Targeting RAS-Raf protein interaction stapled peptide and application thereof in antitumor drugs

Abstract

The invention relates to the field of biological medicine, in particular to a stapled peptide targeting RAS-Raf protein interaction and application of the stapled peptide, the stapled peptide is obtained in the mode that Ac-SLHDCLMKALKVR-NH2 and Ac-SLHDCLMKALKVRGL-NH2 serve as peptide chain templates, amino acid at the i site and amino acid at the (i + 4) site are replaced with S5 and cyclized, and the nitrogen terminal is further modified with stearic acid. The stapled peptide can effectively inhibit proliferation and migration of tumor cells; the serum stability is high, and meanwhile, the cell transmembrane capability is excellent, so that target protein in cells can be better targeted, and the biological function of the target protein can be exerted. Therefore, the alphaH helical conformation of the RBD of the RAF can be stabilized through the stapled peptide and a nitrogen-terminal fatty acid modification strategy, the membrane permeability and stability of the RAF are improved, the binding force of the RAF and RAS is further improved, and proliferation and migration of various tumor cells are inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to a stapled peptide targeting the RAS-Raf protein interaction and its application in the preparation of anti-tumor drugs. Background Art

[0002] The RAS protein plays a central regulatory role in cell signal transduction, especially in the MAPK pathway and the PI3K pathway. Both of these pathways are important pathways for regulating cell growth, proliferation, differentiation, and survival, and RAS, as a key upstream signaling molecule, is the common hub of these two pathways. RAS selectively activates the MAPK pathway or the PI3K pathway by interacting with different downstream effector proteins. Specifically, in the MAPK pathway, after activation, RAS first binds to RAF, activates MEK and ERK through a cascade reaction, and finally transmits the signal to the nucleus to promote gene expression and regulate cell proliferation and differentiation. At the same time, in the PI3K pathway, activated RAS directly binds to PI3K, and then activates AKT and mTOR to regulate cell survival and metabolism.

[0003] The interaction between RAS and RAF is the starting point for the activation of the MAPK pathway and an indispensable link in signal transduction. This interaction effectively activates the RAF kinase, triggering a series of cascade reactions and rapidly transmitting the signal on the cell surface to the nucleus. Therefore, the key role of RAS in these two pathways not only maintains the normal physiological functions of cells but is also the main factor driving abnormal cell proliferation in various cancers.

[0004] Analyze the crystal structure of the RAS-RAF complex. RAS and RAF form a tightly bound interface through two regions in the CR1 region of RAF: Interface 1 includes the α1 helix of the RBD (residues 77-91), and Interface 2 involves the η1 helix of the CRD (residues 172-186). These two regions play a key role in the interaction with RAS. The interaction at the KRAS-RBD interface mainly depends on hydrogen bonds and electrostatic interactions, involving basic residues from the β2 strand and α1 helix of the RBD. Specifically, this interface forms 9 hydrogen bonds and 4 salt bridges, among which 3 hydrogen bonds and 3 salt bridges are within the α1 helix of the RBD, significantly enhancing the nanomolar affinity between KRAS and the RBD. Mutation experiments show that the R89L mutation in the α1 helix almost eliminates this interaction. This indicates that polar and charged residues on the RBD are crucial for the high-affinity interaction between KRAS and the RBD, and the α1 helix plays an indispensable role in maintaining this interaction. Different from the KRAS-RBD interface that mainly relies on polar and electrostatic interactions, the KRAS-CRD interface lacks salt bridges and is mainly characterized by a relatively large hydrophobic interaction surface. Overall, KRAS and the CRD form 9 hydrogen bonds, 6 of which are within the η1 helix. The η1 helix is parallel to the β2 strand of KRAS and is located above the α5 helix of KRAS, thus providing surface complementarity at the KRAS-CRD interface. Therefore, the α1 and η1 helix structures are used as the starting point for structure-based RAS mimetic ligand design. Subsequent experiments found that the peptide based on the η1 helix is inactive, so subsequent peptide designs are all based on the α1 helix in the RBD.

[0005] Regulate the balance between the activities of RAS and RAF proteins, which may be used for the inhibition of tumor cell proliferation. According to the natural peptide sequence of the α1 helix, the unnatural amino acid S with an α-alkenyl group is introduced at the i, i+4 positions 5 ((S)-2-Amino-2-methylhept-6-enoic acid) to synthesize a new stapled peptide that inhibits the RAS-RAF interaction, thereby increasing the binding affinity for the RAS protein. Therefore, we carried out structural simplification and optimization on the protein mimetic of RAF, designed and synthesized a series of stapled peptides, observed their α-helicity, stability, membrane permeability, affinity for RAS, investigated the ability to inhibit tumor cell proliferation, selected the stapled peptides with high inhibitory activity, further investigated their effects on downstream signaling pathways and mechanisms of action, and found lead compounds that effectively inhibit tumor cell proliferation, providing new ideas and theoretical basis for the treatment of tumors.

[0006] Studies have shown that constructing all-carbon hydrogen stapled peptides at the i, i+4 positions of polypeptides through olefin metathesis reactions can stabilize the α-helix structure of polypeptides, effectively improving the stability, membrane permeability, and binding affinity to target proteins of polypeptides. With the in-depth study of the RAS protein-related pathways, it is particularly important to develop new targeted drugs, discover new mechanisms of action, and apply them to new diseases. This work is expected to bring new ideas for the treatment of tumors, especially targeted therapy. Summary of the Invention

[0007] The object of the present invention is, relying on the above research, in view of the current situation of RAS-RAF interaction, to provide a novel stapled peptide targeting the RAS-RAF protein interaction, and further provide the application of this stapled peptide in the preparation of drugs for inhibiting the proliferation of tumor cells.

[0008] To achieve the above object, the present invention adopts the following technical solutions: Based on the characteristics of the interaction interface in the crystal structure of the RAS-RAF complex, using the α1 helix in the RBD domain of the RAF protein and the η1 helix structural fragment in the CRD domain as templates (it was found through subsequent activity screening that the η1 helix has no significant activity, so the polypeptide design is mainly based on the α1 helix), with linear polypeptides as controls, a series of stapled peptides were prepared using olefin metathesis reactions. Their secondary structures were determined by circular dichroism spectroscopy, and their membrane permeability was evaluated by flow cytometry. The inhibitory effects of the stapled peptides on the proliferation of various tumor cells were evaluated at the cellular level.

[0009] Based on the above technical solutions, the present invention provides a stapled peptide targeting the RAS-RAF protein interaction, using the linear peptide Ac-SLHDCLMKALKVR-NH 2 (SEQ ID NO.1) as the peptide chain template, replacing the i-th and (i+4)-th amino acids with (s)-2-amino-2-methyl-6-heptenoic acid (S 5 ) and cyclizing to obtain 6 Sraf series stapled peptides (Sraf-1 - Sraf-6); using the linear peptide Ac-SLHDCLMKALKVRGL-NH 2 (SEQ ID NO.2) as the peptide chain template, replacing 5 C and 9 A with S 5 and cyclizing (Sraf-7), and on this basis, the 11 K and 12 VReplace (Sraf-8—Sraf-10) with the hydrophobic residues M or W respectively, or modify the N-terminus of the peptide chain with stearic acid (Sraf-2-1, Sraf-7-1) to obtain 6 stapled peptides of the Sraf series. Among them, i is any integer from 1, 2, 3, 5, 6, and 10, a total of 12 stapled peptides, numbered Sraf-1~Sraf-10, Sraf-2-1, and Sraf-7-1 in sequence. Specifically, it is shown in Table 1 below.

[0010] Table 1. Sequences, molecular weights of stapled peptides targeting RAS protein, and K d value measured by SPR technology

[0011]

[0012] In the first aspect of the present invention, there is provided a stapled peptide targeting the RAS-Raf protein interaction, and the stapled peptide is selected from one of the following:

[0013] Sraf-1: Using Ac-SLHDCLMKALKVR-NH 2 as the peptide chain template, where 6 L and 10 L are replaced by S 5 and cyclized;

[0014] Sraf-2: Using Ac-SLHDCLMKALKVR-NH 2 as the peptide chain template, where 5 C and 9 A are replaced by S 5 and cyclized;

[0015] Sraf-3: Using Ac-SLHDCLMKALKVR-NH 2 as the peptide chain template, where 3 H and 7 M are replaced by S 5 and cyclized;

[0016] Sraf-4: Using Ac-SLHDCLMKALKVR-NH 2 as the peptide chain template, where 2 L and 6 L are replaced by S 5 and cyclized;

[0017] Sraf-5: Using Ac-SLHDCLMKALKVR-NH 2 as the peptide chain template, where 1 S and 5 C are replaced by S 5 and cyclized;

[0018] Sraf-6: Using Ac-SLHDCLMKALKVRS 5 -NH 2 as the peptide chain template, where 10 L is replaced by S 5 and cyclized;

[0019] Sraf-7: Using Ac-SLHDCLMKALKVRGL-NH 2 as the peptide chain template, where 5 C and 9 A are replaced by S 5 and cyclized;

[0020] Sraf-8: Using Ac-SLHDCLMKALKVRGL-NH 2 as the peptide chain template, where 11 K and 12 V are replaced by M, 5 C and 9 A are replaced by S 5 and cyclized;

[0021] Sraf-9: Using Ac-SLHDCLMKALKVRGL-NH 2 as the peptide chain template, where 11 K and 12 V are respectively replaced by W and M, 5 C and 9 A are replaced by S 5 and cyclized;

[0022] Sraf-10: Using Ac-SLHDCLMKALKVRGL-NH 2 as the peptide chain template, where 11 K and 12 V are replaced by W, 5 C and 9 A are replaced by S 5 and cyclized;

[0023] Sraf-2-1: Using SA(Stearic acid)-SLHDCLMKALKVRGL-NH 2 as the peptide chain template, where 5 C and 9 A are replaced by S 5 and cyclized;

[0024] Sraf-7-1: Using SA(Stearic acid)- SLHDCLMKALKVRGL-NH 2 as the peptide chain template, where 5 C and 9 A are replaced by S 5 and cyclized.

[0025] The chemical structural formulas of the above 12 stapled peptides are as Figures 5 - 16 shown.

[0026] In the second aspect of the present invention, there is provided a method for preparing a stapled peptide targeting the RAS-Raf protein interaction as described above, specifically including the following steps:

[0027] A. Coupling the C-terminus of the first amino acid with a solid-phase carrier under the action of a condensing agent;

[0028] B. Using a deprotecting reagent to remove the Fmoc protecting group on the amino acid;

[0029] C. Coupling the next amino acid under the action of a condensing agent;

[0030] D. Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein, the cyclization sites are S 5 substituting the amino acids at positions i and i + 4 respectively;

[0031] E. Acetylating after deprotecting the last amino acid;

[0032] F. Performing olefin metathesis reaction on the S 5 amino acids at positions i and i + 4 under the action of a cyclizing agent to cyclize the peptide chain;

[0033] G. Using a cleavage reagent to cleave the peptide chain from the carrier, and obtaining the corresponding stapled peptide after purification.

[0034] In the third aspect of the present invention, there is provided an application of a stapled peptide targeting the RAS-Raf protein interaction as described above in the preparation of a drug targeting the RAS protein.

[0035] In the fourth aspect of the present invention, there is provided an application of a stapled peptide targeting the RAS-Raf protein interaction as described above in the preparation of an anti-tumor drug.

[0036] Furthermore, the anti-tumor drug is a drug for inhibiting tumor cell proliferation and / or migration.

[0037] Cell experiment results show that the stearic acid-modified stapled peptides Sraf-2-1 and Sraf-7-1 can inhibit the proliferation of tumor cells in a dose-dependent manner; meanwhile, they have excellent cell-penetrating ability and can penetrate through the cell membrane into the cytoplasm, so as to better target the target protein inside the cell to exert their biological functions. In addition, this stapled peptide can also inhibit tumor cell proliferation and migration.

[0038] Further, the tumor is lung cancer, melanoma or breast cancer.

[0039] In a fifth aspect of the present invention, there is provided a pharmaceutical preparation, which uses the stapled peptide targeting the RAS-Raf protein interaction as described above as the sole active ingredient, or contains the stapled peptide targeting the RAS-Raf protein interaction as described above.

[0040] Furthermore, the pharmaceutical preparation can be made into various dosage forms with pharmaceutically common excipients. For example, it can be decoction, powder, pill, intravenous emulsion, liposome preparation, aerosol, prodrug preparation, injection, mixture, oral ampoule, tablet, capsule, etc. The administration methods are not limited to oral administration, injection, etc.

[0041] The advantages and beneficial effects of the present invention are as follows:

[0042] 1. In terms of preparation, the present invention uses amino resin as a carrier, and uses Ac-SLHDCLMKALKVR-NH 2 and Ac-SLHDCLMKALKVRGL-NH 2 as peptide chain templates. Through the Fmoc solid-phase synthesis method, a peptide chain is synthesized. During this process, on the basis of retaining key amino acid residues, S 5 is used to replace the original amino acids at the i, i+4 positions. The linear peptide coupled to the resin undergoes olefin metathesis reaction cyclization in a 1,2-dichloroethane solution of Grubbs1 st reagent and is then cleaved from the resin to obtain the target stapled peptide. After the obtained compound is purified, it is characterized and analyzed by spectra such as HPLC, HR-MS and circular dichroism spectrum, showing that the purity of the obtained stapled peptide is greater than 95%, and showing a typical α-helix conformation, which is beneficial to maintaining the stability of the peptide.

[0043] 2. In terms of effects, the stapled peptide of the present invention can effectively inhibit the proliferation and migration of tumor cells; it has high serum stability, and at the same time has excellent cell-penetrating ability, and can penetrate through the cell membrane into the cytoplasm, so as to better target the target protein inside the cell to exert its biological function. The stapled peptide can down-regulate the expression level of the p-AKT gene in a dose-dependent manner.

[0044] 3. Through the stapled peptide of the present invention and the N-terminal fatty acid modification strategy, the α-helix of Raf-1 is maintained in a stable conformation, enhancing its membrane permeability, improving serum stability, further retaining or even increasing its binding ability to RAS, and exerting the inhibitory ability on the proliferation and migration of various tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figures 1 - 21 are the mass spectrum and high performance liquid chromatography diagram of the purified stapled peptide;

[0046] Figure 22 is the circular dichroism spectrum diagram of the purified stapled peptide in pure water;

[0047] Figure 23 The stability of the polypeptide obtained by HPLC detection in human serum;

[0048] Figure 24 To analyze the anti - proliferative effects of the polypeptide on non - small cell lung cancer cell line A549 and human melanoma cell line A375 using the CCK - 8 assay;

[0049] Figure 25 a is for detecting the entry of stapled peptide into cells and nuclei by fluorescence confocal microscopy; Figure 25 b is for determining the transmembrane ability of the FITC - modified polypeptide by flow cytometry;

[0050] Figure 26 The protein expression of the PI3K signaling pathway detected by Western Blot (WB) experiment;

[0051] Figure 27 To evaluate the ability of the stapled peptide to inhibit cell migration by the cell scratch assay; Specific embodiments

[0052] The following specifically describes the specific embodiments provided by the present invention in conjunction with the examples. The following examples are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples.

[0053] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0054] Example 1: Synthesis of stapled peptide targeting RAS protein

[0055] Taking the stearic acid - modified stapled peptide Sraf - 7 polypeptide as an example, the specific synthesis steps are described as follows:

[0056] (1) Solid - phase synthesis of polypeptide:

[0057] Weigh the Rink amide AM resin and place it in a peptide - coupling tube. Add dichloromethane (DCM) solvent and soak for 30 minutes to fully swell the resin. After draining the solvent, add 20% (v / v) piperidine / N,N - dimethylformamide (DMF) solution to remove the Fmoc protecting group on the resin. Let it stand for 10 minutes and then drain. Then add 20% (v / v) piperidine / DMF and react slowly on a shaker at room temperature for 10 minutes to ensure that the resin completely exposes the free amino group. Then wash the resin 5 times with DMF and DCM respectively.

[0058] Dissolve 5 equivalents of Fmoc-AA-OH, 5 equivalents of Oxyma and 10 equivalents of DIC in N-methylpyrrolidone (NMP), pour it into the peptide coupling tube, and place it in an incubator at 60 °C and shake slowly for 20 minutes. After the reaction is completed, wash the resin 5 times with DMF and DCM respectively. Subsequently, remove the Fmoc protecting group again with 20% (v / v) piperidine / DMF according to the above steps, let it stand for 10 minutes and then drain the solvent, and react with 20% (v / v) piperidine / DMF at room temperature for 10 minutes. Thereafter, add a solution of 5 equivalents of Fmoc-protected amino acid, 5 equivalents of Oxyma and 10 equivalents of DIC dissolved in NMP, and react at 60 °C for 20 minutes. Repeat the above steps until the polypeptide synthesis is completed. The reaction conditions of stearic acid are the same as those of ordinary amino acids, and it is linked to the N-terminus of the last amino acid.

[0059] For the coupling of special amino acids such as S 5 in the polypeptide, the conditions are slightly adjusted. Add a solution of 2.5 equivalents of Fmoc-S5-OH, 2.5 equivalents of Oxyma and 5 equivalents of DIC dissolved in NMP, place it in an incubator at 60 °C and shake slowly for 2 hours. The coupling step of the next amino acid of S 5 is the same as that of other amino acids, and continue to react at 60 °C for 2 hours. The acetylation reaction of the -NH 2 at the terminal amino acid of the polypeptide is carried out with acetic anhydride / DIEA / DMF according to the volume ratio of 1 / 1 / 8 at room temperature for 10 min (this step is not required for the stapled peptide modified with stearic acid at the N-terminus).

[0060] (2) Staple cyclization (olefin metathesis reaction)

[0061] Add a 1,2-dichloroethane solution of Grubbs 1 st with a concentration of 8 mg / mL to the washed resin for the olefin metathesis reaction of the S 5 side chain, 2 h each time, and react 2 times. Then wash with DMF / DCM 10 times each. The reacted polypeptide resin is polycondensed with methanol for 10 min and dried with nitrogen. Soak the resin with the cleavage solution TFA / PhOH / H 2 O / TIPS (87.5:5:5:2.5, v / v / v / v) for 4 h, then collect the filtrate and blow it with argon for 30 min. Utilize the characteristic that the polypeptide is not easily soluble in ether, precipitate and remove other impurities with ice ether, and dissolve the crude polypeptide product with 50% acetonitrile water.

[0062] The crude polypeptide product is separated and purified by semi-preparative HPLC high performance liquid chromatography to obtain a pure polypeptide product, and the molecular weight of the target product is identified by mass spectrometry technology. The structure of the stapled peptide is as Figure 1As shown; the purity of the polypeptide product was identified by analytical high performance liquid chromatography (HPLC) and mass spectrometry. The HPLC chromatogram and mass spectrum are as Figures 2 - 9 shown, and the purity of the stapled peptide synthesized in the present invention is about 96%.

[0063] Experimental Example 2: Circular Dichroism Test of Polypeptides

[0064] The secondary conformation of the polypeptide in aqueous solution was determined by a circular dichroism spectrometer, and the circular dichroism spectrum (CD) of the polypeptide was plotted. The results are shown in Figure 22 . Based on the characteristic negative absorption peaks near 208 nm and 225 nm and the characteristic positive absorption peak near 195 nm in the circular dichroism spectrum, it can be judged that the stapled peptide has a typical α-helix conformation. The CD experimental results show that the stapled peptides Sraf-2-1 and Sraf-7-1 showed a typical α-helix conformation in their CD tests, while Sraf-4 had the worst helicity, which may be related to the side chain length and the position of the stapled structure in the polypeptide. The helicity of the linear peptide Raf-1 was worse than that of the stapled peptides, indicating that the stapling strategy played an important role in stabilizing the α-helix conformation of the polypeptide.

[0065] Experimental Example 3: Stability Test of Sraf Series Polypeptides

[0066] Polypeptide drugs are easily degraded rapidly in vivo, and the half-life of most natural active peptides in vivo is only within a few hours. Therefore, the ability to maintain serum stability is a key factor for polypeptides to exert biological functions in vivo. The linear peptide Raf-1 and the stearic acid-modified stapled peptides Sraf-2-1 and Sraf-7-1 were selected as test objects, and serum was used to simulate the degradation process of the polypeptide in vivo to further explore the serum stability of the all-hydrocarbon stapled peptide.

[0067] The remaining content of the polypeptide at different time points was monitored by HPLC, and the HPLC curve and degradation kinetic curve of Raf-1, Sraf-2-1 and Sraf-7-1 were obtained. As Figure 23 shown, the stapled peptides Sraf-2-1 and Sraf-7-1 showed considerable stability, and the remaining amount of the intact polypeptide reached more than 50% within 24 hours. Under the same conditions, the linear peptide Raf-1 was rapidly degraded within the first 12 hours, leveled off after 12 hours, and was degraded by about 80% after 24 hours. In summary, the all-hydrocarbon stapled cyclization modification can improve the serum stability of polypeptides, and the stapled peptides Sraf-2-1 and Sraf-7-1 showed high serum stability due to their stable conformation.

[0068] Experimental Example 4: Affinity Test of Sraf Polypeptides with RAS

[0069] The SPR technique was used to detect the binding of the linear peptide and the stapled peptide to KRAS respectively G12CThe in vitro binding force between proteins, the SPR fitting curve and the results are shown in Table 1. The SPR experimental results show that the linear peptide Raf-2 has no specific binding trend; the binding force between the linear peptide Raf-0 and KRAS G12C is relatively low. The binding force of the Sraf series of stapled peptides to KRAS G12C proteins has been improved to varying degrees. For the Sraf series of stapled peptides, except for Sraf-3, the binding force of all stapled peptides to KRAS G12C proteins is higher than that of the initial linear peptide Raf-1. Among them, Sraf-2 and Sraf-7 are particularly obvious. The K G12C value of the interaction between Sraf-2 and KRAS d is 1.83 μM, and the binding force is increased by nearly 196 times. The K G12C value of the interaction between Sraf-7 and KRAS d is 0.29 μM, and the binding force is increased by nearly 1236 times, indicating that the stapling strategy plays an important role in stabilizing the α-helix structure and improving the binding force with the target protein.

[0070] Experimental Example 5: CCK-8 experiment to determine cell viability

[0071] To evaluate the effect of these polypeptides on cell proliferation, a CCK-8 activity experiment was conducted to determine cell viability. The DMSO group without drug treatment was used as a control. The experimental group included 12 stapled peptides to study the anti-proliferative effects of the above 12 polypeptides on A549 cells and A375 cells.

[0072] Tumor cells were seeded in 96-well plates at a density of 5000 cells / well and cultured overnight in a humidified sterile incubator at 37 °C and 5% CO 2 . Then the cells were treated with the polypeptides at the specified concentrations (100 μM / 50 μM / 25 μM) for 24 h, and the culture medium was gently aspirated. The CCK-8 reagent solution was dissolved in serum-free medium at a volume ratio of 1:10, and 100 μL was added to each well. Incubate at 37 °C for 1 h, and record the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader.

[0073] The results of the CCK-8 experiment are shown in Figure 24 . The inhibition effects of Sraf-2 and Sraf-7 with the highest binding affinity on A549 cells and A375 cells are also the strongest. Interestingly, although the binding affinity of the N-terminal stearic acid-modified stapled peptides Sraf-2-1 and Sraf-7-1 is slightly lower than that of the prototype peptides Sraf-2 and Sraf-7, their anti-tumor cell proliferation ability is several times higher than that of the prototype peptides. In addition, Sraf-2-1 and Sraf-7-1 have strong inhibitory effects on several other tumor cell lines (SK-MEL-2, B16-F10, MCF-7).

[0074] Example 6: Flow cytometry experiment to determine the cell-penetrating ability of polypeptides

[0075] Due to problems such as the molecular size, polarity, hydrophilicity, and charge of polypeptides, it is difficult for them to rapidly cross the cell membrane like small molecule drugs. The lack of cell membrane permeability greatly limits the development of polypeptide drugs. Effective intracellular regulation requires efficient uptake of stapled peptides by cells and cytoplasmic localization. We selected the A549 cell line and used flow cytometry to detect the amounts of linear peptide Raf-1, stapled peptides Sraf-2 and Sraf-7, and stearic acid-modified stapled peptides Sraf-2-1 and Sraf-7-1 entering the cells.

[0076] Seed A549 cells in a 6-well plate at a density of 1×10 6 cells / well and culture them overnight in a humidified sterile incubator at 37 °C and 5% CO 2 until the cells are completely adherent. Starve the cells for 2 h by replacing the complete culture medium with serum-free basal medium. Treat A549 cells with 2.5 μM, 5 μM, 10 μM FITC-labeled polypeptides in the dark for 4 h, then wash away the excess FITC-labeled polypeptides with PBS, digest the cells with trypsin for 1 min, collect the cells into a 1.5 mL EP tube with cold basal medium, and centrifuge at 4 °C and 500 rpm for 10 min. Finally, discard the supernatant, resuspend the cells with PBS, and immediately perform flow cytometry analysis. The experimental results show that, as Figure 25 shown in b, at 2.5 μM, the fluorescence intensities of stearic acid-modified stapled peptides Sraf-2-1 and Sraf-7-1 in A549 cells are significantly higher than those of linear peptide Raf-1 and stapled peptides Sraf-2 and Sraf-7, and show a concentration-dependent manner, indicating that Sraf-2-1 and Sraf-7-1 are most likely to cross the cell membrane and be taken up by cells. The flow cytometry experimental results prove that Sraf-2-1 and Sraf-7-1 can efficiently cross the cell membrane and enter the cells.

[0077] We directly observed the intracellular distribution of FITC-Sraf-2-1 and FITC-Sraf-7-1 by fluorescence microscopy. Figure 25The blue part in a is the nucleus stained by Hoechst 33342, and the green is the polypeptide labeled with FITC fluorescence. Under the fluorescence microscope, it can be clearly seen that A549 cells effectively uptake Sraf-2-1 and Sraf-7-1 into the cytoplasm, laying a foundation for the next step of accurately targeting the RAS protein to exert its intracellular biological functions. In summary, stearic acid modification and the all-hydrocarbon stapling strategy can improve the cell membrane penetration of polypeptides, and the stapled peptides Sraf-2-1 and Sraf-7-1 can quickly cross the A549 cell membrane and localize in the cytoplasm.

[0078] Example 7: The stapled peptide downregulates the phosphorylation (p-AKT) level of the RAS downstream target gene AKT

[0079] When the upstream of the PI3K signaling pathway is inactivated and AKT is not phosphorylated, it will not dissociate from the cell membrane and further enter the nucleus. To evaluate the effect of the peptide on the RAS downstream signaling pathway, we detected the effect of Sraf-2-1 and Sraf-7-1 on the PI3K pathway by Western blot analysis.

[0080] Seed A549 cells in a 6-well plate at a density of 1*10 7 cells / well and culture them overnight at 37 °C and 5% CO 2 in a humidified sterile incubator until the cells are completely adherent. Use cells treated with DMSO as a control, and different concentrations of stearic acid-modified stapled peptides Sraf-2-1 and Sraf-7-1 as the experimental groups (100 μM / 75 μM / 50 μM). Extract proteins 4 hours after drug administration and analyze the protein expression by Western Blot (WB). The results of the WB experiment show that ( Figure 26 ) Sraf-2-1 and Sraf-7-1 can bind to the RAS protein, inhibit the phosphorylation of AKT in the PI3K pathway, and inhibit the proliferation of tumor cells, which is statistically significant. It indicates that the inhibitory effect of the polypeptide on tumor cell proliferation may be related to its regulation of the PI3K pathway.

[0081] Example 8: Detection of the polypeptide's ability to promote migration by cell scratch assay

[0082] We used the scratch assay to detect the cell migration ability. The experimental groups were the DMSO control group and the drug administration group. Take A549 cells in the logarithmic growth phase, after starvation treatment with serum-free medium for 24 h, digest the cells with trypsin and resuspend the cells to make a cell suspension. Gently pipette the cell suspension, count under the microscope, and adjust the cell density to 5×10 5Cells / ml were inoculated into 6-well plates and incubated at 37 °C for 24 h to starve overnight. The confluent cells were scratched with the tip of a pipette. Image acquisition was performed at 0 h and 24 h after drug treatment. Cell migration was quantified as the difference in the area of two cell-free wounds. All experiments were independently repeated three times.

[0083] The results of the scratch assay are shown as Figure 27 follows. After administration of Sraf-7-1, the migration of A549 cells could be inhibited; compared with the DMSO control group, the migration distance became shorter. By statistically analyzing the migration distance using Image J software, it was found that Sraf-7-1 could inhibit the migration of A549 cells, showing a statistically significant difference ( Figure 27 ).

[0084] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A stapled peptide targeting RAS-Raf protein interaction, characterized in that: The stapling peptide is selected from one of the following: Sraf-1: Ac-SLHDCLMKALKVR-NH2 is used as peptide chain template, of which 6 L and 10 L replaced by S5 and cyclized; Sraf-2: Ac-SLHDCLMKALKVR-NH2 is used as peptide chain template, 5 C and 9 A replaced by S5 and cyclized; Sraf-3: Ac-SLHDCLMKALKVR-NH2 is used as peptide chain template, in which 3 H and 7 M replaced by S5 and cyclized; Sraf-4: Ac-SLHDCLMKALKVR-NH2 is used as peptide chain template, 2 L and 6 L replaced by S5 and cyclized; Sraf-5: Ac-SLHDCLMKALKVR-NH2 is used as peptide chain template, in which 1 S and 5 C replaced by S5 and cyclized; Sraf-6: Ac-SLHDCLMKALKVRS5-NH2 was used as peptide chain template, of which 10 L replaced by S5 and cyclized; Sraf-7: Ac-SLHDCLMKALKVRGL-NH2 is used as peptide chain template, 5 C and 9 A replaced by S5 and cyclized; Sraf-8: Ac-SLHDCLMKALKVRGL-NH2 is used as peptide chain template, in which 11 K and 12 V Replaced by M, 5 C and 9 A replaced by S5 and cyclized; Sraf-9: Ac-SLHDCLMKALKVRGL-NH2 is used as peptide chain template, of which 11 K and 12 V Replaced by W and M respectively, 5 C and 9 A replaced by S5 and cyclized; Sraf-10: Ac-SLHDCLMKALKVRGL-NH2 is used as peptide chain template, in which 11 K and 12 V Replaced by W, 5 C and 9 A replaced by S5 and cyclized; Sraf-2-1: SA (Stearic acid)-SLHDCLMKALKVR-NH2 is used as peptide chain template, in which 5 C and 9 A replaced by S5 and cyclized; Sraf-7-1: SA (Stearic acid)- SLHDCLMKALKVRGL-NH2 is used as peptide chain template, in which 5 C and 9 A Replaced by S5 and cyclized.

2. A method for preparing a stapled peptide targeting RAS-Raf protein interaction as claimed in claim 1, characterized in that: The steps include: A. The C-terminus of the first amino acid is coupled to the solid phase carrier under the action of a condensing agent; B. Use a deprotection reagent to remove the Fmoc protecting group on the amino acid; C. Coupling the next amino acid under the action of a condensing agent; D. Repeat the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site replaces the amino acid at position i and i+4 with S5 respectively; E. The last amino acid is deprotected and then acetylated; F. Under the action of a cyclizing agent, the amino acids at positions i and i+4 undergo olefin metathesis reaction to cyclize the peptide chain; G. Use a cutting reagent to cut the peptide chain from the carrier and obtain the corresponding stapled peptide after purification.

3. Use of the stapled peptide targeting RAS-Raf protein interaction as claimed in claim 1 in the preparation of a drug targeting RAS protein.

4. Use of the stapled peptide targeting RAS-Raf protein interaction as claimed in claim 1 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The anti-tumor drug is a drug that inhibits the proliferation and / or migration of tumor cells.

6. The use according to claim 4, characterized in that: The tumor is lung cancer, melanoma or breast cancer.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation has the stapled peptide targeting the RAS-Raf protein interaction as claimed in claim 1 as the only active ingredient, or contains the stapled peptide targeting the RAS-Raf protein interaction as claimed in claim 1.

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