A pinned peptide, its preparation method and application

Abstract

This invention discloses a stapled peptide, its preparation method, and its applications. The method involves using an amino resin as a solid-phase support, synthesizing a peptide chain in a condensation system according to the linear peptide template LNWGAILKHIIK-NH2 via Fmoc solid-phase synthesis. While retaining key amino acid residues, S5 is used to replace the original amino acid at positions i and i+4, where i = 1-7, resulting in a linear peptide linked to the resin. This linear peptide undergoes an olefin metathesis reaction under the catalysis of Grubbs I reagent, followed by cyclization and cleavage from the resin to obtain the target stapled peptide. This method is simple and easy to implement, yielding a stapled peptide with a purity greater than 95% and a high yield. The synthesized stapled peptide significantly improves protein hydrolysis stability and cell membrane permeability, and can significantly inhibit the growth and proliferation of human breast cancer cells and human liver cancer cells, showing potential application value in the treatment of tumor diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a pinned peptide, its preparation method, and its application. Background Technology

[0002] Breast cancer and liver cancer are two major malignant tumors with continuously rising incidence and mortality rates worldwide, posing a serious threat to public health. However, current treatment options for these two cancers, including surgery, radiotherapy, and chemotherapy, often result in significant side effects, severely impacting patients' quality of life and physical health. Therefore, developing effective anti-breast cancer or anti-liver cancer drugs with fewer side effects is of significant clinical importance. In recent years, peptide drugs have attracted widespread attention due to their high specificity, tunable quality, and high biological activity, offering unparalleled advantages in targeted therapy and reducing off-target effects compared to small molecule and protein drugs.

[0003] Literature review revealed that Panurgines (PNGs) isolated from the venom of the wild bee Panurgus calcaratus had a certain inhibitory effect on MCF-7 breast cancer cells and HepG2 liver cancer cells. Sabína,Slaninová 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 developing novel anti-breast cancer drugs or anti-liver cancer drugs using PNGs. However, linear peptide PNGs face many challenges in penetrating cell membranes and are easily hydrolyzed by proteins. To address these issues, chemical modification of PNGs to develop a highly efficient, stable, and low-side-effect PNG peptide therapeutic strategy is crucial. Summary of the Invention

[0004] Given the above background, the purpose of this invention is to improve the structure of PNGs, thereby enhancing their proteolytic stability and cell membrane permeability, and thus developing a novel polypeptide derivative with potential therapeutic effects against breast cancer and liver cancer. Based on existing literature, this invention comprehensively studies the application of various hydrocarbon pinning strategies in the design and synthesis of PNG derivatives. Utilizing hydrocarbon pinning strategies, a series of new PNG derivatives with excellent proteolytic stability were designed and synthesized. Through a series of experimental investigations and optimizations, this invention screened out PNG5, a polypeptide derivative with the best inhibitory effect on tumor cell proliferation, which has potential therapeutic effects against breast cancer and liver cancer.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A type of stapled titanium, selected from one of the following:

[0007] a) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 1L and 5A are replaced and cyclized with S5 (2-amino-2-methylhept-6-enoic acid);

[0008] b) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 2N and 6I are replaced by S5 and cyclized;

[0009] c) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 3W and 7L are replaced by S5 and cyclized;

[0010] d) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 4G and 8K are replaced by S5 and cyclized;

[0011] e) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 5A and 9H are replaced by S5 and cyclized;

[0012] f) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 6I and 10I are replaced by S5 and cyclized;

[0013] g) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 7L and 11I are replaced by S5 and cyclized.

[0014] The present invention also provides the use of the pinning peptide in the preparation of a medicament for treating breast cancer.

[0015] The present invention also provides the use of the pinning peptide in the preparation of a medicament for treating liver cancer.

[0016] This invention also provides a method for preparing the pinned peptide, which uses an amino resin as a solid-phase carrier and synthesizes a peptide chain in a condensation system according to the amino acid sequence of the linear peptide template LNWGAILKHIIK-NH2 via Fmoc solid-phase synthesis. While retaining key amino acid residues, the original amino acids are replaced with S5 at positions i and i+4, where i = 1-7, resulting in a linear peptide linked to the resin. This linear peptide undergoes an olefin metathesis reaction under the catalysis of Grubbs I reagent, followed by cyclization, and is then cleaved from the resin to obtain the target pinned peptide.

[0017] Specifically, the following steps are included:

[0018] (1) The first amino acid at the C-terminus is coupled to the solid support under the action of the condensing agent;

[0019] (2) Use a deprotection reagent to remove the Fmoc protecting group on the amino acid;

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

[0021] (4) Repeat the deprotection-coupling operation to synthesize peptide chains according to the amino acid sequence; wherein, the cyclization site is replaced by S5 for amino acids at positions i and i+4, respectively.

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

[0023] (6) Under the action of the cyclizing agent, the S5 at positions i and i+4 undergoes an olefin metathesis reaction, cyclizing the peptide chain;

[0024] (7) Use a cleavage reagent to cut the peptide chain from the carrier to obtain the corresponding pinned peptide.

[0025] The advantages of this invention are:

[0026] 1. This invention uses amino resin as a carrier and synthesizes peptide chains in a condensation system according to the amino acid sequence of the template using the Fmoc solid-phase synthesis method. During this process, while retaining key amino acid residues, S5 replaces the original amino acid at a specific position and is attached to the resin. The linear peptide undergoes olefin metathesis reaction on Grubbs I reagent for cyclization and is then cleaved from the resin to obtain the target pinned peptide.

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

[0028] 3. This invention designs and synthesizes a pinned peptide, and experiments have confirmed that compared with the LNWGAILKHIIK-NH2 linear peptide, it significantly improves protein hydrolysis stability and cell membrane penetration ability, and can significantly inhibit the growth and proliferation of human breast cancer cells and human liver cancer cells, and has potential application value in the treatment of breast cancer, liver cancer and other related tumor diseases. Attached Figure Description

[0029] Figure 1 The synthetic route for the pinned peptide PNG-4 is shown below, where:

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

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

[0032] Figure 2 Circular dichroism chromatograms of PNG and PNG-derived pinned peptides.

[0033] Figure 3 A comparison of the trypsin hydrolysis stability of PNG and PNG-derived pinning peptides.

[0034] Figure 4 Fluorescent micrographs of MCF-7 cell membrane penetration for PNG, pinning peptides PNG-4 and PNG-5.

[0035] Figure 5 Studies on the inhibition of MCF-7 cell colony formation by PNG, pinning peptide 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 treated with PNG, PNG-4, and PNG-5 compared to the number of untreated cell colonies.

[0036] Figure 6 Inhibition of MCF-7 cell apoptosis by hydrocarbon peptides: (A) Apoptosis of MCF-7 cells treated with different peptide preparations. (B) Quantitative analysis of cell apoptosis levels using ImageQuant (Molecular Dynamics, USA).

[0037] Figure 7 The RP-HPLC chromatogram of the stapled peptide PNG-4 in Example 1 is shown.

[0038] Figure 8 The image shows the electrospray ionization mass spectrometry (ESI-MS) spectrum of the pinned peptide PNG-4 from Example 1. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] This invention uses the template linear peptide PNG: LNWGAILKHIIK-NH2 (SEQ ID NO.1) and employs the i, i+4 strategy to pin the peptide, introducing S5 at positions i, i+4 of the peptide backbone, where i = 1-7. Then, an olefin ring-closure metathesis reaction is performed using Grubbs I reagent. The specific steps include:

[0041] (1) The first amino acid at the C-terminus is coupled to the Fmoc protecting group and the solid support under the action of the condensing agent;

[0042] (2) Use a deprotection reagent to remove the Fmoc protecting group on the amino acid;

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

[0044] (4) Repeat the deprotection-coupling operation to synthesize peptide chains according to the amino acid sequence; wherein, the cyclization site is replaced by S5 for amino acids at positions i and i+4, respectively.

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

[0046] (6) Under the action of the cyclizing agent, the S5 at positions i and i+4 undergoes an olefin metathesis reaction, cyclizing the peptide chain;

[0047] (7) Use a cleavage reagent to cut the peptide chain from the carrier to obtain the pinned peptide.

[0048] This method successfully yielded seven PNG-derived pinning peptides. When i = 1, 2, 3, 4, 5, 6, 7, the obtained pinning peptides were numbered PNG-1, PNG-2, PNG-3, PNG-4, PNG-5, PNG-6, and PNG-7, respectively.

[0049] The experimental materials involved came from the following sources:

[0050] The 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 pinned peptide PNG-4

[0054] like Figure 1 As shown, linear peptides were synthesized using Rink Amide MBHA resin as a solid-phase support via the Fmoc solid-phase peptide synthesis method (SPPS), specifically including the following steps:

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

[0056] Add 20% piperidine-DMF solution (0.1M Oxyme) until the resin is completely submerged, shake at 25°C for 5 min × 2 to remove Fmoc from the resin, and wash the resin with DCM and DMF 3 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 in 6 ml NMP and added to the resin. The mixture was shaken at 60 °C for 20 min (the reaction of one amino acid after S5 was 2 h). The resin was washed with DMF, DCM and DMF 5, 5 and 2 times in sequence.

[0058] Repeat steps (1) and (2). According to the polypeptide sequence, Fmoc amino acids (0.5 mmol), Oxyme (71 mg), and DIC (75 μL) are dissolved in 6 ml of NMP and added to the resin. The mixture is shaken at 60 °C for 20 min. The process of deprotection → condensation → deprotection is repeated until all amino acids are linked. After the last amino acid is deprotected, 10 ml of a DIEA:acetic anhydride:DMF (1:1:8) mixture is added and the mixture is shaken at 25 °C for 20 min. The resin is then washed three times each with DCM, DMF, and anhydrous diethyl ether, and finally dried under vacuum.

[0059] After the resin is completely dry, add 6 ml of dichloroethane solution containing Grubbs I (58 mg) reagent, and shake the reaction twice at 25 °C for 2 h each time. After the reaction is complete, wash the resin three times each with DCM, DMF and anhydrous diethyl ether, and then dry the resin under vacuum.

[0060] Wash and dry the resin, then add a mixed solution of TIPS, TFA, H2O, and phenol in a volume ratio of 2:88:5:5 (V / V / V / V) 10 mL. Shake at room temperature for 4 hours, filter, wash the resin with a small amount of TFA, and collect the filtrate. Bubble away excess TFA with nitrogen, pour in ice-cold ether to precipitate, centrifuge, discard the supernatant, and continue washing and centrifuging with ice-cold ether three times. Dry with nitrogen to obtain the crude pinned peptide.

[0061] The crude peptide was dissolved in acetonitrile and water, and then 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 elution 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, the purity of PNG-4 was confirmed to be greater than 95% after purification by RP-HPLC and electrospray ionization mass spectrometry (ESI-MS).

[0064] Example 2

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

[0066] Experiment 1: Compound Characterization and Related Bioactivity Studies

[0067] After synthesizing pure samples of the above-mentioned stapling peptides, the properties of these PNG-derived stapling peptides were investigated.

[0068] 1.1 Characterization of α-helicity

[0069] First, the secondary structure (α-helicity) of the stapled peptide was characterized using circular dichroism (CD). Figure 2 The results showed that the α-helicality of the peptide was significantly improved after pinning.

[0070] 1.2 Protein hydrolysis stability test

[0071] Subsequently, the proteolytic stability of these peptides to 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) Take 100 μL of polypeptide solution and add it to 900 μL of enzyme solution. Incubate at 37°C and take samples at 0h, 2h, 4h, 6h and 8h respectively. Quench trypsin with 20 μL of HCl.

[0075] (4) Centrifuge, take the supernatant, inject 30-50 μL, detect by HPLC, calculate the peak area to evaluate the remaining amount of peptide, and thus obtain the degradation rate of peptide.

[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 approximately 1.1 hours, while the stapled peptide was not completely degraded after 8 hours, with a half-life increased to ≥3.9 hours. This demonstrates that, compared to linear peptides, stapled peptides exhibit superior proteolytic stability.

[0077] 1.3 Cell membrane penetration experiment

[0078] To assess the membrane permeability of the peptides, we labeled peptides PNG, PNG-4, and PNG-5 using the FITC isothiocyanate isomer pattern and evaluated the membrane permeability of the pinned peptides using fluorescence microscopy. MCF-7 cells were seeded at a density of 5 × 10⁵ cells / well and cultured overnight in 6-well plates. Cells were then treated with 10 μM FITC-labeled peptides (without FBS) for 6 h. After washing with PBS, cells were fixed with 4% (w / v) paraformaldehyde for 10 min, and the nuclei were stained with DAPI. The membrane permeability of the peptides was then analyzed and evaluated using fluorescence microscopy (CKX53, Olympus, Japan).

[0079] Figure 4 The results showed that the linear peptide PNG exhibited poor membrane permeability, barely crossing 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] Cell viability was detected using the CCK8 (Cell Counting Kit-8) method, and IC50 was measured after 72 hours. 50Specifically, HepG2 and MCF-7 cells were seeded at a density of 3000 cells / well in 96-well plates and cultured overnight at 37°C and 5% CO2. Next, the cells were treated with different concentrations of peptides for 72 hours. After treatment, the medium was replaced with fresh medium containing 10% CCK8, and incubation continued for 1 hour. Optical density (OD) was measured at 450 nm using a Cytation5 Cell Imaging Multimodal Detector (Bio-Tek, Vermont, USA). Nonlinear regression analysis was performed using Graph Pad Prism to determine the IC50 value of the peptide. 50 value.

[0082] Pinning the linear peptide PNG significantly enhanced the inhibitory activity of the pinned peptide against both MCF-7 and HepG2 cell lines. The IC50 values ​​of these pinned peptides were... 50 The values ​​are all below 10 μM, far exceeding PNG (IC). 50 >20 μM (Table 1). The activity enhancement was most significant at PNG-4 and PNG-5. These findings indicate that the pinning modification sites significantly affect the antitumor activity of the peptides. Subsequent experiments selected PNG-4 and PNG-5 to further investigate their biological activity.

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

[0084]

[0085] 1.5 Cell colony formation experiment

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

[0087] like Figure 5 As shown in Figure A, compared with the untreated control group, treatment with PNG-4 and PNG-5 resulted in a concentration-dependent reduction in the number of colonies formed by MCF-7 cells, indicating their potential to inhibit tumor cell proliferation. Figure 5 B is the result of using ImageQuant (Molecular Dynamics, USA) to... Figure 5Quantitative analysis of the cell colonies after treatment A yielded the percentage of treated cell colonies relative to the number of untreated cell colonies. Notably, PNG-5 exhibited a more significant anti-tumor effect, effectively and significantly reducing colony formation compared to PNG-4, highlighting its superior potential for tumor treatment. Figure 5 B).

[0088] 1.6 Apoptosis Detection

[0089] Induction of apoptosis is an important mechanism by which anticancer drugs exert their effects. To evaluate the apoptosis-inducing ability of pinned peptides, Annexin V / PI double staining and flow cytometry analysis were performed. MCF-7 cells were seeded at a density of 5 × 10^5 cells / well in 6-well plates and cultured overnight, followed by treatment with gradient concentrations of peptide solutions for 24 hours. After discarding the supernatant, cells were collected after trypsin digestion, washed with pre-chilled PBS, and centrifuged. The pellet was resuspended in 300 μL of binding buffer. 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added to each tube, and the cells were incubated at room temperature in the dark for 30 min. Finally, apoptosis was detected in the samples using flow cytometry (Cytoflex, Beckman Coulter, USA).

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

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

Claims

1. The use of a stapled peptide in the preparation of a medicament for treating breast cancer or liver cancer, wherein the stapled peptide is selected from one of the following: a) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 1L and 5A are replaced by S5 and cyclized; b) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 2N and 6I are replaced by S5 and cyclized; c) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 3W and 7L are replaced by S5 and cyclized; d) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 4G and 8K are replaced by S5 and cyclized; e) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 5A and 9H are replaced by S5 and cyclized; f) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 6I and 10I are replaced by S5 and cyclized; g) Using LNWGAILKHIIK-NH2 as a peptide template, amino acid residues 7L and 11I are replaced by S5 and cyclized; S5 is 2-amino-2-methyl-6-enoic acid.

2. The application according to claim 1, characterized in that, The method for preparing the pinned peptide involves using an amino resin as a solid-phase support and synthesizing the peptide chain in a condensation system according to the amino acid sequence of the linear peptide template LNWGAILKHIIK-NH2 via the Fmoc solid-phase synthesis method. While retaining key amino acid residues, S5 is used to replace the original amino acid at positions i and i+4, where i = 1-7, resulting in a linear peptide linked to the resin. This linear peptide undergoes an olefin metathesis reaction under the catalysis of Grubbs I reagent, cyclizes, and is then cleaved from the resin to obtain the target pinned peptide.

3. The application according to claim 2, characterized in that, The preparation method of the pinned peptide specifically includes the following steps: (1) The first amino acid at the C-terminus is coupled to the solid support under the action of the condensing agent; (2) Use a deprotection reagent to remove the Fmoc protecting group on the amino acid; (3) The next amino acid is linked under the action of a condensing agent; (4) Repeat the deprotection-coupling operation to synthesize peptide chains according to the amino acid sequence; wherein, the looping sites are replaced by S5 for amino acids at positions i and i+4, respectively. (5) The last amino acid is deprotected and then acetylated; (6) Under the action of the cyclizing agent, the S5 at positions i and i+4 undergoes an olefin metathesis reaction, cyclizing the peptide chain; (7) Use a cleavage reagent to cut the peptide chain from the carrier to obtain the corresponding pinned peptide.

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