CN107474117B

CN107474117B - Antitumor cyclopeptide and preparation method and medical application thereof

Info

Publication number: CN107474117B
Application number: CN201710667566.3A
Authority: CN
Inventors: 张娜; 唐珊; 汪杨俊琦; 齐晓倩; 赵丽娇; 钟儒刚
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology
Priority date: 2017-08-07
Filing date: 2017-08-07
Publication date: 2020-08-07
Anticipated expiration: 2037-08-07
Also published as: CN107474117A

Abstract

An antitumor cyclic peptide, a preparation method and medical application thereof, belonging to the field of biological pharmacy. An antitumor cyclic peptide with the amino acid sequence of

Description

Antitumor cyclopeptide and preparation method and medical application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to an anti-tumor cyclopeptide, and a preparation method and medical application thereof.

Background

In recent years, polypeptide drugs synthesized by modern biotechnology have become one of hot spots in drug research and development, and have wide application prospects in prevention, diagnosis and treatment of relevant major diseases such as tumors and the like due to wide adaptation, high safety and obvious curative effect.

As polypeptide drugs become one of the indispensable important fields for the development of modern drugs, their development is also faster and faster. More than 300 polypeptide drugs are currently in clinical trials, more than 600 are in preclinical trials, and more than 60 are on the market, including polypeptide drugs for the treatment of tumors, such as Mepect for the treatment of resectable myeloma, isotadax for the treatment of cutaneous T-cell lymphoma, and Adcetris for the treatment of CD30 positive hodgkin lymphoma, among others. The polypeptide drug has small molecular structure, easy modification and synthesis, and less waste discharged in the production process, and belongs to green pharmacy, so the polypeptide drug is one of the drugs with the most development prospect in the 21 st century.

Disclosure of Invention

The invention provides an anti-tumor cyclopeptide, the amino acid sequence of which is

Wherein two Cys form a disulfide bond.

The antitumor cyclic peptide has a molecular weight of 1442.71, can be purified by reverse phase high performance liquid chromatography, and can be purified in such a way that the purity thereof reaches 96.78%.

The invention provides an anti-tumor composition, which comprises the anti-tumor cyclic peptide.

The invention further discloses a preparation method of the anti-tumor cyclopeptide, and the anti-tumor cyclopeptide is prepared by a solid phase synthesis method. The solid phase synthesis method can be achieved by the following measures:

selecting CTC high molecular resin, connecting Fmoc-Gly-OH at the C end, namely the carboxyl end, of the antitumor cyclic peptide to the CTC resin in a covalent bond mode, removing Fmoc protection groups, taking the amino group of Gly combined on the high molecular resin as a synthesis starting point, sequentially carrying out condensation reaction to form peptide bonds according to the sequence of an amino acid sequence CGHFKFGY L RCG from the C end, namely the carboxyl end, to the N end, namely the amino end of the antitumor cyclic peptide, removing Fmoc protection, carrying out condensation to form the peptide bonds, continuously and repeatedly circulating the steps until the sequences are completely coupled and the Fmoc protection is removed, then cutting the resin with methanol to obtain a crude polypeptide product, further purifying by using a reversed-phase high-performance liquid chromatography, treating to enable two Cys to form disulfide bonds to form rings, and purifying by using the reversed-phase high-performance liquid chromatography to obtain the target cyclic peptide with the purity of 96.78%.

The invention provides application of the antitumor cyclopeptide in preparation of antitumor drugs. The tumor is selected from liver cancer, cervical cancer and lung cancer. The proliferation inhibition rates of the anti-tumor cyclopeptide to liver cancer cell HepG-2 and cervical cancer cell Hela within the concentration range of 5-500 mu M are 2.6-100% and 3.9-99.9%, respectively, the proliferation inhibition rate of the anti-tumor cyclopeptide to lung cancer cell A549 within the concentration range of 9-900 mu M is 16.5-99.3%, and the IC of the anti-tumor cyclopeptide is IC₅₀The values were 36.90. mu.M, 61.41. mu.M and 39.87. mu.M, respectively.

The invention provides application of the antitumor cyclopeptide in preparing a medicament for promoting tumor cell apoptosis.

In one embodiment, the tumor cell is selected from the group consisting of a liver cancer cell, a cervical cancer cell, and a lung cancer cell.

In one embodiment, the tumor cell is a lung cancer cell. When the concentration of the antitumor cyclopeptide is 40 mu M, the apoptosis rate of the antitumor cyclopeptide on lung cancer cells A549 is 40.49%.

Compared with the prior art, the invention has the following advantages and technical effects: the invention synthesizes the anti-tumor cyclopeptide for the first time, and adopts a CCK-8 method to determine the anti-tumor activity of the cyclopeptide, and the anti-tumor cyclopeptide has obvious capacity of inhibiting the proliferation of tumor cells. In addition, the inventor also adopts the flow cytometry to detect the apoptosis-promoting effect of the cyclic peptide on the tumor cells, and the result shows that the anti-tumor cyclic peptide has a remarkable apoptosis-promoting effect on the tumor cells.

Drawings

FIG. 1 antitumor cyclopeptides

HP L C of (1).

FIG. 2 antitumor cyclopeptides

MS diagram of (1).

FIG. 3 is a graph showing the apoptosis of lung cancer cell A549 by the anti-tumor cyclopeptide of the present invention at a concentration of 40. mu.M.

Detailed Description

The following examples are given as specific embodiments of the present invention, but the present invention is not limited to the following examples.

EXAMPLE 1 solid phase Synthesis of antitumor Cyclic peptides

In the solid phase synthesis process of the antitumor cyclic peptide, a Fmoc scheme is adopted, CTC resin is selected and treated to expose active sites of the resin, Fmoc-Gly-OH is connected to the CTC resin in a covalent bond mode, the resin is washed by DMF, 25% piperidine/DMF solution with volume concentration is added to remove Fmoc protective groups, HBTU, N-methylmorpholine and Fmoc-Cys (Trt) -OH are added in a standard solid phase synthesis process (SPPS), Fmoc-Cys (Trt) -OH are subjected to dehydration condensation reaction with Gly connected to the CTC resin through peptide bonds, ninhydrin is used for detecting whether the reaction is complete, 25% piperidine/DMF solution with volume concentration is added to remove the Fmoc protective groups, the steps are adopted, the Fmoc protective groups are sequentially connected according to the sequence of CGHFKF L RCG until the sequence is completely coupled, the Fmoc protective groups are removed, the resin is washed by methanol to be cut, the resin is transferred to a cutting pipe, and the existing technology comprising TFA and H and the TFA and the prior art are added₂Precipitating with diethyl ether, and cutting to obtain polypeptide crude product. Further purifying by reverse phase high performance liquid chromatography, dissolving in pure water, and adding 1% of I₂MeOH solution (1g I)₂Dissolved in 100ml MeOH) was slowly added dropwise to the solution, allowing the two Cys to form disulfide bonds to form a ring. The aqueous solution was extracted with dichloromethane and purified by reverse phase high performance liquid chromatography to obtain antitumor cyclic peptide with a purity of 96.78%.

Example 2 measurement of inhibitory Effect of Cyclic peptides on proliferation of liver cancer cell HepG-2, cervical cancer cell Hela and lung cancer cell A549 by CCK-8 method

Suspending the liver cancer cell HepG-2 cell suspension (1.5 × 10)⁴M L), cervical cancer cell Hela cell suspension (2 × 10)⁴Pieces/m L) and lung cancer cell A549 cell suspension (2 × 10)⁴Pieces/m L) were added to 96-well plates at a volume of 200. mu. L per well, and CO was maintained at a constant temperature of 37 ℃₂Culturing in an incubator. After 24h, the cells grow adherently, the old culture solution is sucked out, the antitumor cyclic peptides are prepared into 5-500 mu M and 9-900 mu M liquid medicines according to the concentrations in the table 1 and the table 2 respectively, the liquid medicines are added into corresponding 96-well plates respectively, a negative control and a blank control are set, and the temperature is kept constant at 37 ℃ by CO₂And (5) incubating the incubator. After 48h, sucking out the liquid medicine, adding the prepared CCK-8 solution, and keeping the temperature of the CO constant at 37 DEG C₂Culturing in an incubator. After 2h, the absorbance at a wavelength of 450nm was measured on a microplate reader. As shown in Table 1, the in vitro inhibition rate of the antitumor cyclopeptide on the hepatoma cell HepG-2 is 2.6-100% under the action of 5-500 μ M concentration, and the IC of the antitumor cyclopeptide is₅₀The value is 36.90 mu M, the inhibition rate of the cervical cancer cell Hela is 3.9-99.9%, and the IC is₅₀The value was 61.41. mu.M. As shown in Table 2, the antitumor cyclopeptide has an inhibition rate of 16.5-99.3% against lung cancer cell A549 at a concentration of 9-900. mu.M, and its IC₅₀The value was 39.87. mu.M.

TABLE 1 inhibitory Effect of antitumor cyclopeptides on tumor cell HepG-2 and HeLa cell proliferation

TABLE 2 inhibitory Effect of antitumor cyclopeptides on the proliferation of tumor cells A549

Example 3 detection of the apoptotic Effect of Cyclic peptides on Lung cancer cell A549 by flow cytometry

Lung cancer cell A549 is selected as a research object, the apoptosis promoting effect of the cyclopeptide on tumor cells is detected, and the lung cancer cell A549 cell suspension (1.75 × 10) in logarithmic growth phase is used⁴Pieces/m L) were added to a 6-well plate at a volume of 2m L per well, and CO was maintained at a constant temperature of 37 ℃₂Culturing in an incubator. After 24h, cells grow on the wall, anti-tumor cyclopeptide liquid with the final concentration of 40 mu M is added, negative control of only adding culture medium and cells without adding drugs and positive control of single staining with FITC/PI are set, and CO is kept at the constant temperature of 37 DEG C₂Culturing in an incubator. After 48h, the cell culture broth was carefully handled into a centrifuge tube for use, the cells were digested with trypsin without EDTA, and the cells were carefully blown up by adding the previously collected broth. The cells were pelleted by centrifugation, washed with 4 ℃ pre-chilled PBS and pelleted again by centrifugation.

Resuspending the cells in a prepared concentration of binding buffer to a concentration of 1-5 × 10⁶And/m L, putting 100 mu L of cell suspension into a 5m L flow tube, adding Annexin V/FITC, uniformly mixing, incubating at room temperature in a dark place for 5min, adding an propidium iodide solution (PI) and PBS, and performing flow detection on a flow cytometer.

The detection result is shown in fig. 3, and it can be seen from the figure that the cyclic peptide has an apoptosis-promoting effect on lung cancer cell a 549. The final result shows that the apoptosis rate of the cyclic peptide to the lung cancer cell A549 is 40.49% under the concentration of 40 mu M.

In a similar way, the result of the same test method shows that the antitumor cyclopeptide also has the effect of promoting apoptosis on liver cancer cells HepG-2 and cervical cancer cells Hela.

Claims

1. An anti-tumor cyclopeptide, characterized in that the amino acid sequence of the anti-tumor cyclopeptide is

Wherein two Cys form a disulfide bond.

2. An anti-tumor composition comprising the anti-tumor cyclic peptide of claim 1.

3. The method for preparing the anti-tumor cyclopeptide according to claim 1, wherein the anti-tumor cyclopeptide is prepared by a solid phase synthesis method.

4. The use of the anti-tumor cyclopeptide according to claim 1 for the preparation of an anti-tumor medicament, wherein the tumor is selected from the group consisting of liver cancer, cervical cancer and lung cancer.

5. Use of the anti-tumor cyclopeptide according to claim 1 for the preparation of a medicament for promoting apoptosis of tumor cells, said tumor cells being lung cancer cells.