A linear polypeptide and a cyclic peptide thereof having angiogenesis-inhibiting effect

Abstract

The present invention discloses a linear polypeptide and a cyclic peptide thereof that have the ability to inhibit angiogenesis. The linear polypeptide is composed of 18 amino acids, has a molecular weight of 2127.35, an isoelectric point of 4.44, and an amino acid sequence of ECRIQERNQQQDPLVCPA. The linear polypeptide of the present invention is derived from the enzymatic hydrolysate of geckos and can be prepared by solid-phase synthesis. The linear polypeptide of the present invention has a good inhibitory effect on the growth of vascular endothelial cells (VECs). The further modified cyclic peptide can enhance its structural stability in the digestive tract and can be used for the preparation and development of digestive tract tumor drugs, with good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a linear polypeptide and a cyclic peptide thereof having an angiogenesis-inhibiting effect. Background Art

[0002] The active ingredients of geckos are primarily composed of primary metabolites such as proteins, peptides, amino acids, and sugars. They also contain fatty acids, nucleosides, and macro- and trace elements. Current research reports indicate that peptides are the primary inhibitors of angiogenesis. However, geckos are mostly used as whole powder for medicinal purposes. After oral administration, they rely on nonspecific degradation by gastrointestinal enzymes, resulting in a complex peptide composition within the digestive tract and inefficient release of active ingredients. Therefore, new separation technologies are urgently needed to enhance their clinical efficacy.

[0003] In recent years, the incidence of gastric cancer has steadily increased, posing a serious threat to human health. Numerous clinical studies have shown that the growth and metastasis of gastric cancer are linked to angiogenesis, and therefore, inhibiting angiogenesis can inhibit tumor growth and metastasis. Antiangiogenic drugs can inhibit angiogenesis and also reorganize the tumor vasculature, reducing microvessel density. Currently, inhibiting some angiogenesis in tumors has become a hot topic in the treatment of gastric cancer.

[0004] Currently, there are synthetic angiogenesis inhibitors that have good angiogenesis inhibitory effects. However, they have significant side effects, such as teratogenicity, orthostatic hypotension, fever, rash, edema, dry mouth, dry skin, and constipation.

[0005] Therefore, it is urgent to develop new drugs with less toxic side effects and strong angiogenesis inhibitory activity. Summary of the Invention

[0006] The present invention overcomes the shortcomings and deficiencies of existing technologies and provides a linear polypeptide with angiogenesis-inhibiting properties. This invention obtains the linear polypeptide by enzymatically hydrolyzing geckos with artificial gastric fluid, analyzing the hydrolysis products through mass spectrometry and screening for cell viability. Research results demonstrate that the linear polypeptide exhibits a potent inhibitory effect on vascular endothelial cells.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A linear polypeptide, the amino acid sequence of which is as follows: ECRIQERNQQQDPLVCPA (Glu-Cys-Arg-Ile-Gln-Glu-Arg-Asn-Gln-Gln-Gln-Asp-Pro-Leu-Val-Cys-Pro-Ala, SEQ ID No: 1).

[0009] The linear polypeptide is derived from the artificial gastric juice enzymatic hydrolysis product of the dried whole body of Gekko swinhonis Gunther, Gekko japonicus Dumeril et Bibron or Gekko subpalmatus Guenther, which are animals of the Gekkonidae family.

[0010] Another object of the present invention is to provide a cyclic peptide modified from the aforementioned linear peptide, wherein the sulfhydryl groups of two cysteine ​​residues in the amino acid sequence of the linear peptide are linked by a disulfide bond. The modification is intended to improve the stability of the cyclic peptide in gastric fluid relative to the linear peptide.

[0011] The above-mentioned linear polypeptide or cyclic peptide can be obtained by solid phase synthesis using conventional techniques in the art.

[0012] Another object of the present invention is to provide the use of the linear polypeptide or cyclic peptide in the preparation of vascular endothelial cell inhibitors.

[0013] Another object of the present invention is to provide the use of the linear polypeptide or cyclic peptide in the preparation of a drug for treating cancer. Preferably, the cancer is gastric cancer.

[0014] The present invention has the following advantages and effects over the existing technology:

[0015] The invention has good vascular endothelial cell inhibitory effect.

[0016] The linear polypeptide of the present invention is easy to modify in structure, easy to synthesize, has strong activity, and has obvious application value.

[0017] The linear polypeptide of the present invention is derived from geckos and has high safety. It is enzymatically hydrolyzed by artificial gastric juice to simulate the human stomach environment. Artificial gastric juice is a reagent specified in pharmacopoeias and also has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Chromatogram of solid-phase synthesized linear peptide.

[0019] Figure 2 Mass spectra of solid-phase synthesized linear peptides.

[0020] Figure 3 Analysis of the results of the inhibitory effect of different concentrations of linear peptides on VEC.

[0021] Figure 4 Chromatogram of the cyclic peptide after the linear peptide is modified.

[0022] Figure 5 Mass spectrum of the cyclic peptide after linear peptide modification.

[0023] Figure 6Analysis of the results of the inhibitory effect of different concentrations of linear peptides on VEC.

[0024] Figure 7 Analysis of the stability results of cyclic peptides and linear peptides in artificial gastric fluid.

[0025] Figure 8 Analysis of the results of cyclic peptide inhibition of gastric cancer tumor sphere growth. DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0027] Example 1 Preparation of gecko-derived linear polypeptides

[0028] Preparation of artificial gastric juice: According to the provisions of the 2020 edition of the Chinese Pharmacopoeia, take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, and dilute with water to 1000 mL.

[0029] The webless gecko Gekko swinhonis Gunther was ground into powder and passed through a 200-mesh sieve. The powder was mixed with 20 mL of artificial gastric juice at a ratio of 1 g. The mixture was shaken in a 37°C water bath shaker to simulate the human gastric environment for 2 hours. After the enzymatic hydrolysis was completed, the supernatant was centrifuged at 12,000 rpm to obtain the supernatant.

[0030] The supernatant was identified by Nano LC-MS / MS to obtain the peptides shown in Table 1.

[0031] Table 1 Identification of peptide components

[0032]

[0033]

[0034] Example 2 Investigation of the activity of different linear peptides from geckos

[0035] VEC cells were taken, and after the cells were stably passaged, different linear polypeptides prepared in Example 1 were added to the culture medium to prepare a 50 μg / mL solution, which was added to the culture dish of VEC cells. After co-culture with the solution for 24 hours, the cell viability was determined by CCK8.

[0036] Cell inhibition rate (%) = (1-absorbance of experimental group / absorbance of control group) × 100%. The results are shown in Table 2.

[0037] Table 2

[0038]

[0039]

[0040] The results showed that the linear peptide No. 3 had the highest inhibition rate of vascular endothelial cells and the best effect. Further research was conducted on this linear peptide. Figure 3 The results showed that compared with the blank group, the linear peptide had a significant inhibitory effect on VEC cells at a concentration of 50 μg / mL 24 hours after administration (p < 0.05); and the linear peptide had a significant inhibitory effect on VEC cells at a concentration of 50 μg / mL 48 hours after administration (p < 0.01). The above results indicate that the linear peptide of the present invention has a good inhibitory effect on VEC cells.

[0041] Example 3 Preparation of the Cyclic Peptide of the Present Invention

[0042] According to the structural characteristics of linear polypeptides, the cysteines at both ends are connected to form disulfide bonds through solid phase synthesis, and the linear polypeptide is further cyclized into a cyclic peptide.

[0043] The specific synthesis steps are as follows:

[0044] 1. Select an appropriate amount of modified chlorine resin according to the product amount to start peptide synthesis, add 20% pip / DMF to the reactor, and shake the reactor for 20 minutes.

[0045] 2. Filter to remove the solvent, fill the system with DMF, shake the reactor for 1 minute, and filter to remove the liquid; this operation is carried out three times.

[0046] 3. Take 150 μL of detection reagents A and B and a little resin and add them to the detection tube. Place the detection tube at 100℃ for 30 seconds and check whether the resin changes color. If the color changes, it means that Fmoc removal is successful.

[0047] 4. Add the prepared amino acid solution to the reactor and add 1 mL of DIC / DMF solution. Oscillate the reactor for 1 hour.

[0048] 5. Repeat the steps in step 3. If the color does not change, the coupling is successful.

[0049] 6. Repeat step 2 to wash the resin.

[0050] 7. Repeat the above steps and add the corresponding amino acids until the last amino acid is condensed.

[0051] 8. After amino acid condensation is complete, the peptide is cleaved from the resin and the crude product is analyzed to determine the retention time of the target peptide.

[0052] 9. Determine the peptide solubilization method based on its hydrophobicity and isoelectric point, and dissolve the peptide. Filter the solution through a 0.22 μm organic filter membrane into a 50 mL centrifuge tube.

[0053] 10. The preparation method of the peptide was determined based on its quantity, purity, and isoelectric point. The solution was injected into the instrument, and fractions were collected based on HPLC and purity. The fractions were analyzed for quality by MS and HPLC (Inertsil ODS-3 column, 4.6 x 250 mm; mobile phase A: 0.065% trifluoroacetic acid in water; mobile phase B: 0.05% acetonitrile in water; gradient elution program: 0.01-25 min, mobile phase B 5-65%; 25-25.01 min, mobile phase B 65-95%; 25.01-27 min, mobile phase B 95-95%; 27-35 min, mobile phase B 95-5%; 35-35.01 min, mobile phase B 5-5%; detection wavelength 220 nm). Qualified fractions were collected and lyophilized to obtain lyophilized powder.

[0054] The chromatogram of the cyclic peptide after the linear peptide is modified is as follows Figure 4 shown.

[0055] The structure of the synthesized cyclic peptide was identified by Maldi-TOF-MS. Figure 5 The results showed that the synthesis was successful.

[0056] Example 4 VEC cell inhibitory activity of the cyclic peptide of the present invention

[0057] VEC cells were taken, and after the cells were stably passaged, the cyclic peptide was added to the culture medium to prepare drug solutions of different concentrations, which were added to the culture dishes of VEC cells, and the cell viability was determined by CCK8.

[0058] The results are as follows Figure 6 The results showed that compared with the blank group, the cyclic peptide had a significant inhibitory effect on VEC cells at a concentration of 10 μg / mL after 24 hours of administration (p<0.05); the cyclic peptide had a significant inhibitory effect on VEC cells at a concentration of 5 μg / mL after 48 hours of administration (p<0.01). The above results indicate that the cyclic peptide has a good inhibitory effect on VEC cells.

[0059] Example 5 Stability Study of the Linear and Cyclic Peptides of the Present Invention

[0060] The cyclic peptide and linear peptide were dissolved in artificial gastric juice respectively, and placed in a 37-degree Celsius water bath shaker to simulate the human gastric environment and shaken for 2 hours. Samples were taken at different time points for content determination.

[0061] The results are as follows Figure 7 As shown, the results showed that according to the peak areas of cyclic peptides and linear peptides at different time points in artificial gastric juice measured by HPLC, the stability of cyclic peptides in the artificial gastric juice environment was significantly improved compared with linear peptides.

[0062] Example 6

[0063] Gastric cancer MKN45 cells were co-cultured with vascular endothelial cells to form 3D tumor spheres. Cyclic peptide was added to the culture medium to prepare different concentrations of drug solutions, which were then added to the culture dishes of the 3D tumor spheres to measure the growth size of the tumor spheres.

[0064] The results are as follows Figure 8 As shown, the cyclic peptide can effectively inhibit the growth of 3D tumor spheres (fluorescence microscopy was used to observe and photograph for 3 consecutive days after administration. The results showed that the growth of gastric cancer tumor spheres was significantly inhibited with the increase of drug concentration. The results of live and dead cell staining (red for apoptotic cells and green for live cells) showed that: with the increase of drug concentration, the apoptotic cells in gastric cancer tumor spheres increased significantly).

Claims

1. A linear polypeptide, characterized in that The amino acid sequence is as follows: ECRIQERNQQQDPLVCPA.

2. A cyclic peptide formed based on the linear polypeptide according to claim 1, characterized in that The sulfhydryl groups on two cysteine ​​residues in the amino acid sequence of a linear peptide are connected by a disulfide bond to form a cyclic peptide.

3. Use of the cyclic peptide according to claim 2 in the preparation of a drug for treating gastric cancer.

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