CN113845571A

CN113845571A - Active polypeptide for inhibiting growth of liver cancer cells and preparation method and application thereof

Info

Publication number: CN113845571A
Application number: CN202111327144.4A
Authority: CN
Inventors: 王小明; 李杉; 张雷
Original assignee: South China University of Technology SCUT; Zhongshan Institute of Modern Industrial Technology of South China University of Technology
Current assignee: South China University of Technology SCUT; Zhongshan Institute of Modern Industrial Technology of South China University of Technology
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2021-12-28
Anticipated expiration: 2041-11-10
Also published as: CN113845571B; WO2023082803A1

Abstract

The invention discloses an active polypeptide for inhibiting the growth of liver cancer cells, a preparation method and application thereof, wherein the amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK. The active polypeptide synthesized by the invention can be used for preparing medicines for preventing or treating hepatitis, liver cirrhosis and hepatic fibrosis.

Description

Active polypeptide for inhibiting growth of liver cancer cells and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to an active polypeptide for inhibiting growth of liver cancer cells, and a preparation method and application thereof.

Background

In recent years, hepatitis has been one of the world-wide major public health problems, and recent data from the world health organization shows that there are 20 million people infected with hepatitis virus worldwide, about 5 million people infected with chronic hepatitis, and about 100 million people died each year. Liver-related diseases including various hepatitis, cirrhosis and liver fibrosis are serious diseases which disturb human health, and a large amount of liver cells are necrotized in hepatitis, cirrhosis and liver fibrosis, so that liver failure and even life are threatened.

Active polypeptides have attracted strong interest to scholars as a new protein drug. The unique amino acid composition and the amphipathy in the structure of the active polypeptide enable the polypeptide to be combined with macromolecules in a cell nucleus such as nucleic acid, protein and the like and components with negative charges on the surface of viruses or bacteria, and further destroy the cell membrane structure or the intracellular macromolecules to disturb the normal functions of cells and cause cell death. In recent years, many advances have been made in the treatment of liver diseases with polypeptide drugs, but effective therapeutic strategies and effective drugs for treating liver diseases are still lacking to date.

Therefore, it is an urgent problem to provide an active polypeptide capable of inhibiting DNA synthesis of cancer cells, especially inhibiting regeneration of liver cancer cells.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an active polypeptide for inhibiting the growth of liver cancer cells, a preparation method and application thereof.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, an active polypeptide for inhibiting the growth of liver cancer cells is provided, wherein the amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK.

In a second aspect, there is provided a method of preparing an active polypeptide as defined in the first aspect, comprising the steps of:

(1) the method comprises the following steps of (1) taking chlorotrityl chloride resin as an initial raw material, taking 9-fluorenylmethyloxycarbonyl protected amino acid as a monomer to perform a synthesis reaction, and grafting the chlorotrityl chloride resin with the amino acid to synthesize a polypeptide solution;

(2) after the synthesis reaction is finished, adding a cutting fluid, and cutting the polypeptide solution from the chlorotrityl chloride resin;

(3) and (3) precipitating the polypeptide solution by using ether and/or blowing dry the polypeptide solution by using nitrogen, and purifying by adopting preparative HPLC (high performance liquid chromatography) to obtain the active polypeptide.

Further, the synthesis direction of the synthesis reaction is as follows: and sequentially condensing from the C end to the N end according to the amino acid sequence of the active polypeptide.

Further, the cutting fluid comprises the following components in percentage by mass: TFA 95%, water 1%, EDT 2% and TIS 2%.

In a third aspect, there is provided a use of the active polypeptide of the first aspect in preparing a medicament for preventing or treating hepatitis, liver cirrhosis and liver fibrosis.

As described above, the medicament of the present invention can prevent or treat hepatitis, liver cirrhosis and liver fibrosis, and generally, the active polypeptide of the present invention can be formulated as an active ingredient in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated drug may be administered by conventional routes including, but not limited to, oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

When the medicament is used for oral administration, the medicament contains a safe and effective amount of the active polypeptide and pharmaceutically acceptable carriers and/or auxiliary agents, the orally administered medicament can be prepared into common formulations such as tablets, powder, granules, capsules and the like, and the used excipient can be at least one of starch, lactose, sucrose, mannose and hydroxymethyl cellulose; the disintegrant can be at least one of cross-linked polyvinyl pyrrolidone and sodium carboxymethyl starch; the binder may be at least one of gelatin and polyethylene glycol. The medicine for oral administration may be formulated into emulsion, syrup, etc. in addition to the above-mentioned dosage forms.

The medicine of the present invention may be prepared into injection, and the injection may be prepared with water for injection, physiological saline and glucose solution and through conventional process.

In addition, the active polypeptide of the present invention may be used in combination with other medicine for treating hepatitis, hepatocirrhosis and liver fibrosis.

Compared with the prior art, the invention has the following beneficial effects:

the active polypeptide provided by the invention can target liver organs, obviously inhibit DNA synthesis of tumor cells, inhibit proliferation and growth of liver cancer cells, accelerate liver tissue repair and restore liver functions.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:

FIG. 1 is a high performance liquid chromatogram of an active polypeptide provided by the present invention;

FIG. 2 is a mass spectrum of an active polypeptide provided by the present invention;

FIG. 3 is a graph showing the inhibitory effect of the active polypeptides of the present invention on SMMC-7721, LO-2, Chang lever, HepG-2 and Huh-7 cell growth.

Detailed Description

For a fuller understanding of the technical aspects of the present invention, reference should be made to the following detailed description taken together with the accompanying drawings; it is to be understood that the described embodiments are merely a subset of the embodiments of the invention, and not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The features, benefits and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure.

All percentages, parts and ratios are based on the total weight of the composition of the present invention, unless otherwise specified. The term "weight content" herein may be represented by the symbol "%".

The use of "including," "comprising," "containing," "having," or other variations thereof herein, is meant to encompass the non-exclusive inclusion, as such terms are not to be construed. The term "comprising" means that other steps and ingredients can be added that do not affect the end result. The term "comprising" also includes the terms "consisting of and" consisting essentially of. The compositions and methods/processes of the present invention can comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any of the additional or optional ingredients, components, steps, or limitations described herein.

In the scheme of the invention, DCM is dichloromethane; HBTU is benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate; DIEA is N, N-diisopropylethylamine; DMF is N, N-dimethylformamide; TFA is trifluoroacetic acid; EDT is 1, 2-ethanedithiol; fmoc is 9-fluorenylmethyloxycarbonyl; TIS is triisopropylsilane.

Example 1

This example provides a method for preparing an active polypeptide, comprising the steps of:

(1) 3g of chlorotrityl chloride resin (the degree of substitution is 1.03mmol/g) is put into a reaction tube, 45mL of DCM is added, and the mixture is shaken for 30 min;

(2) to the first amino acid

Filtering off the solvent through a sand core, adding 10mmol of Fmoc-Gly amino acid, adding DMF for dissolving, adding 30mmol of DIEA, and oscillating for 60 min;

(3) deprotection of the amino acid

Removing DMF, adding 45mL of 20% piperidine DMF solution, and reacting for 5 min; removing and adding 20% piperidine DMF solution for 15 min;

(4) washing machine

Adding 30mL of DMF for washing twice, adding 30mL of DCM for washing twice, and finally adding 30mL of DMF for washing twice;

(5) condensation of

Taking 12mmol of protected amino acid and 13mmol of HBTU, dissolving with 15mL of DMF, adding into a reaction tube, immediately adding 13mmol of DIEA, and reacting for 30 min;

(6) washing machine

Adding 30mL of DMF for washing once, adding 30mL of DCM for washing twice, and adding 30mL of DMF for washing twice;

7) repeating the steps 3-6, and sequentially accessing the amino acids in the sequence from right to left until the end;

8) cleaning resins

After all amino acids in the resin access sequence are pumped, adding 30mL of DMF for cleaning twice, adding 30mL of methanol for cleaning twice, and adding 30mL of DMF for cleaning twice; adding 30mL of DCM for washing twice, and pumping for 10 min;

9) cleaving the polypeptide from the resin to obtain a polypeptide solution

Preparing cutting fluid: TFA 95%, water 1%, EDT 2%, TIS 2%;

the dosage is as follows: 30 mL; cutting time: 120 min;

10) purification of

And drying the cut polypeptide liquid by using nitrogen, washing the polypeptide liquid for 6 to 8 times by using diethyl ether, drying the polypeptide liquid at normal temperature, and purifying a crude product by using a preparative HPLC (high performance liquid chromatography) chromatogram to obtain the active polypeptide.

The amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK.

Example 2

The active polypeptide obtained in example 1 was subjected to liquid chromatography under the conditions:

mobile phase A: 0.1% aqueous TFA; mobile phase B: 0.1% TFA in acetonitrile;

gradient: 20% -35% of mobile phase B for 10 min;

a chromatographic column: kromasil 100-5C 184.6X 300mm (5 um);

wavelength: 220 nm;

flow rate: 1.0 mL/min.

The high performance liquid chromatogram thereof is shown in FIG. 1; the purity of the sample was measured to be 98.42%.

Mass spectrum (ESI): the mass spectrum is shown in FIG. 2; the molecular ion peak of the active polypeptide was 2103.47.

Example 3

This example further demonstrates the inhibitory effect of the active polypeptide of example 1 on SMMC-7721 cell growth, and the specific procedures are as follows:

the active polypeptide prepared in example 1 was collected and cultured in RPMI-1640 medium to prepare a solution containing 100. mu.g of active polypeptide per 1 mL. Culturing SMMC-7721 cells in 10% calf serum culture solution to logarithmic growth stage, wherein the SMMC-7721 cells are commercially purchased cells; digesting the logarithmic growth-promoting SMMC-7721 cells with 0.25% trypsin-disodium edetate digestive juice, adding 10% calf serum culture solution, and diluting to 2.5 × 10 per 1mL⁴～5×10⁴The cell suspension was plated on a 96-well cell plate at 100. mu.L per well.

Adding 100 mu L of test solution into each well of a test sample group, making 3 wells of each batch of test samples, adding 100 mu L of RPMI-1640 culture solution into each well of a cell control group, culturing in an incubator at 37 ℃ for 48 hours in which 5% carbon dioxide saturated water vapor is contained, taking out the culture plate 4 hours before finishing the culture, absorbing the culture solution, adding 0.01mol/L phosphate buffer solution (pH7.3) into each well, washing once, adding 100 mu L of the phosphate buffer solution (pH7.3) and 20 mu L of MTT solution into each well, and continuing the culture.

After the completion of the culture, the culture medium was aspirated, 100. mu.L of dimethyl sulfoxide was added to each well, the mixture was shaken, and the absorbance was measured at a wavelength of 550nm on a microplate reader, and the results of the measurement are shown in FIG. 3.

As a result: the cell control group is 0.0631 + -0.0003, the test sample group is 0.0203 + -0.0009, the significant difference between the test sample group and the cell control group is that p is less than or equal to 0.01.

And (4) conclusion: the polypeptide of the invention has obvious promotion effect on the growth of SMMC-7721 liver cells.

Example 4

This example further demonstrates the inhibitory effect of the active polypeptide of example 1 on the growth of LO-2, Chang lever, HepG-2 and Huh-7 cells, based on example 3.

SMMC-7721 cells were replaced with LO-2, Chang lever, HepG-2 and Huh-7 cells and their absorbance was measured according to the method in example 3; wherein LO-2, Chang lever, HepG-2 and Huh-7 are all commercially available cells.

The test results are shown in FIG. 3, and the test results in FIG. 3 show that there is a significant difference between the test sample group and the cell control group, and p is less than or equal to 0.01.

The results of the tests combining example 3 and example 4 can give: the active polypeptide of the invention has obvious inhibition effect on the growth of SMMC-7721, LO-2, Chang lever, HepG-2 and Huh-7 liver cells.

The active polypeptide of the invention can target liver organs, obviously inhibit DNA synthesis of tumor cells, inhibit proliferation and growth of liver cancer cells, accelerate liver tissue repair and restore liver function.

In addition, according to the above test results, it can be concluded that the active polypeptide of the present invention can be used for the development of drugs for preventing or treating hepatitis, liver cirrhosis, and liver fibrosis.

It is to be noted that the pharmaceutical composition of the present invention, which contains the active polypeptide of example 1 as a main active ingredient, does not exclude variations in formulation systems and administration methods, combinations of derivatives and multiple active substances obtained by simply modifying and modifying the active polypeptide, and the like.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.

Claims

1. An active polypeptide for inhibiting the growth of liver cancer cells, wherein the amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK.

2. A method for preparing an active polypeptide according to claim 1, comprising the steps of:

3. The method according to claim 2, wherein the synthesis direction of the synthesis reaction is: and sequentially condensing from the C end to the N end according to the amino acid sequence of the active polypeptide.

4. The preparation method according to claim 2, wherein the cutting fluid comprises the following components in percentage by mass: TFA 95%, water 1%, EDT 2% and TIS 2%.

5. Use of the active polypeptide according to claim 1 for the preparation of a medicament for the prevention or treatment of hepatitis, cirrhosis and liver fibrosis.

6. Use according to claim 5, wherein the medicament is an injectable solution.

7. The use according to claim 5, wherein the medicament is a medicament for oral administration.

8. Use according to claim 7, wherein the medicament for oral administration further comprises a pharmaceutically acceptable carrier and/or adjuvant.