Nail-connected peptide as well as preparation method and application thereof

Abstract

The invention discloses a nailing peptide as well as a preparation method and application thereof. The preparation method of the nail-connected peptide comprises the steps that amino resin serves as a solid-phase carrier, a peptide chain is synthesized in a condensation system through an Fmoc solid-phase synthesis method according to the amino acid sequence of a straight-chain peptide template LNWGAILKHIIK-NH2, on the basis that key amino acid residues are reserved, the original amino acid is replaced by S5 at i and i + 4 sites, i = 1-7, the straight-chain peptide connected to the resin is obtained, and the nail-connected peptide is obtained. The straight-chain peptide is subjected to olefin metathesis reaction cyclization under the catalytic action of a GrubbsI reagent and then is cut off from resin to obtain the target nail-connected peptide. The method is simple and easy to implement, the purity of the obtained nail-connected peptide is greater than 95%, and the yield is high. The synthesized nail-connected peptide has the advantages that the protein hydrolysis stability and the cell membrane penetrating power are obviously improved, the growth and reproduction of human breast cancer cells and human liver cancer cells can be obviously inhibited, and the potential application value in tumor disease treatment is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a stapled peptide and a preparation method and application thereof. Background Art

[0002] Breast cancer and liver cancer are two major malignant tumors with increasing morbidity and mortality rates worldwide, posing a serious threat to public health. However, current treatment options for these two cancers, including surgery, radiotherapy, and chemotherapy, can cause significant side effects, seriously affecting the quality of life and physical health of patients. Therefore, the development of effective anti-breast cancer or anti-liver cancer drugs with few side effects is of great clinical significance. In recent years, peptide drugs have attracted widespread attention due to their high specificity, adjustable quality, and high biological activity, which are unmatched by small molecule and protein drugs in targeted therapy and reducing off-target effects.

[0003] After consulting the literature, it was found that Panurgines (PNGs) isolated from the venom of wild bee Panurgus calcaratus had a certain inhibitory effect on MCF-7 breast cancer cells and HepG2 liver cancer cells ( Sabina, Slanino Monincová Lenka, et al. Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae). [J]. Amino acids, 2013, 45 (1): 143-57.). This discovery provides a theoretical basis for the development of new anti-breast cancer drugs or anti-liver cancer drugs using PNGs. However, linear peptides PNGs face many challenges in penetrating cell membranes and are easily hydrolyzed by proteins. In order to solve these problems, it is crucial to chemically modify PNG to develop an efficient, stable and less side-effect PNG peptide therapeutic strategy. Summary of the invention

[0004] In view of the above background, the purpose of the present invention is to improve the structure of PNGs, improve their proteolytic stability and cell membrane penetration ability, so as to develop a new type of polypeptide derivative with potential anti-breast cancer and anti-liver cancer therapeutic effects. On the basis of existing literature, the present invention comprehensively studies the application of various hydrocarbon stapling strategies in the design and synthesis of PNG derivatives, and uses hydrocarbon stapling strategies to design and synthesize a series of new PNG derivatives with excellent proteolytic stability. After a series of experimental explorations and optimizations, the present invention screened out the polypeptide derivative PNG5 with the best effect of inhibiting tumor cell proliferation, which has potential therapeutic effects on the treatment of breast cancer and liver cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A nailed titanium selected from the following:

[0007] a) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5A were replaced by S5 (2-amino-2-methylhept-6-enoic acid, 2-amino-2-methyl-6-enoic acid) and cyclized;

[0008] b) LNWGAILKHIIK-NH2 was used as a peptide chain template, in which amino acid residues 2N and 6I were replaced by S5 and cyclized;

[0009] c) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 3W and 7L were replaced by S5 and cyclized;

[0010] d) LNWGAILKHIIK-NH2 was used as a peptide chain template, in which amino acid residues 4G and 8K were replaced by S5 and cyclized;

[0011] e) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 5A and 9H were replaced by S5 and cyclized;

[0012] f) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 6I and 10I were replaced by S5 and cyclized;

[0013] g) LNWGAILKHIIK-NH2 was used as the peptide chain template, in which amino acid residues 7L and 11I were replaced by S5 and cyclized.

[0014] The present invention also provides the use of the stapled peptide in preparing a drug for treating breast cancer.

[0015] The present invention also provides the use of the stapled peptide in preparing a medicine for treating liver cancer.

[0016] The present invention also provides a method for preparing the stapled peptide, which uses an amino resin as a solid phase carrier, and synthesizes a peptide chain in a condensation system according to the linear peptide template LNWGAILKHIIK-NH2 amino acid sequence by Fmoc solid phase synthesis method, and on the basis of retaining key amino acid residues, replaces the original amino acid with S5 at positions i and i+4, wherein i=1-7, to obtain a linear peptide connected to the resin, and the linear peptide is subjected to olefin metathesis reaction cyclization under the catalytic action of GrubbsⅠ reagent, and then cut off from the resin to obtain the target stapled peptide.

[0017] The specific steps include:

[0018] (1) coupling the first amino acid at the C-terminus to the solid phase carrier under the action of a condensing agent;

[0019] (2) removing the Fmoc protecting group on the amino acid using a deprotection reagent;

[0020] (3) Connecting the next amino acid under the action of a condensing agent;

[0021] (4) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively;

[0022] (5) the last amino acid is deprotected and then acetylated;

[0023] (6) Under the action of a cyclizing agent, the i and i+4 positions S5 undergo olefin metathesis reaction to cyclize the peptide chain;

[0024] (7) Use a cleavage agent to cut the peptide chain from the support to obtain the corresponding stapled peptide.

[0025] The present invention has the advantages that:

[0026] 1. The present invention uses amino resin as a carrier and synthesizes a peptide chain by Fmoc solid phase synthesis in a condensation system according to the amino acid sequence of the template. On the basis of retaining the key amino acid residues, S5 is used to replace the original amino acid at a specific position. The linear peptide connected to the resin undergoes olefin metathesis reaction on Grubbs I reagent for cyclization and is then cut from the resin to obtain the target stapled peptide.

[0027] 2. The method of the present invention is simple and easy to implement, and the purity of the obtained stapled peptide is greater than 95% with high yield.

[0028] 3. The present invention designs and synthesizes stapled peptides, and experiments have confirmed that compared with the LNWGAILKHIIK-NH2 straight-chain peptide, it significantly improves the protein hydrolysis stability and cell membrane penetration ability, can significantly inhibit the growth and reproduction of human breast cancer cells and human liver cancer cells, and has potential application value in the treatment of related tumor diseases such as breast cancer and liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The synthetic route of the stapled peptide PNG-4 is shown below:

[0030] a is 20% piperidine / DMF, b is Fmoc-Aa-OH / Oxym / Dic,

[0031] c is Fmoc-S5-OH / Oxyme / Dic, d is GrubbsⅠ / DCE, and e is TIPS / TFA / H2O / phenol.

[0032] Figure 2 Circular dichroism spectra of PNG and PNG-derived stapled peptides.

[0033] Figure 3 Comparative chart of the trypsin hydrolysis stability of PNG and PNG-derived stapled peptides.

[0034] Figure 4 Fluorescence microscopic images of the MCF-7 cell membrane permeability of PNG, stapled peptides PNG-4, and PNG-5.

[0035] Figure 5 Figure 3. Study on the inhibition of MCF-7 cell colony formation by PNG, stapled peptides PNG-4 and PNG-5: (A) MCF-7 cell colonies treated with PNG, PNG-4 and PNG-5, respectively; (B) The percentage of cell colonies after treatment with PNG, PNG-4 and PNG-5 to the number of untreated cell colonies.

[0036] Figure 6 The inhibitory effect of hydrocarbon peptides on apoptosis of MCF-7 cells: (A) The apoptosis of MCF-7 cells treated with different peptide preparations. (B) The level of apoptosis was quantitatively analyzed using ImageQuant (Molecular Dynamics, USA).

[0037] Figure 7 This is the RP-HPLC spectrum of the stapled peptide PNG-4 of Example 1.

[0038] Figure 8 This is the electrospray ionization mass spectrum (ESI-MS) of the stapled peptide PNG-4 in Example 1. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0040] The present invention adopts the i, i+4 strategy to staple the polypeptide according to the template linear peptide PNG: LNWGAILKHIIK-NH2 (SEQ ID NO.1), introduces S5 at the i, i+4 position of the peptide backbone, wherein i=1-7, and then uses the Grubbs I reagent to perform olefin ring-closing metathesis reaction, specifically comprising the following steps:

[0041] (1) coupling the first amino acid at the C-terminus with the Fmoc protecting group and the solid phase support respectively under the action of a condensing agent;

[0042] (2) removing the Fmoc protecting group on the amino acid using a deprotection reagent;

[0043] (3) Connecting the next amino acid under the action of a condensing agent;

[0044] (4) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively;

[0045] (5) the last amino acid is deprotected and then acetylated;

[0046] (6) Under the action of a cyclizing agent, the i and i+4 positions S5 undergo olefin metathesis reaction to cyclize the peptide chain;

[0047] (7) Use a cleavage agent to cut the peptide chain from the support to obtain the stapled peptide.

[0048] The method successfully obtained 7 PNG-derived stapled peptides. When i=1, 2, 3, 4, 5, 6, 7, the obtained stapled peptides were numbered PNG-1, PNG-2, PNG-3, PNG-4, PNG-5, PNG-6, PNG-7, respectively.

[0049] The sources of experimental materials involved are as follows:

[0050] Amino acids were purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0051] Rink Amide MBHA resin was purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0052] Trypsin was purchased from Shanghai Titan Technology Co., Ltd.

[0053] Example 1: Preparation of stapled peptide PNG-4

[0054] like Figure 1 As shown, Rink Amide MBHA resin is used as a solid phase carrier and Fmoc solid phase peptide synthesis (SPPS) is used to synthesize linear polypeptides, which specifically includes the following steps:

[0055] (1) Take 500 mg of Rink Amide MBHA resin (loading amount is 0.30 mmol / g) and add it to a solid phase synthesis reaction tube. Soak it in DCM for 20 min to allow the resin to swell fully, and then drain it for later use.

[0056] Add 20% piperidine-DMF solution (0.1 M Oxyme) until the resin is completely submerged, shake at 25°C for 5 min×2 to remove Fmoc on the resin, and wash the resin with DCM and DMF three times each.

[0057] (2) Fmoc-Lys-OH amino acid (1 mmol), Oxyme (142 mg, 1 mmol), and DIC (155.0 μL, 1 mmol) were mixed and dissolved in 6 ml of NMP, added to the resin and shaken at 60 °C for 20 min (one amino acid after S5 reacts for 2 h), and the resin was washed 5, 5, and 2 times with DMF, DCM, and DMF, respectively.

[0058] Repeat steps (1) and (2), dissolve Fmoc amino acid (0.5 mmol), Oxyme (71 mg) and DIC (75 μL) in 6 ml NMP according to the peptide sequence, add to the resin, shake at 60°C for 20 min, repeat deprotection → condensation → deprotection until all amino acids are connected. After the last amino acid is deprotected, add 10 ml of a mixture of DIEA: acetic anhydride: DMF (1:1:8) and shake at 25°C for 20 min. Wash the resin with DCM, DMF and anhydrous ether for 3 times each, and then dry the resin in vacuum.

[0059] After the resin was completely dried, a dichloroethane solution (6 ml) of Grubbs I (58 mg) reagent was added and the mixture was shaken at 25°C for two times, each time for 2 h. After the reaction was completed, the resin was washed with DCM, DMF and anhydrous ether for three times each, and the resin was dried in vacuo.

[0060] The resin was washed and dried, and a mixed solution of TIPS, TFA, H2O and phenol was added in a volume ratio of 2:88:5:5 (V / V / V / V) 10mL, and the mixture was shaken at room temperature for 4h, filtered, and the resin was washed with a small amount of TFA, and the filtrate was collected. The excess TFA was blown away by nitrogen bubbling, and after centrifugation with ice ether, the supernatant was discarded, and the mixture was washed repeatedly with ice ether and centrifuged three times, and the crude spiked peptide was obtained by nitrogen drying.

[0061] The crude peptide was dissolved in acetonitrile and water and purified by preparative RP-HPLC. The separation conditions were as follows:

[0062] Chromatographic column: YMC-Pack ODS-AQ column; mobile phase: mobile phase A is 0.1% TFA / water, mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 25% B elution 0-5 min, 25% B-45% B, 5-60 min; flow rate is 15 ml / min, injection volume is 5 ml, detection wavelength is 214 nm.

[0063] like Figure 7-8 As shown, after purification, PNG-4 purity was confirmed to be greater than 95% by RP-HPLC and electrospray ionization mass spectrometry (ESI-MS).

[0064] Example 2

[0065] Referring to the method of Example 1 above, the cyclization site of S5 was changed to obtain stapled peptides numbered PNG-1, PNG-2, PNG-3, PNG-5, PNG-6, and PNG-7, respectively.

[0066] Test 1 Compound characterization and related biological activity studies

[0067] After the pure products of the above stapled peptides were synthesized, the properties of these PNG-derived stapled peptides were investigated.

[0068] 1.1 Characterization of α-helicity

[0069] First, circular dichroism (CD) spectroscopy was used to characterize the secondary structure (α-helicity) of the stapled peptides. Figure 2 The results showed that the α-helicity of the peptide was significantly improved after stapling.

[0070] 1.2 Protein hydrolysis stability test

[0071] Subsequently, the proteolytic stability of these peptides towards trypsin was investigated.

[0072] (1) Dissolve 1 mg of trypsin in 10 mL of PBS buffer solution and dilute the solution to a concentration of 0.5 μg / mL;

[0073] (2) Dissolve the polypeptide in pure water to prepare a 1 mmol / L solution;

[0074] (3) 100 μL of the peptide solution was added to 900 μL of the enzyme solution, incubated at 37°C, and samples were taken at 0 h, 2 h, 4 h, 6 h, and 8 h, respectively, and trypsin was quenched with 20 μL of HCI;

[0075] (4) Centrifuge, take the supernatant, inject 30-50 μL, detect by HPLC, calculate the peak area, and use it to evaluate the remaining amount of the polypeptide, thereby obtaining the degradation rate of the polypeptide.

[0076] Figure 3 The results showed that after the addition of protease, the linear peptide PNG was almost completely degraded after 8 hours, with a half-life of about 1.1 hours, while the stapled peptide was not completely degraded at 8 hours, and the half-life was increased to ≥3.9 hours. This shows that compared with the linear peptide, the stapled peptide has excellent proteolytic stability.

[0077] 1.3 Cell membrane permeability experiment

[0078] To evaluate the membrane permeability of the peptides, we labeled the peptides PNG, PNG-4, and PNG-5 with isothiocyanate isomers (FITC) mode and analyzed them using fluorescence microscopy to evaluate the membrane permeability of the stapled peptides. MCF-7 cells were seeded in 6-well culture plates at a density of 5×10^5 / well and cultured overnight. The cells were then treated with 10μM FITC-labeled peptides (without FBS) for 6h. After washing the cells with PBS, the cells were fixed with 4% (w / v) paraformaldehyde for 10min, and finally the nuclei were stained with DAPI. The membrane permeability of the peptides was then analyzed and evaluated by fluorescence microscopy (CKX53, Olympus, Japan).

[0079] Figure 4 The results showed that the linear peptide PNG showed poor membrane permeability and barely passed through the cell membrane. In contrast, the stapled peptides PNG-4 and PNG-5 showed strong cell membrane permeability in MCF-7 cells.

[0080] 1.4 Cell viability assay

[0081] The cell activity was detected by CCK8 (Cell Counting Kit-8) method, and IC was determined after 72 h. 50Specifically, HepG2 and MCF-7 cells were seeded in 96-well plates at a density of 3000 cells / well and cultured overnight at 37°C and 5% CO2. Next, the cells were treated with different concentrations of peptides for 72 hours. After the treatment, the culture medium was replaced with a new culture medium containing 10% CCK8 and incubated for another hour. The optical density (OD) was measured at 450nm using a Cytation5 cell imaging multimode detector (Bio-Tek, Vermont, USA). Nonlinear regression analysis was performed using Graph Pad Prism to obtain the IC value of the peptide. 50 value.

[0082] After the linear peptide PNG was stapled, the inhibitory activity of the stapled peptides against MCF-7 and HepG2 cell lines was significantly improved. 50 The values ​​were all lower than 10 μM, far exceeding PNG (IC 50 >20μM) (Table 1). Among them, PNG-4 and PNG-5 had the most obvious activity enhancement. These findings indicate that the stapled modification site significantly affects the anti-tumor activity of the peptide. Subsequent experiments selected PNG-4 and PNG-5 to further investigate their biological activities.

[0083] Table 1 Characterization of α-helicity and antitumor activity (HepG2 and MCF-7) of PNG and its derived stapled peptides

[0084]

[0085] 1.5 Cell colony formation assay

[0086] To evaluate the in vitro antitumor activity of the stapled peptides, the effects of PNG-4 and PNG-5 on the colony-forming ability of MCF-7 cells were examined. Single-cell suspensions of MCF-7 cells were prepared and plated in 6-well plates at a density of 1000 cells per well. After overnight cell attachment, the cells were treated with fresh culture medium containing different concentrations of peptides. The medium was changed every 3 days, and cell clones were clearly visible after 14 days of culture. The supernatant was discarded, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes, followed by crystal violet staining. Cells with a colony number greater than 50 were counted, and the results were expressed as mean ± standard deviation (SD).

[0087] like Figure 5 As shown in A, treatment with PNG-4 and PNG-5 resulted in a concentration-dependent decrease in the number of colonies formed by MCF-7 cells compared with the untreated control group, indicating that they have the potential to inhibit tumor cell proliferation. Figure 5 B is the use of ImageQuant (Molecular Dynamics, USA) Figure 5After quantitative analysis of the cell colonies after A treatment, the percentage of the number of treated cell colonies to the number of untreated cell colonies was obtained. It is worth noting that PNG-5 showed a more obvious anti-tumor effect and significantly reduced colony formation more effectively than PNG-4, highlighting its superior tumor treatment potential ( Figure 5 B).

[0088] 1.6 Cell apoptosis detection

[0089] Inducing apoptosis is an important mechanism for anticancer drugs to work. In order to evaluate the ability of stapled peptides to induce apoptosis, Annexin V / PI double staining and flow cytometry analysis were performed. MCF-7 cells were seeded in 6-well culture plates at a density of 5×10^5 / well and cultured overnight, and then treated with gradient concentrations of peptide solutions for 24 hours. The supernatant was discarded, and the cells were collected after trypsin digestion, washed with pre-cooled PBS and centrifuged, and the precipitate was resuspended in 300μL binding buffer. 5μL Annexin V-FITC and 5μL propidium iodide (PI) were added to each tube and incubated at room temperature in the dark for 30min. Finally, the samples were tested for apoptosis using a flow cytometer (Cytoflex, Beckman, USA).

[0090] like Figure 6 As shown, PNG-4 and PNG-5 caused a significant increase in the rate of cell apoptosis compared with the linear peptide PNG.

[0091] After PNG treatment for 24 h, the apoptosis rate (including early apoptosis and late apoptosis) was 8.53%, while after PNG-4 and PNG-5 treatment for 24 h, the apoptosis rate (including early apoptosis and late apoptosis) increased to 32.29% and 48.92%, respectively.

Claims

1. A nailed titanium, characterized in that: Select one of the following: a) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5A were replaced by S5 and cyclized; b) LNWGAILKHIIK-NH2 was used as a peptide chain template, in which amino acid residues 2N and 6I were replaced by S5 and cyclized; c) LNWGAILKHIIK-NH2 was used as a peptide chain template, in which amino acid residues 3W and 7L were replaced by S5 and cyclized; d) LNWGAILKHIIK-NH2 was used as a peptide chain template, in which amino acid residues 4G and 8K were replaced by S5 and cyclized; e) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 5A and 9H were replaced by S5 and cyclized; f) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 6I and 10I were replaced by S5 and cyclized; g) LNWGAILKHIIK-NH2 was used as a peptide chain template, wherein amino acid residues 7L and 11I were replaced by S5 and cyclized; The S5 is 2-amino-2-methyl-6-enoic acid.

2. A method for preparing the stapled peptide according to claim 1, characterized in that: The method uses amino resin as a solid phase carrier, and synthesizes a peptide chain in a condensation system by Fmoc solid phase synthesis method according to the linear peptide template LNWGAILKHIIK-NH2 amino acid sequence, and on the basis of retaining key amino acid residues, replaces the original amino acid with S5 at positions i and i+4, wherein i=1-7, to obtain a linear peptide connected to the resin, and the linear peptide is cyclized by olefin metathesis reaction under the catalysis of GrubbsⅠ reagent and then cut off from the resin to obtain the target stapled peptide.

3. The method according to claim 2, characterized in that The specific steps include: (1) coupling the first amino acid at the C-terminus to the solid phase carrier under the action of a condensing agent; (2) removing the Fmoc protecting group on the amino acid using a deprotection reagent; (3) Connecting the next amino acid under the action of a condensing agent; (4) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S5 to replace the amino acid at position i and i+4 respectively; (5) the last amino acid is deprotected and then acetylated; (6) Under the action of a cyclizing agent, the i and i+4 positions S5 undergo olefin metathesis reaction to cyclize the peptide chain; (7) Use a cleavage agent to cut the peptide chain from the support to obtain the corresponding stapled peptide.

4. Use of the stapled peptide according to claim 1 in the preparation of a drug for treating breast cancer.

5. Use of the stapled peptide according to claim 1 in the preparation of a drug for treating liver cancer.

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