CN114957399B - A kind of polypeptide, polypeptide derivative and its application in the preparation of antitumor drug - Google Patents

Abstract

The invention discloses a polypeptide, a polypeptide derivative and application thereof in preparing an anti-tumor medicament, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1 or SEQ ID NO. 2, and the polypeptide derivative is chimeric peptide formed by connecting the polypeptide with cell penetrating peptide. The polypeptide and the polypeptide derivative provided by the invention can effectively block the connection of FAM83A protein and beta-catenin protein, so as to achieve the effect of inhibiting the activity of Wnt channels; and the compound can inhibit proliferation, migration, invasion and other abilities of pancreatic cancer cells, so that the compound has great application potential in preparation of antitumor drugs.

Description

A polypeptide, a polypeptide derivative and its application in preparing anti-tumor drugs

Technical Field

The present invention belongs to the field of pharmaceutical technology, and specifically relates to a polypeptide and a polypeptide derivative, and application thereof in the preparation of anti-tumor drugs.

Background Art

The Wnt signaling pathway is widely present in invertebrates and vertebrates and is a highly conserved signaling pathway in the process of species evolution. Wnt signaling plays a vital role in the early development of animal embryos, organ formation, tissue regeneration and other physiological processes. If the key proteins in this signaling pathway mutate or are abnormally expressed, resulting in abnormal activation of the signal, it may induce cancer.

The Wnt signaling pathway includes the classical Wnt signaling pathway and the non-classical Wnt signaling pathway. In the classical pathway, namely the Wnt-β-catenin signaling pathway, the Wnt factor inhibits the phosphorylation and degradation of free β-catenin protein in the cell by activating the Frizzle/LRP5/6 co-receptor on the cell membrane. When the β-catenin protein level in the cytoplasm increases, the β-catenin protein will be nuclear translocated, resulting in an increase in the β-catenin protein in the nucleus. The β-catenin protein in the nucleus can form a complex with the TCF/LEF-1 transcription factor family in conjunction with Pygo2, Bcl-9 and FoxM1 proteins and activate the transcriptional activation of downstream target genes in the Wnt signaling pathway. Therefore, designing specific protein drugs, especially peptide drugs, for this signaling pathway to treat tumors has important value and application prospects.

More and more studies have shown that the family with sequences similarity 83 member A gene (FAM83A) is associated with tumor invasion and metastasis, and FAM83A is abnormally highly expressed in lung cancer, breast cancer and pancreatic cancer, and the upregulation of this gene is closely correlated with tumor formation, prognosis and other indicators. However, the clinical significance of FAM83A in cancer and its mechanism of action in cancer are still unclear.

Summary of the invention

In response to the problems in the prior art, the present invention, through multi-sample clinical results and biological means, found that FAM83A protein can directly interact with β-catenin protein and thus promote the abnormal activation of the Wnt signaling pathway in cells, and further determined the protein interval where the interaction occurs. Based on this, a polypeptide that can block the interaction between FAM83A and β-catenin in cells was obtained. The experimental results show that the polypeptide molecule can significantly inhibit the activation of the Wnt signaling pathway in cells and has a significant inhibitory effect on the growth of pancreatic cancer cells.

The technical solution of the present invention is specifically as follows:

The present invention first provides a polypeptide or a polypeptide derivative, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the polypeptide derivative is a chimeric peptide formed by connecting the above polypeptide with a cell-penetrating peptide.

Furthermore, in the above technical solution, the cell-penetrating peptide is connected to the N-terminus of the polypeptide; further, the sequence of the cell-penetrating peptide is shown in SEQ ID NO:3.

Furthermore, in the above technical solution, the N-terminus of the polypeptide or polypeptide derivative is acetylated and the C-terminus is amidated.

The present invention also provides an anti-tumor pharmaceutical composition, the active ingredient of which contains a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a derivative of the polypeptide.

Furthermore, in the above technical solution, the pharmaceutical composition also includes a drug carrier.

The present invention further provides the use of the above polypeptide, or polypeptide derivative, or anti-tumor pharmaceutical composition in the preparation of anti-tumor drugs.

Furthermore, in the above technical solution, the tumor is pancreatic cancer.

Furthermore, in the above technical solution, an effective amount of the drug is administered to an individual. Furthermore, the administration method is injection.

The beneficial effects of the present invention are as follows: based on the direct contact area between the FAM83A protein and the β-catenin protein, the polypeptide and polypeptide derivative provided by the present invention can effectively block the connection between the FAM83A protein and the β-catenin protein, thereby achieving the effect of inhibiting the activity of the Wnt pathway. The test results also show that the polypeptide of the present invention can inhibit the proliferation, migration and invasion of pancreatic cancer cells, thereby being able to exert an anti-tumor function well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the screening results of the interaction region between FAM83A protein and β-catenin protein;

FIG2 is a diagram showing the binding strength of FaP1 and FaP2 polypeptides to β-catenin protein;

FIG3 is a schematic diagram of the binding principle of the FaP2 polypeptide and the β-catenin protein structure;

FIG4 is a graph showing the effect of the short peptide CP-FaP2 on the expression of target genes AXIN2, C-myc and CyclinD1 in pancreatic cancer cells;

FIG5 is a graph showing the detection result of the short peptide CP-FaP2 inhibiting the proliferation of pancreatic cancer cells;

FIG6 is a graph showing the detection results of the ability of the short peptide CP-FaP2 to inhibit the invasion and migration of pancreatic cancer cells;

FIG. 7 is a graph showing the detection results of the short peptide CP-FaP2 inhibiting pancreatic cancer tumor growth and metastasis in mice.

DETAILED DESCRIPTION

In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

Example 1

In this embodiment, the polypeptide is obtained by the following steps:

(1) PCR technology was used to construct a vector expressing FAM83A truncation.

Specifically, as shown in Figure 1, the DUF1669 domain of the FAM83A protein is first designed to be truncated every 50 amino acids. When truncating or mutating the FAM83A protein, the primer design should be centered on the mutated amino acid, with about 15bp added before and after, with a total length of about 33bp, and a pair of complementary primers should be synthesized at the same time. The annealing temperature during amplification is about 60°C. When the primer specifically binds to the template, it will replace the original base and recognize the replaced base, and the formed circular plasmid is still intact. Because the DNA obtained by the new PCR does not have methylation modification, and the plasmid extracted from Escherichia coli has methylation modification, the original template plasmid can be digested by using the methylation digestion enzyme DpnI, and the remaining plasmids are all successfully mutated. The specific process is as follows:

① PCR amplification of mutant plasmid: Use the successfully constructed plasmid as a template, and the amplified length is the total length of the target fragment and the vector. Because additional mutations are more likely to occur over a long period of time, a high-fidelity enzyme should be selected for the amplification experiment to prevent new mutations.

② Recover the product after PCR using a gel recovery kit and finally elute with 43 μL ddH ₂ O.

③The recovered DNA product needs to be digested with DpnI and digested in a 37℃ water bath for 1h. The enzyme digestion system is shown in the following table:

④ After enzyme digestion, the enzyme digestion products should be recovered according to the enzyme digestion product kit and eluted with _30μLddH2O .

⑤Finally, the purified product obtained in step ④ was transformed into Escherichia coli, and a single colony was picked and shaken, and then sequenced directly. The strain with the correct sequencing result and successful mutation was preserved with glycerol or directly expanded and cultured to extract the plasmid for subsequent experimental use.

(2) Determination of the interaction region between FAM83A and β-catenin

The truncated forms obtained by (1) were co-transfected with the β-catenin expression vector into HEK293T cells, and the binding between the truncated forms of FAM83A and the β-catenin protein was identified by immunoprecipitation and western blotting.

Immunoprecipitation technology is a common method for verifying the interaction between proteins in cells based on the specific binding between antigens and antibodies; western blotting is a method of detecting target proteins by using antibodies to recognize proteins. Both are existing technologies and will not be described in detail here.

The final test results are shown in Figure 1: In the DUF1669 domain of the FAM83A protein, the 150-250 amino acid region is the direct contact area between the FAM83A protein and the β-catenin protein.

(3) Determination of peptide sequence

The I-TASSER database was used to predict the secondary structure of the FAM83A protein in the 140-210 amino acid range, and two α-helical peptides (with amino acid regions of 145-157 and 171-184) were screened out as the basis for designing peptide sequences. The two obtained peptides are:

FaP1, whose amino acid sequence is: NNIRDLVRRCITR (SEQ ID NO: 1);

FaP2, the amino acid sequence of which is: DVEIFCDILEAAN (SEQ ID NO: 2).

FaP1, FaP2, and a control peptide (denoted as FaPC, with an amino acid sequence of YIQAQAREPPCPPD (SEQ ID NO: 4)) were fused with GFP fluorescent protein to construct a fusion expression vector. After transfection into HEK293T cells, immunoprecipitation and western blotting techniques were used to verify their binding with the target protein β-catenin. It was found that FaP2 had a stronger interaction with β-catenin than FaP1 (Figure 2).

The online small molecule docking software was further used to predict the docking results of the short peptide FaP2 and the protein structure in the 530-666 region of β-catenin. The results showed that the short peptide FaP2 binds to the minor groove formed by the secondary structure of the β-catenin protein (Figure 3).

As can be seen from Figures 2 and 3, the polypeptide FaP2 can target and bind to the β-catenin protein.

Example 2

Based on the polypeptide FaP2 obtained in Example 1, the cell-penetrating peptide TAT was connected to its N-terminus, and the sequence of the peptide was YGRKKRRQRRR (SEQ ID NO: 3), and the N-terminus of the polypeptide was acetylated and the C-terminus was amidated. The obtained short peptide with the cell-penetrating peptide was named CP-FaP2.

The anti-tumor function of CP-FaP2 was verified as follows:

(1) Effects of CP-FaP2 on the Wnt signaling pathway

Pancreatic cancer PANC-1 cells were treated with CP-FaP2 for 24 hours at a final concentration of 5 μM. Cell lysates were collected and western blotting was performed to detect the expression levels of Wnt signaling pathway target genes AXIN2, C-myc, and CyclinD1.

The results are shown in Figure 4: Compared with the normal polypeptide (i.e., the control peptide connected with the same cell-penetrating peptide, denoted as CP-FaPC) treatment, CP-FaP2 treatment can significantly reduce the expression level of downstream target proteins of the Wnt signaling pathway, that is, CP-FaP2 can affect the activation of the Wnt pathway.

(2) Effect of CP-FaP2 on the proliferation of pancreatic cancer cells

The Edu (5-ethynyl-2'-deoxyuridine) cell proliferation assay was used for detection (this example was performed according to the instructions of the BeyoClick ^™ EdU-488 Cell Proliferation Detection Kit). After adding 5 μM CP-FaP2 to pancreatic cancer AsPC-1 cells for 24 hours, the newly synthesized DNA in the cells was stained with Edu dye, and the effect on cell proliferation was determined by the number of green-stained cells.

The results are shown in Figure 5: CP-FaP2 can indeed inhibit the proliferation of pancreatic cancer AsPC-1 cells.

(3) Effect of CP-FaP2 on the migration and invasion of pancreatic cancer cells.

The trans-well chamber is placed in a culture plate. The inner chamber is called the upper chamber, and the inner chamber is called the lower chamber. The upper chamber contains the upper culture fluid (2% serum concentration), and the lower chamber contains the lower culture fluid (20% serum concentration). The upper and lower culture fluids are separated by a polycarbonate membrane. When cells are placed in the upper chamber of the trans-well, due to the lack of serum concentration, the cells tend to pass through the polycarbonate membrane and enter the culture medium with high serum in the lower chamber. The migrated cells are stained with crystal violet for easy statistics. This method is used to detect the migration of cells. If a layer of artificial basement membrane is placed in the upper chamber, the process of cells passing through the artificial basement membrane and then entering the lower chamber can be simulated. This method is used to detect the invasiveness of cells.

Based on the above principle, after pancreatic cancer PANC-1 cells were plated in the upper chamber, the cells were treated with the short peptide CP-FaP2 for 24 hours. It was found that the number of cells migrating or invading to the lower chamber was significantly reduced, as shown in Figure 6, indicating that CP-FaP2 has the ability to inhibit the invasion and migration of pancreatic cancer cells.

Example 3

This example directly studies the effect of the short peptide CP-FaP2 on the growth of pancreatic cancer in animals. The specific process is as follows:

First, 3×10 ⁶ pancreatic cancer cells were injected subcutaneously and into the tail vein of 4-week-old female nude mice, respectively. Two weeks later, CP-FaP2 (at a concentration of 2 mg/kg/3d) was injected into the nude mice through the tail vein. Four weeks later, the volume of subcutaneous tumors formed in the nude mice and the number of tumors metastasized to the lungs and colon of the nude mice were weighed.

The results are shown in FIG7 : CP-FaP2 can significantly inhibit the growth of pancreatic cancer cells in nude mice ( FIG7 A, B ) and their metastasis to the lung ( FIG7 C ) and colon ( FIG7 D ).

In summary, the polypeptides FaP1, FaP2 and their derivatives CP-FaP2 provided by the present invention block the connection between FAM83A protein and β-catenin protein by acting on the direct contact area between FAM83A protein and β-catenin protein, thereby regulating the activity of the Wnt pathway. Moreover, experiments have shown that the above-mentioned polypeptides and their derivatives can inhibit the proliferation, migration and invasion of pancreatic cancer cells, and in vivo animal experiments have shown that they can inhibit the volume growth and metastasis of pancreatic cancer tumors. Therefore, the polypeptides and their derivatives provided by the present invention have great application potential in the preparation of anti-tumor drugs.

The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Sequence Listing

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Claims (9)

1. A polypeptide or a polypeptide derivative, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the polypeptide derivative is a chimeric peptide formed by connecting the polypeptide with a cell-penetrating peptide. 2. The polypeptide or polypeptide derivative according to claim 1, characterized in that the cell-penetrating peptide is connected to the N-terminus of the polypeptide. 3. The polypeptide or polypeptide derivative according to claim 1, characterized in that the N-terminus of the polypeptide or polypeptide derivative is acetylated and the C-terminus is amidated. 4. The polypeptide or polypeptide derivative according to claim 1, characterized in that the sequence of the cell-penetrating peptide is shown in SEQ ID NO: 3. 5. An anti-tumor pharmaceutical composition, characterized in that its active ingredient contains the polypeptide or polypeptide derivative according to any one of claims 1 to 4. 6. The pharmaceutical composition according to claim 5, characterized in that it further comprises a drug carrier. 7. Use of a polypeptide derivative in the preparation of an anti-tumor drug, characterized in that the polypeptide derivative is a chimeric peptide formed by connecting the polypeptide shown in SEQ ID NO: 2 and the cell-penetrating peptide shown in SEQ ID NO: 3, and the tumor is pancreatic cancer. 8. The use according to claim 7, characterized in that an effective amount of the drug is administered to an individual. 9. The use according to claim 8, characterized in that the administration method is injection.

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