30 mL of DCM was added for washing twice, and then draining was performed for 10 min. (9) A polypeptide was cut from the resin to obtain a polypeptide solution Preparation of a cutting solution: 95% of TFA, 1% of water, 2% of EDT, and 2% of TIS; use amount: 30 mL; and cutting time: 120 mm.
(1(J) Purification The cut polypeptide solution was blow-dried with nitrogen, washed with ether for 6-8 times and dried at room temperature, and then a crude product was purified by preparative H PLC to obtain the active polypeptide.
The active polypeptide has an amino acid sequence of VNNSSLIGLGYTQTLKPCTIC. Example 2 The active polypeptide obtained in Example 1 was analyzed by liquid chromatography under the following conditions: mobile phase A: a 0.1% TFA aqueous solution; mobile phase B: a 0.1% TFA acetonitrile solution; gradient: 20%-35% of the mobile phase B. 10 min; chromatographic column: Kromasil 100-5 C18 4.6*300 mm (5 urn); wavelength: 220 nm; and flow rate: 1.0 mL/min.
A high-performance liquid chromatogram is shown in FIG. 1; and a sample is measured to have a purity of 98.42%.
Mass spectrometry (ES!): a mass spectrogram is shown in FIG. 2; and the active polypeptide has a molecular ion peak of 2103.47.
Example 3
In this example, an inhibition effect of the active polypeptide in Example 1 on growth of SMMC-7721 cells is further verified, and specific operation steps are as follows.
The active polypeptide prepared in Example 1 was taken and prepared into a solution containing 100 pg of the active polypeptide per 1 mL by using an RPMI-1640 culture solution. The SMMC-7721 cells were cultured to a logarithmic growth phase with a 10% calf serum culture medium, where the SMMC-7721 cells were commercially purchased cells. The SMMC-7721 cells in the logarithmic growth phase were digested with a 0.25% trypsin-disodium edetate digestion solution. Then, the solution was diluted to contain 2.5* 104 to 5*104 cells per 1 mL by adding a 10% calf serum culture medium, and a cell suspension obtained was plated on a 96-well cell plate with 100 jaL per well In a test sample group, 100 pL of a test sample solution was added to each well, and each test sample was added to 3 wells. In a cell control group, 100 [IL of an RPMI-1640 culture solution was added to each well. The cells were cultured in an incubator containing 5% carbon dioxide saturated water vapor at 37°C for 48 hours, a culture plate was taken out 4 hours before the culture was completed, and the culture solution was sucked off. A 0.01 mol/L phosphate buffer (with a pH of 7.3) was added to each well for washing once, and then 100 [IL of the above phosphate buffer (with the pH of 7.3) and 20!IL of an IMTT solution were added to each well for continuous culture.
After the culture was completed, the culture solution was sucked off. 100 pt of dimethyl sulfoxide, was added to each well and shaken uniformly, and the absorbance was measured by using a microplate reader at a wavelength of 550 nm. Test results are shown in FIG. 3.
According to the results, the cell control group has an absorbance of 0.0631±0.0003, the test sample group has an absorbance of 0.0203±0.0009, and there is a significant difference between the test sample group and the cell control group, p<0.01.
In conclusion, the polypeptide of the present invention has a significant inhibition effect on the growth of the SMMC-7721 liver cells.
Example 4
In this example, an inhibition effect of the active polypeptide in Example 1 on growth of LO-2, Chang liver, HepG-2, and Huh-7 cells is further verified on the basis of Example 3.
According to the method in Example 3, the SMMC-7721 cells were replaced with the LO-2. Chang liver, HepG-2, and Huh-7 cells, and the absorbance was measured, where the LO-2, Chang liver, HepG-2, and Huh-7 cells were all commercially purchased cells.
Test results are shown in FIG. 3. According to the test results in HG. 3, it can be seen that there is a significant difference between the test sample group and the cell control group, p<0.01.
By combining the test results in Example 3 and Example 4, it can be seen that the active polypeptide of the present invention has a significant inhibition effect on the growth of the SMMC-7721, LO-2, Chang liver, HepG-2, and Huh-7 liver cells.
The active polypeptide in the present invention can target the liver organ, significantly inhibit synthesis of DNA of tumor cells, inhibit proliferation and growth of liver cancer cells, accelerate repair of liver tissues, and restore functions of the liver.
In addition, according to the above test results, it can be seen that the active polypeptide of the present invention can be used for research and development of a drug for preventing or treating of hepatitis, liver cirrhosis, and liver fibrosis.
It is worth noting that the drug described in the present invention takes the active polypeptide in Example 1 as a main active ingredient, and does not exclude changes in preparation systems and administration ways, derivatives of the active polypeptide obtained after simple chemical modifications and adjustments as well as combinations of various active substances.
The technical solutions provided in the embodiments of the present invention are described above in detail. Although the principles and implementations of the embodiments of the present invention are described by using specific examples in this specification, the descriptions of the foregoing embodiments are merely applicable to help understand the principles of the embodiments of the present invention. Meanwhile, a person of ordinary skill in the art may make modifications to the specific implementations and application range according to the embodiments of the present invention. In conclusion, the content of this specification should not be construed as a limit on the present invention.
Claims (8)
- CLAIMSWhat is claimed is: 1. An active polypeptide for inhibiting growth of liver cancer cells, wherein the active polypeptide has an amino acid sequence of VNNSSLIGLGYTQTLKPGIK.
- 2. A method for preparing the active polypeptide according to claim 1, comprising the following steps: (1) carrying out a synthesis reaction with chlorotrityl chloride resin as a starting raw material and a 9-fluorenylmethoxycarhonyl protected amino acid as a monomer for connecting the chlorotrityl chloride resin with the amino acid to synthesize a polypeptide solution; (2) after the synthesis reaction is completed, adding a cutting solution to cut the polypeptide solution from the chlorotrityl chloride resin; and (3) precipitating the polypeptide solution with ether and/or blow-drying the polypeptide solution with nitrogen, and then performing purification by preparative HPLC to obtain the active polypeptide.
- 3. The preparation method according to claim 2, wherein the synthesis reaction is carried out in a synthesis direction of sequentially performing condensation from a C terminal to an N terminal according to the amino acid sequence of the active polypeptide.
- 4. The preparation method according to claim 2, wherein the cutting solution comprises, by mass percentage, 95% of TEA, 1% of water, 2% of EDT, and 2% of TIS.
- 5. Use of the active polypeptide according to claim 1 in preparation of a drug for preventing or treating hepatitis, liver cirrhosis, and liver fibrosis.
- 6. The use according to claim 5, wherein the drug is an injectable solution.
- 7. The use according to claim 5, wherein the drug is a drug for oral administration.
- 8. The use according to claim 7, wherein the drug for oral administration further comprises a pharmaceutically acceptable carrier and/or adjuvant.
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| CN202111327144.4A CN113845571B (en) | 2021-11-10 | 2021-11-10 | Active polypeptide for inhibiting liver cancer cell growth and preparation method and application thereof |
| PCT/CN2022/117155 WO2023082803A1 (en) | 2021-11-10 | 2022-09-06 | Active polypeptide for inhibiting growth of liver cancer cells, and preparation method therefor and use thereof |
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| WO2007134328A2 (en) * | 2006-05-15 | 2007-11-22 | Van Andel Research Institute | Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein |
| US20120087862A1 (en) * | 2006-08-09 | 2012-04-12 | Homestead Clinical Corporation | Organ-specific proteins and methods of their use |
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| WO2019169697A1 (en) * | 2018-03-09 | 2019-09-12 | 广东药科大学 | Hepatoma cell-targeting antimicrobial peptide chimera m27-39-htpp and use therof |
| CN113845571A (en) * | 2021-11-10 | 2021-12-28 | 华南理工大学 | Active polypeptide for inhibiting growth of liver cancer cells and preparation method and application thereof |
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- 2022-09-06 GB GB2215958.6A patent/GB2621640A/en active Pending
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| WO2007134328A2 (en) * | 2006-05-15 | 2007-11-22 | Van Andel Research Institute | Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein |
| US20120087862A1 (en) * | 2006-08-09 | 2012-04-12 | Homestead Clinical Corporation | Organ-specific proteins and methods of their use |
| US20180372754A1 (en) * | 2015-12-23 | 2018-12-27 | Polyquant Gmbh | Bladder cancer biomarker proteins |
| WO2019169697A1 (en) * | 2018-03-09 | 2019-09-12 | 广东药科大学 | Hepatoma cell-targeting antimicrobial peptide chimera m27-39-htpp and use therof |
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