A cyclic polypeptide and its use in the preparation of an anti-tumor drug

Abstract

The application discloses a cyclic polypeptide and application thereof in preparation of an anti-tumor drug. The amino acid sequence of the cyclic polypeptide is as follows: CKGKFKRPPLKRVRMSADAMLC. The application further provides a preparation method of the cyclic polypeptide, which comprises the following steps: cloning a DNA sequence encoding the cyclic polypeptide into a prokaryotic expression vector without trace, then transforming E. coli DH5alpha, inducing expression by IPTG, and removing a label by SUMO protease treatment to obtain the cyclic polypeptide. The prepared cyclic polypeptide has improved enzymatic stability and a longer half-life, that is, the cyclic polypeptide can remain in the body for a longer time. The application further finds through experiments that the cyclic polypeptide can inhibit the growth and proliferation of various tumor cells, and therefore, the cyclic polypeptide can be used in the aspect of anti-tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a cyclic polypeptide and its application in the preparation of antitumor drugs. Background Technology

[0002] Liver cancer is one of the six most common cancers worldwide, with hepatocellular carcinoma (HCC) being the most prevalent subtype causing liver cancer death. Despite advances in diagnosis and treatment, most patients with advanced HCC have a survival rate of less than one year. Currently, there are no specific drugs for HCC in clinical practice; therefore, the development of novel treatments remains a hot topic.

[0003] Peptide drugs are peptides with specific therapeutic effects obtained through chemical synthesis, genetic recombination, or extraction from plants and animals. They represent the specific applications of peptides in the pharmaceutical field. Peptide drugs possess unique advantages in the pharmaceutical industry. Compared to small-molecule chemical drugs, peptide drugs offer advantages such as high bioactivity and targeting, strong specificity, higher efficacy, and greater safety. Furthermore, since peptides are compounds composed of amino acids, their metabolites are also amino acids, resulting in fewer side effects on the human body. Compared to protein drugs, peptide drugs also offer numerous advantages such as better stability, higher purity, lower production costs, and low or no immunogenicity. Therefore, based on these unique advantages, peptide drugs are widely used in the treatment of diabetes, cancer, hepatitis, and other diseases. For example, in cancer treatment, the water-soluble nonapeptide leuprorelin can stimulate the pituitary gland to secrete gonadotropins, used to treat or alleviate various sex hormone-dependent diseases such as prostate cancer. In addition, silengiptide, widely used in cancer treatment, exerts its anti-tumor effect by blocking the binding of integrins to the extracellular matrix, inhibiting integrin-mediated signal transduction processes, and thereby blocking cell proliferation, survival, and migration. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a cyclic polypeptide.

[0005] Another object of the present invention is to provide the use of the cyclic polypeptide in the preparation of antitumor drugs.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A cyclic polypeptide with the following amino acid sequence: CKGKFKRPPLKRVRMSADAMLC (SEQ ID NO.1).

[0008] The DNA sequence and nucleotide sequence of the cyclic polypeptide are shown in SEQ ID NO.2.

[0009] A recombinant expression vector containing the DNA sequence of the aforementioned cyclic polypeptide.

[0010] A recombinant bacterium was prepared by transforming Escherichia coli competent cells with the above-mentioned recombinant expression vector.

[0011] The method for preparing the cyclic polypeptide includes the following steps:

[0012] (1) The DNA sequence of the above-mentioned cyclic polypeptide was cloned into the prokaryotic expression vector pET-N-His-PreScission-SUMO without leaving a trace to obtain the prokaryotic expression plasmid;

[0013] (2) The prokaryotic expression plasmid obtained in step (1) was transformed into Escherichia coli, induced to express by isopropyl-β-D-thiogalactoside (IPTG), and then the tag was removed by SUMO protease to obtain a cyclic polypeptide.

[0014] The Escherichia coli mentioned in step (2) is Escherichia coli DH5α.

[0015] The induction concentration of isopropyl-β-D-thiogalactoside (IPTG) in step (2) is 1 mmol / L.

[0016] The preferred induction conditions in step (2) are: induction at 37°C for 4 hours.

[0017] The method for preparing the cyclic polypeptide further includes a step of further purifying the cyclic polypeptide obtained in step (2); the purification is achieved through the following steps: after inducing expression of isopropyl-β-D-thiogalactoside (IPTG), the supernatant is taken after ultrasonic disruption, and purified sequentially by metal chelate affinity chromatography and ion exchange chromatography. Then, the SUMO-His tag is removed by SUMO enzyme treatment, and then purified by metal chelate affinity chromatography to obtain the purified cyclic polypeptide.

[0018] The application of the cyclic polypeptide in the preparation of antitumor drugs.

[0019] The tumors mentioned include liver cancer.

[0020] The application of the cyclic polypeptide in the preparation of drugs that inhibit the growth / proliferation of liver cancer cells.

[0021] The effective concentration of the cyclic polypeptide is 12.5–100 μmol / L; preferably 50 μmol / L.

[0022] The liver cancer cells include at least one of human liver cancer cells SMMC-7721, HepG2, Huh7, MHCC97H, and MHCC97L; preferably human liver cancer cells MHCC97L.

[0023] The drug may contain one or at least two pharmaceutically acceptable carriers.

[0024] The carrier is preferably a slow-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant, and lubricant.

[0025] The dosage forms of the drug are decoction, tablets, pills, capsules, injections (such as powder injections), granules (powders), oral liquids, or syrups.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] 1. The cyclic polypeptide (named ctSAIF) in this invention has improved enzymatic stability, a longer half-life, and can be retained in vivo for a longer period of time.

[0028] 2. The cyclic polypeptide ctSAIF in this invention has a significant inhibitory effect on a variety of tumor cells, including human liver cancer cell lines (7721, HepG2, Huh7, MHCC97H, MHCC97L).

[0029] 3. The cyclic polypeptide ctSAIF in this invention inhibits the development of liver cancer xenografts in mice.

[0030] 4. The cyclic polypeptide ctSAIF in this invention can be synthesized through bioengineering, without damaging the ecological environment, and is easy to industrialize, making it a very promising anti-tumor drug resource.

[0031] 5. The cyclic polypeptide ctSAIF in this invention has anti-tumor effects at both the cellular and animal levels. Therefore, it can be used to prepare anti-tumor drugs for the treatment of liver cancer. Attached Figure Description

[0032] Figure 1 This is a design concept diagram of the ctSAIF peptide in this invention; wherein, A is the HPLC chromatogram of the SAIF in vitro serum degradation experiment; B is the SAIF in vitro serum degradation curve; C is the total ion chromatogram of the SAIF in vitro serum degradation experiment; and D is a summary table of amino acid sequences.

[0033] Figure 2This is a process diagram of the bioengineering synthesis of ctSAIF peptide in this invention; wherein, A is the SDS-PAGE results before and after induction (lane 1: Marker; lane 2: whole bacteria before induction; lane 3: whole bacteria after induction; lane 4: supernatant of bacterial lysate after induction; lane 5: precipitate of bacterial lysate after induction); B is the SDS-PAGE results before and after purification (lane 1: bacterial cell lysate; lane 2: Ni column flow-through buffer; lanes 3 and 4: two elution peaks of Ni column; lane 5: 30Q flow-through buffer; lanes 6 and 7: two elution peaks of 30Q; lane 8: protein molecular weight standard); C is the SDS-PAGE results before and after purification (lane 1: Marker; lane 2: B2C-SUMO; lane 3: Mix after enzyme digestion; lane 4: Ni column flow-through buffer; lane 5: Ni column elution peak); D is the mass spectrum result of ctSAIF; E is the in vitro serum degradation curve of cyclic peptide ctSAIF and SAIF.

[0034] Figure 3 This is a graph showing the results of the ctSAIF polypeptide in this invention inhibiting the growth of liver cancer cell lines in vitro; where A represents the effect of the two polypeptides on the viability of human hepatocytes 7701 and human liver cancer cell lines (7721, MHCC97H, HepG2, Huh7, MHCC97L); B represents the inhibitory effect of the two polypeptides on the colony formation of human hepatocytes 7701 and human liver cancer cells (7721, MHCC97H, HepG2, Huh7, MHCC97L); and C represents the statistical histogram of cell colonies.

[0035] Figure 4 This is a graph showing the results of the ctSAIF polypeptide in this invention inhibiting liver cancer xenografts in mice; where A is a visual graph of tumor size; B is a curve of tumor volume; C is the tumor weight 14 days after SAIF administration; D is the weight change of nude mice during drug treatment; and E is Ki67 staining of tumor sections from nude mice. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0037] Example 1: Design concept and production method of ctSAIF peptide

[0038] 1.1 Experimental Design

[0039] In our in vitro serum stability analysis of the peptide SAIF (sequence KEIDDLNLKVFDLKGKFKRPPLKRVRMSADAML, synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) (method described in 1.4), HPLC revealed poor stability with a half-life of only 2–3 h. However, the HPLC chromatogram showed several new peaks with retention time drift, which we speculated were degradation peaks. These new peaks persisted even after 12 h, suggesting that the stability of the degradation peaks might be improved. Figure 1 A, Figure 1 B Figure 1 C); We performed mass spectrometry analysis, which revealed a degradation peak. This peak remained at a high level even after 12 hours of co-incubation with serum in vitro. The molecular weight, determined by primary mass spectrometry, was 2395.38. We named this sequence ctSAIF. Based on the SAIF sequence, we enumerated the molecular weights of all possible degradable peptides, thus identifying the corresponding amino acid sequence at this molecular weight as: CKGKFKRPPLKRVRMSADAMLC( Figure 1 D).

[0040] 1.2 Preparation of ctSAIF

[0041] 1.2.1 Overview

[0042] The DNA sequence of ctSAIF (GTGAGCAGATTGGAGTGCAAGGGCAAGTTCAAGAGGCCCCCCCTGAAGAGGGTGAGGATGAGCGCCGACGCCATGCTGTGCGATCCGAATTCGAGCT) (SEQ ID NO. 2) was obtained by the amino acid sequence of ctSAIF. After the DNA sequence was chemically synthesized by Shanghai Sangon Biotech Co., Ltd., it was cloned into the prokaryotic expression vector pET-N-His–SUMO (which has been published in the reference: Xie Q, Yao S, Chen X, Xu L, Peng W, Zhang L, Zhang Q, Liang XF, Hong A: A polypeptide from shark troponin I can inhibit angiogenesis and tumor growth. Molecular biology reports 2012, 39(2): 1493-1501.). This is a prokaryotic expression plasmid for expressing a target protein with both a His tag and a SUMO tag at the N-terminus. The plasmid was then transformed into *E. coli* DH5α and induced with isopropyl-β-D-thiogalactoside (IPTG) (final IPTG concentration 1 mM, temperature 37℃, induction for 4 h) to obtain the fusion protein ctSAIF-SUMO-His. After further treatment with SUMO protease (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P2312S) to remove the tags and purify the protein, precise expression of the peptide was achieved, which was verified using mass spectrometry.

[0043] 1.2.2 Process and Results

[0044] Depend on Figure 2 Lanes 2 and 3 of swim chevron A show that after transforming the vector containing the ctSAIF sequence into *E. coli* DH5α, compared with the control without IPTG induction, a large amount of protein with a molecular weight of approximately 21 kDa was expressed after induction. After resuspending the cells (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA; pH 7.5), and after sonication to disrupt the bacterial count, [the cells were then...]. Figure 2 As can be seen from lanes 4 and 5 of A, most of the inducible proteins exist in the supernatant in a soluble form.

[0045] The lysed supernatant was loaded onto a Ni Sepharose 6Fast Flow metal chelate affinity chromatography system. After elution (eluent: 20 mM Tris, 300 mM imidazole, pH 8.0), it was loaded onto a Source 30Q ion exchange chromatography system and eluted (eluent: 20 mM Tris, 300 mM sodium chloride, pH 8.0). Figure 2 Lane B, 6, showed a single band of ctSAIF-SUMO-His with a molecular weight of approximately 21 kDa. The purified ctSAIF-SUMO-His was then treated with SUMOase to remove the SUMO-His tag, followed by digestion at 4°C for 16 hours. The results are as follows... Figure 2 As shown in lane C, ctSAIF-SUMO-His was separated into approximately 18 kDa SUMO-His (lane 2) and approximately 3 kDa single-chain ctSAIF (lane 3). The enzymatic digest was then subjected to metal chelate affinity chromatography (NiSepharose 6Fast Flow, eluent: 20 mM Tris, 300 mM imidazole, pH 8.0). The flow-through (20 mM Tris, pH 8.0) was collected (lane 4) to obtain relatively pure single-chain ctSAIF. All purification processes were performed at room temperature using an AKTAavant instrument (catalog number 28930842). All chromatography columns were purchased from GlaxoSmithKline Biotechnology Co., Ltd., with the NiSepharose 6Fast Flow packing material catalog number 17531805 and the Source 30Q packing material catalog number 17127302, both at a flow rate of 1 mL / min.

[0046] 1.3 Preparation of cyclic polypeptide ctSAIF

[0047] To obtain the cyclic peptide, we oxidized the single-chain ctSAIF to dehydrogenate the first and last cysteine ​​residues to form disulfide bonds. The specific steps were as follows: Dimethyl sulfoxide (DMSO) was added to the purified single-chain ctSAIF solution to a final concentration of 5% (v / v), the pH was adjusted to 8.5 with 1M NaOH solution, and the mixture was magnetically stirred at room temperature for 8 hours. During this time, the free cysteine ​​residues were monitored using Ellman's reagent until no free cysteine ​​residues were detected, indicating that the oxidation was complete.

[0048] Subsequent mass spectrometry analysis of the oxidation products verified whether ctSAIF molecules would form dimers. The results showed that, with a molecular weight of 2534.20, no dimers formed between ctSAIF molecules. Figure 2 D) indicates that the cyclization has yielded the cyclic polypeptide ctSAIF.

[0049] 1.4 In vitro stability experiment of cyclic polypeptide ctSAIF

[0050] Rat serum (freshly collected from 8-week-old SD rats, purchased from Guangdong Yaokang Biotechnology Co., Ltd.) was mixed with peptides at a ratio of 9:1 (w / v) to achieve a peptide concentration of 1 mg / mL. The peptide-serum mixture was incubated in a 37°C water bath for different times. After reaching the predetermined time, pure trifluoroacetic acid was added to each sample to achieve a final trifluoroacetic acid concentration of 10% (v / v). After shaking, the mixture was centrifuged at 13000 rpm for 10 min, and 10 μL of the supernatant was collected into an HPLC sample vial for later use.

[0051] HPLC-UV analysis was performed using the method in Table 1 (instrument model: Waters ACQUITY Arc Bio). A C18 analytical column (Thermo Acclaim300 C18) was used. The target peak was determined based on the retention time, its peak area was recorded, and a degradation curve was plotted.

[0052] Following the pretreatment and chromatographic methods in Table 1, trifluoroacetic acid (TFA) was replaced with formic acid, and high-resolution mass spectrometry (ABSCIEX 500R) was used for analysis to obtain a total ion chromatogram and a first-order mass spectrum. The instrument calculated the molecular weight of the main peak based on the mass-charge ratio.

[0053] result Figure 2 As shown in E, the half-life of ctSAIF is extended from about 2 hours for SAIF to about 6 hours.

[0054] Table 1

[0055] 0min 95％ 5％ 27min 75％ 25％ 28min 5％ 95％ 33min 5％ 95％ 40min 95％ 5％ 45min 95％ 5％

[0056] Example 2: Effects of cyclic polypeptide ctSAIF on the growth of liver cancer cells in vitro

[0057] 2.1 Experimental Materials

[0058] Cells: Since the goal of this invention is to inhibit tumor formation, five human liver cancer cell lines were selected: SMMC-7721 (abbreviated as 7721), HepG2, Huh7, MHCC97H (abbreviated as 97H), and MHCC97L (abbreviated as 97L), with normal liver cells QSG-7701 (abbreviated as 7701) as the control. All six cell lines were purchased from the ATCC cell bank.

[0059] Reagents: cyclic polypeptide ctSAIF, SAIF polypeptide, and DMEM culture medium prepared in Example 1.

[0060] 2.2 Experimental Methods

[0061] Cells in good growth phase, in the logarithmic growth phase, were seeded at a density of 2500 cells per well in 96-well plates and incubated at 37°C in a 5% (v / v) CO2 incubator for 24 hours. Then, the old culture medium was removed, and the cells were starved by treatment with DMEM medium (containing 0.5% (v / v) fetal bovine serum) for 24 hours. The cyclic peptide ctSAIF was diluted to different concentrations (12.5, 25, 50, 100 μM) in DMEM medium containing 0.5% (v / v) fetal bovine serum, with 100 μL added to each well. After returning to the 37°C incubator for another 48 hours, the culture medium was removed, and 100 μL / well of CCK-8 working solution was added, followed by incubation at 37°C in a 5% (v / v) CO2 incubator for 1 hour. Finally, the absorbance of each well at 450 nm was measured using a multi-mode microplate reader. Three replicates were performed, with no peptide drug as a blank control and SAIF peptide as a control.

[0062] 2.3 Experimental Results

[0063] To determine whether ctSAIF has the same tumor-suppressive effect as SAIF, we used liver cancer cells as a cell model for analysis. The results are as follows: Figure 3 As shown, the inhibitory effects of SAIF and ctSAIF cyclic peptide on human HCC cell proliferation were first verified by cell proliferation assays. It was found that after treatment with different concentrations of SAIF or ctSAIF cyclic peptide for 48 hours, both SAIF and ctSAIF cyclic peptide reduced cell viability in a concentration-dependent manner. Figure 3 A). Simultaneously, the clonogenic assay ( Figure 3 B) also indicates that SAIF and B2 peptide have a long-term inhibitory effect on HCC cell proliferation. Furthermore, the human normal hepatocyte cell line 7701 was used to preliminarily evaluate the toxicity of SAIF and ctSAIF cyclic peptide, and the results showed that neither peptide had significant toxicity to the normal hepatocyte cell line.

[0064] Example 3: In vivo antitumor activity of the cyclic polypeptide ctSAIF

[0065] 3.1 Experimental Materials

[0066] Animals: 5-week-old BALB / c-nude mice (average weight 25g, 12 mice in total), purchased from Guangdong Yaokang Biotechnology Co., Ltd., product number D000521.

[0067] Reagents: cyclic polypeptides ctSAIF and SAIF polypeptide prepared in Example 1.

[0068] 3.2 Experimental Methods

[0069] Male BALB / c-nude mice were fed diet and purified water at room temperature in an SPF laboratory. MHCC97L cells (0.1 mL of physiological saline containing 1×10⁻⁶ cells) were added. 7 (1 cell) was subcutaneously injected into the axilla of a 5-week-old male BALB / c-nude. Tumor volume and body weight were measured every 2 days, and tumor volume was calculated using the following formula: (short diameter)² × (long diameter) / 2. When the tumor volume reached approximately 50 mm, the tumor was considered complete. 3 These mice were randomly divided into three groups: a model group (as a control), a SAIF treatment group (25 mg / kg), and a ctSAIF cyclic peptide treatment group (25 mg / kg). SAIF and ctSAIF cyclic peptide were administered via tail vein injection at a volumetric dose of 0.1 mL / 20 g. Mice were sacrificed 14 days after administration, and tumors were collected for histopathological examination.

[0070] 3.3 Experimental Results

[0071] The results are as follows Figure 4 As shown, compared with the control group, the mean tumor volume (MTBV) was observed after treatment with ctSAIF. Figure 4 (A and 4B) and average tumor weight ( Figure 4 C) significantly reduced. During the treatment, there was no significant change in the body weight of the nude mice in any group. Figure 4 D). Meanwhile, Ki-67 staining of tumor tissue further confirmed that both SAIF and ctSAIF cyclic peptide could inhibit tumor cell growth. Figure 4 E).

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A cyclic polypeptide, characterized in that, The amino acid sequence is shown below: CKGKFKRPPLKRVRMSADAMLC.

2. The use of the cyclic polypeptide according to claim 1 in the preparation of antitumor drugs, characterized in that: The tumor in question is liver cancer.

3. The use of the cyclic polypeptide of claim 1 in the preparation of a drug for inhibiting the growth / proliferation of liver cancer cells.

4. The application according to claim 3, characterized in that: The effective concentration of the cyclic polypeptide is 12.5–100 μmol / L.

5. The application according to claim 4, characterized in that: The effective concentration of the cyclic polypeptide is 500 μmol / L.

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