CN118255843A - Tapelin targeting TEAD-VGL4 interaction and application thereof in skin repair - Google Patents

Abstract

The present invention relates to the field of biomedicine, specifically a stapled peptide targeting TEAD-VGL4 interaction and its application in the preparation of a drug for promoting skin wound repair, with the straight-chain peptides Ac-DPVVEEHFRRSLGK- _NH2 and Ac-SVDDHFAKALGDTWLQIKAA- _NH2 shown in PDB ID:4LN0 as peptide chain templates, the amino acids at positions i and i+4 are replaced with 2-amino-2-methylhept-6-enoic acid ( _S5 ) and cyclized to obtain. The stapled peptide of the present invention can maintain a stable alpha helical conformation, improve its binding force to TEAD, promote the proliferation and migration of multiple cells, especially promote the proliferation of human dermal fibroblasts HDF-α; it has good serum stability and excellent cell membrane penetration ability, can pass through the cell membrane into the cytoplasm, target the target protein in the cell and exert its biological function.

Description

A stapled peptide targeting TEAD-VGL4 interaction and its application in skin repair

Technical Field

The present invention relates to the field of biomedicine, and in particular to a stapled peptide targeting TEAD-VGL4 interaction and an application thereof in the preparation of a drug for promoting skin wound repair.

Background technique

Wound healing is a complex process of skin tissue repair. For some chronic or large wounds, there are more injury factors, the wound healing process is disordered, and it is difficult for the body to heal itself, thus affecting the normal function and structure recovery, and medical treatment is required. Promoting the functional regeneration of damaged skin tissue is one of the important ways to repair skin wounds. It can quickly close the wound, restore the skin's defense function, and reduce the occurrence of complications such as infection. Skin wound repair and healing treatment has been a research hotspot in recent years.

The Hippo signaling pathway is one of the important regulatory pathways for cell growth, proliferation, and migration. Studies have found that the core element of the Hippo signaling pathway, the YAP/TAZ complex, is involved in the growth regulation of fibroblasts; the key effector transcription factor TEAD downstream of the Hippo pathway plays an important role in regulating normal organ size, cell proliferation and apoptosis. The activation of the Hippo signaling pathway depends on a series of kinase reactions. When stimulated by upstream signals, MST1/2 will be phosphorylated by upstream kinases, and the activated MST1/2 will bind to the regulatory protein SAV1, further activating LATS1/2 and MOB1A/B. After the combination of the two, the downstream transcription cofactor YAP/TAZ is induced to be phosphorylated, and then continues to bind to the 14-3-3 protein, and finally stays in the cytoplasm and is degraded by ubiquitination, inhibiting the pro-proliferation and anti-apoptotic activities of YAP/TAZ, thereby negatively regulating organ development, preventing cell proliferation and promoting cell apoptosis. If the upstream Hippo signaling pathway is inactivated, YAP/TAZ cannot be phosphorylated. Unphosphorylated YAP/TAZ will migrate into the cell nucleus and bind to the TEAD transcription factor to form a complex, activating the transcription of cell cycle and cell survival genes (such as CTGF, CYR61), initiating a pro-proliferation program, and thereby promoting cell proliferation, invasion, and metastasis.

As a transcriptional co-activator, YAP/TAZ does not have a DNA binding domain and needs to bind to the transcription factor TEAD to regulate the expression of downstream genes. Studies have found that VGL4 competes with YAP for binding to TEAD through the TDU domain, thereby inhibiting the activity of the YAP-TEAD transcription complex and inhibiting the expression of YAP downstream target genes. VGL4 can regulate both the stability of TEAD and its interaction with YAP. The TEAD-VGL4 interaction can promote the degradation of TEAD1 by cysteine proteases, reduce the level of TEAD1 protein, and thus reduce cell proliferation and survival. Many biological experiments have shown that inhibiting the TEAD-VGL4 interaction can promote tissue repair and cell regeneration. At present, there are few reports of effective inhibitors for this interaction, so this target has attracted much attention.

Hélène Adihou et al. reported a protein mimetic 4E designed based on the double helix structure of the transcriptional corepressor VGL4, which is intended to bind to the TEAD transcription factor. X-ray crystal structure verified that the protein mimetic maintains a certain conformation to bind TEAD and inhibit TEAD-VGL4 interaction. Experiments have found that the protein mimetic stimulates the expression of TEAD target genes in human cardiomyocytes, and promotes YAP nuclear translocation and cell cycle activity in young rat cardiomyocytes, which are central features required for cardiomyocyte proliferation. However, the protein mimetic has low membrane permeability and needs to be linked to TAT to increase intracellular activity. The affinity with the target can be further improved, and its role and mechanism in other diseases related to cell proliferation and regeneration also need to be studied.

Summary of the invention

The purpose of the present invention is to provide a novel stapled peptide targeting the TEAD-VGL4 protein interaction, and further provide the use of the stapled peptide in the preparation of a drug for promoting cell proliferation.

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

Analysis of the crystal structure of the mTEAD-VGL4 complex shows that the binding sequence of VGL4 to TEAD mainly consists of three fragments, including the N-terminal α1-helix (green), the C-terminal α2 and α3 helical structures (blue), and the β-chain (yellow) connecting the two parts. The α1 helix of VGL4 can bind to TEAD monomer a, while the β-chain, helix α2 and α3 can bind to TEAD monomer b. Studies have shown that the full binding sequence has the highest affinity for TEAD, the affinity of the middle β-chain is very low (Kd>100μM), the N-terminal α1-helix sequence has a medium affinity, and the C-terminal α2 and α3 helical structures have a higher affinity. The α2 and α3 helical structures have also been reported to be crucial for the binding of VGL4 to TEAD.

Regulating the balance between the activities of VGL4, TEAD, and YAP, or inhibiting the degradation of TEAD by cysteine proteases caused by TEAD-VGL4 interaction, may be used for wound repair or promoting fibroblast proliferation. Therefore, we simplified and optimized the α2 and α3 helical parts of VGL4 to generate linear peptides Hip1 and Hip2; introduced the non-natural amino acid S ₅ (S-2-amino-2-methylhept-6-enoic acid) with α-olefin groups at positions i and i+4 of the linear peptides, designed and synthesized a series of novel stapled peptides targeting TEAD-VGL4 interaction, observed their α-helicity, stability, membrane permeability, and affinity with TEAD, investigated their ability to promote the proliferation of skin fibroblasts, selected stapled peptides with high pro-proliferation activity, further investigated their effects and mechanisms on YAP target genes and downstream signaling pathways, and discovered lead compounds that effectively promote fibroblast proliferation and skin regeneration, providing new ideas and theoretical basis for functional regeneration and repair of damaged skin tissue.

Studies have shown that constructing a fully hydrogenated stapled peptide at the i and i+4 positions of a polypeptide through an olefin metathesis reaction can stabilize the α-helical structure of the polypeptide, improve the stability, membrane permeability, and binding affinity of the polypeptide to the target protein. The peptides currently developed are mainly targeted at the YAP/TEAD interaction, and most of them act on tumor inhibition. They have problems such as poor stability, low membrane permeability, and low binding affinity. With the deepening of research on the Hippo signaling pathway, 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 to the treatment of skin tissue regeneration, especially wound repair.

The present invention uses the linear peptide Ac-DPVVEEHFRRSLGK-NH ₂ (SEQ ID NO.1) shown in PDB ID: 4LN0 as a peptide chain template, replaces the amino acids at positions i and i+4 with (s)-2-amino-2-methylhept-6-enoic acid (S ₅ ) and cyclizes to obtain 5 SHip1 series stapled peptides; uses the linear peptide Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ (SEQ ID NO.2) as a peptide chain template, replaces the amino acids at positions i and i+4 with (s)-2-amino-2-methyl-6-heptenoic acid (S ₅ ) and cyclizes to obtain 8 SHip2 series stapled peptides. There are 13 stapled peptides in total, and the structural formula is as follows:

In a first aspect of the present invention, a stapled peptide targeting TEAD-VGL4 interaction is provided, wherein the stapled peptide is selected from one of the following:

SHip1-1: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 1 _D and 5 _E were replaced by S ₅ and cyclized;

SHip1-2: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 3 _V and 7 _H were replaced by S ₅ and cyclized;

SHip1-3: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 4 _V and 8 _F were replaced by S ₅ and cyclized;

SHip1-4: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 7 _H and 11 _S were replaced by S ₅ and cyclized;

SHip1-5: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 8 _F and 12 _L were replaced by S ₅ and cyclized;

SHip2-1: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 3 _D and 7 _A were replaced by S ₅ and cyclized;

SHip2-2: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 4 _D and 8 _K were replaced by S ₅ and cyclized;

SHip2-3: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 7 _A and 11 _G were replaced by S ₅ and cyclized;

SHip2-4: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 11 _G and 15 _L were replaced by S ₅ and cyclized;

SHip2-5: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 12 _D and 16 _Q were replaced by S ₅ and cyclized;

SHip2-6: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 13 _T and 17 _I were replaced by S ₅ and cyclized;

SHip2-7: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 14 _W and 18 _K were replaced by S ₅ and cyclized;

SHip2-8: Ac-SVDDHFAKALGDTWLQIKAA- _NH2 was used as the peptide chain template, in which _15L and _19A were replaced by _S5 and cyclized.

In a second aspect of the present invention, a method for preparing a stapled peptide targeting TEAD-VGL4 interaction as described above is provided, which specifically comprises the following steps:

A) coupling the C-terminus of the first amino acid to a solid phase carrier under the action of a condensing agent;

B) removing the Fmoc protecting group on the amino acid using a deprotection reagent;

C) coupling the next amino acid under the action of a condensing agent;

D) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S ₅ to replace the amino acid at position i and i+4 respectively;

E) The last amino acid is deprotected and then acetylated;

F) causing the S ₅ amino acids at positions i and i+4 to undergo olefin metathesis reaction under the action of a cyclizing agent to cyclize the peptide chain;

G) Using a cleavage agent to cut the peptide chain from the carrier, and purifying the corresponding stapled peptide.

The third aspect of the present invention provides a use of the stapled peptide targeting TEAD-VGL4 interaction as described above in the preparation of a drug targeting TEAD protein.

The results of cell experiments showed that the stapled peptide SHip2-5 can promote the proliferation and migration of human skin fibroblasts in a dose-dependent manner; at the same time, it has excellent cell penetrating ability and can pass through the cell membrane into the cytoplasm, thereby better targeting the target protein in the cell to exert its biological function.

A fourth aspect of the present invention provides a use of the stapled peptide targeting TEAD-VGL4 interaction as described above in the preparation of a drug for promoting skin wound repair.

In a fourth aspect, the present invention provides a drug for promoting skin wound repair, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the stapled peptide targeting the TEAD-VGL4 interaction as described above as the only active ingredient, or comprises the stapled peptide targeting the TEAD-VGL4 interaction as described above.

Furthermore, the drug can be made into various dosage forms with commonly used pharmaceutical excipients, such as decoctions, powders, pills, intravenous emulsions, liposome preparations, aerosols, prodrug preparations, injections, mixtures, oral ampoules, tablets, capsules, etc. The administration method is not limited to oral administration, injection, etc.

The advantages and beneficial effects of the present invention are:

Based on the crystal structure of the TEAD-VGL4 complex (PDB ID: 4LN0), the present invention uses the α-helical fragment helixα1 at the N-terminus of the VGL4 protein and the C-terminal double helical fragment helixα2/3 as templates to design and synthesize a series of novel stapled peptides targeting the interaction of TEAD/VGL4 proteins, determine their secondary structures by circular dichroism, evaluate their membrane permeability by flow cytometry, and evaluate the proliferation-promoting effect of the stapled peptides on various cells at the cellular level. The stapled peptides of the present invention can maintain a stable α-helical conformation, improve their binding force to TEAD, promote the proliferation and migration of various cells, especially promote the proliferation of human dermal fibroblasts HDF-α; have good serum stability and excellent cell penetration ability, can pass through the cell membrane into the cytoplasm, target the target protein in the cell and exert its biological function.

1. In terms of preparation, the present invention uses amino resin as a carrier, Ac-DPVVEEHFRRSLGK-NH ₂ and Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ as peptide chain templates, and synthesizes a peptide chain by Fmoc solid phase synthesis. On the basis of retaining key amino acid residues, the original amino acid is replaced by S ₅ at a specific position. The linear peptide coupled to the resin undergoes olefin metathesis reaction and cyclization in a 1,2-dichloroethane solution of Grubbs ^1st reagent, and then is cut from the resin to obtain the target stapled peptide. After the obtained compound is purified, it is characterized and analyzed by HPLC, HR-MS and circular dichroism. The obtained stapled peptide has a purity of more than 95% and retains a typical α-helical conformation.

2. In terms of effect, the stapled peptide SHip2-5 can promote the migration of human skin fibroblasts in a dose-dependent manner; at the same time, it has excellent cell penetration ability and can pass through the cell membrane into the cytoplasm, thereby better targeting the target protein in the cell to exert its biological function. The stapled peptide can upregulate the expression of the YAP gene and the mRNA levels of its downstream target genes hCTGF, hCYR61 and hANKRD1.

3. The stapled peptide strategy of the present invention maintains a stable conformation of the α-helix of Hip2, enhances its membrane permeability, improves serum stability, further retains or even improves its binding ability to TEAD, and exerts its ability to promote proliferation and migration of various cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a circular dichroism spectrum of the stapled peptide in pure water after purification;

FIG2 is a CCK-8 assay to analyze the effect of peptides on the proliferation of human skin fibroblasts;

FIG3 is a flow cytometry experiment to determine the membrane penetration ability of FITC-modified polypeptides; and a fluorescence confocal experiment to detect the membrane penetration of stapled peptides;

Figure 4 shows the dual luciferase reporter gene experiment to detect the expression of YAP and the qRT-PCR experiment to detect the expression of YAP downstream target genes hCTGF, hCYR61 and hANKRD1;

FIG5 is a cell scratch assay to evaluate the ability of stapled peptides to promote cell migration;

Figures 6-7 are the mass spectra and HPLC chromatograms of Hip1 and Hip2, respectively;

Figures 8-12 are the mass spectrometry and HPLC analysis of SHip1-1, SHip1-2, SHip1-3, SHip1-4, and SHip1-5, respectively;

Figures 13-20 are mass spectrometry and HPLC analyses of SHip2-1, SHip2-2, SHip2-3, SHip2-4, SHip2-5, SHip2-6, SHip2-7, and SHip2-8, respectively;

Figures 21-24 are the mass spectrometry and HPLC analysis of FITC-Hip1, FITC-Hip2, FITC-Hip2-5, and FITC-Hip2-6, respectively.

Detailed ways

The specific implementation methods provided by the present invention are described in detail below in conjunction with the examples. The following examples are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified.

Example 1: Synthesis of stapled peptides targeting TEAD proteins

Taking the stapled peptide SHip2-5 polypeptide as an example, the specific synthesis steps are described as follows:

(1) Solid phase synthesis of peptides:

Weigh Rink amide MBHA resin into a peptide tube, add reagent dichloromethane (DCM) to the peptide tube to soak and swell the resin for 30 minutes. After draining the solvent, use 20% (v/v) piperidine/N, N-dimethylformamide (DMF) to remove the Fmoc protecting group on the resin, let it stand for 5 minutes and drain the solvent, then add 20% (v/v) piperidine/DMF, and shake slowly in a shaker at room temperature for 10 minutes to completely expose the free amino groups of the resin.

Wash with DMF/DCM alternately for 5 times. Dissolve 5eq Fmoc-AA-OH, 5eq Oxyma, and 10eq DIC in N-methylpyrrolidone NMP reagent and pour into the peptide tube, place it in a 60℃ constant temperature box and shake it slowly for 20 minutes. Then wash it alternately with DMF/DCM for 5 times. Then use 20% (v/v) piperidine/DMF to remove the Fmoc protecting group on the resin, let it stand for 5 minutes, then drain the solvent, add 20% (v/v) piperidine/DMF, and shake it slowly at room temperature in a shaker for 10 minutes. Then add NMP solution containing 5eq Fmoc-protected amino acids but exposed carboxyl groups, 5eq Oxyma, and 10eq DIC, and react at 60℃ for 20 minutes. Repeat this cycle until the peptide is connected. The reaction conditions of the special amino acid (S ₅ ) and the next amino acid in the peptide chain are slightly different. Add NMP solution containing 2.5eq Fmoc-S ₅ -OH, 2.5eq Oxyma, and 5eq DIC to the resin and place it in a 60℃ constant temperature box for 2 hours. The amount of the next amino acid of S ₅ is the same as that of other amino acids, and the reaction is carried out at 60℃ for 2 hours. The acetylation reaction of the side chain -NH ₂ of the terminal amino acid of the polypeptide is carried out with acetic anhydride/DIEA/DMF in a volume ratio of 1/1/8 at room temperature for 10 minutes.

(2) Olefin metathesis reaction

Add 8 mg/mL of Grubbs 1 ^st in 1,2-dichloroethane to the washed resin to perform olefin metathesis of the S ₅ side chain, 2 h/time, 2 times. Then wash with DMF/DCM alternately 10 times. The peptide resin after the reaction is completed is condensed with methanol for 10 min and blown dry with nitrogen. Soak the resin with cutting liquid 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 with argon for 30 min. Taking advantage of the fact that the peptide is not easily soluble in ether, ice ether precipitation is used to remove other impurities, and the crude peptide is dissolved in 50% acetonitrile water.

The crude polypeptide product is separated and purified by preparative HPLC to obtain a pure polypeptide product, and the molecular weight of the target product is identified by high-resolution mass spectrometry. The structure of the stapled peptide is shown in Table 1; the purity of the polypeptide product is identified by analytical high-performance liquid chromatography HPLC and mass spectrometry, and the HPLC spectrum and mass spectrum are shown in Figures 6 to 24. The purity of the stapled peptide polypeptide synthesized by the present invention is about 95% or more.

Table 1. Sequences and molecular weights of all peptides

Hip1 (DPVVEEHFRRSLGK), white powder after freeze-drying. Purity: 96.0923%. HR-MS: C ₇₅ H ₁₂₀ N ₂₄ O ₂₂ , Calc. for 1708.9000, found (M+2H) ²⁺ : 855.4606

Hip2 (SVDDHFAKALGDTWLQIKAA), white powder after freeze-drying. Purity: 95.0744%. HR-MS: C ₁₀ 1H ₁₅₅ N ₂₇ O ₃₀ , Calc. for 2226.14 found (M+2H) ²⁺ : 1114.5832

SHip1-1 (S ₅ PVVS ₅ EHFRRSLGK), white powder after freeze-drying. Purity: 99.7852%. HR-MS: C ₈₀ H ₁₃₀ N ₂₄ O ₁₈ , Calc. for 1714.9995, found (M+2H) ²⁺ : 859.0145

SHip1-2 (DPS ₅ VEES ₅ FRRSLGK), white powder after freeze-drying. Purity: 96.1205%. HR-MS: C ₇₈ H ₁₂₆ N ₂₂ O ₂₂ , Calc. for 1722.9417, found (M+2H) ²⁺ : 862.4807

SHip1-3 (DPVS ₅ EEHS ₅ RRSLGK), white powder after freeze-drying. Purity: 99.6155%. HR-MS: C ₇₅ H ₁₂₄ N ₂₄ O ₂₂ , Calc. for 1712.9322, found (M+2H) ²⁺ : 857.4764

SHip1-4 (DPVVEES ₅ FRRS ₅ LGK), white powder after freeze-drying. Purity: 98.7719%. HR-MS: C ₈₀ H ₁₃₀ N ₂₂ O ₂₁ , Calc. for 1734.9781, found (M+2H) ²⁺ : 869.0001

SHip1-5 (DPVVEEHS ₅ RRSS ₅ GK), white powder after freeze-drying. Purity: 97.5935%. HR-MS: C ₇₄ H ₁₂₂ N ₂₄ O ₂₂ , Calc. for 1698.9166, found (M+2H) ²⁺ : 850.4663

SHip2-1 (SVS ₅ DHFS ₅ KALGDTWLQIKAA), white powder after freeze-drying. Purity: 97.3495%. HR-MS: C ₁₀₈ H ₁₆₇ N ₂₇ O ₂₈ , Calc. for 2290.2474, found (M+2H) ²⁺ : 1146.6399

SHip2-2 (SVDS ₅ HFAS ₅ ALGDTWLQIKAA), white powder after freeze-drying. Purity: 97.7301%. HR-MS: C ₁₀₅ H ₁₆₀ N ₂₆ O ₂₈ , Calc. for 2233.19, found (M+2H) ²⁺ : 1116.0887

SHip2-3 (SVDDHFS ₅ KALS ₅ DTWLQIKAA), white powder after freeze-drying. Purity: 97.5935%. HR-MS: C ₁₁₀ H ₁₆₉ N ₂₇ O ₃₀ , Calc. for 2348.2529, found (M+2H) ²⁺ : 1175.6444

SHip2-4 (SVDDHFAKALS ₅ DTWS ₅ QIKAA), white powder after freeze-drying. Purity: 96.5342%. HR-MS: C ₁₀₇ H ₁₆₃ N ₂₇ O ₃₀ , Calc. for 2306.2100, found (M+2H) ²⁺ : 1154.6136

SHip2-5 (SVDDHFAKALGS ₅ TWLS ₅ IKAA), white powder after freeze-drying. Purity: 97.0400%. HR-MS: C ₁₀₆ H ₁₆₄ N ₂₆ O ₂₇ , Calc. for 2233.2259, found (M+2H) ²⁺ : 1118.1299

SHip2-6 (SVDDHFAKALGDS ₅ WLQS ₅ KAA), white powder after freeze-drying. Purity: 95.8999%. HR-MS: C ₁₀₅ H ₁₅₉ N ₂₇ O ₂₉ , Calc. for 2262.1797, found (M+2H) ²⁺ : 1132.6059

SHip2-7 (SVDDHFAKALGDTS ₅ LQIS ₅ AA), white powder after freeze-drying. Purity: 99.0433%. HR-MS: C ₉₈ H ₁₅₅ N ₂₅ O ₃₀ , Calc. for 2162.1372, found (M+2H) ²⁺ 1082.5847

SHip2-8 (SVDDHFAKALGDTWS ₅ QIKS ₅ A), white powder after freeze-drying. Purity: 96.7048%. HR-MS: C ₁₀₆ H ₁₆₁ N ₂₇ O ₃₀ , Calc. for 2292.19, found (M+2H) ²⁺ : 1147.6135

FITC-Hip1 (FITC-βA-DPVVEEHFRRSLGK), white powder after freeze-drying. Purity: 97.5935%. HR-MS: C ₉₇ H ₁₃₄ N ₂₆ O ₂₇ S Calc.for2126.96, found(M+2H) ²⁺ :1064.9938

FITC-Hip2 (FITC-βA-SVDDHFAKALGDTWLQIKAA), white powder after freeze-drying. Purity: 98.0248%. HR-MS: C ₁₂₃ H ₁₆₉ N ₂₉ O ₃₅ S, Calc. for 2644.21, found (M+2H) ²⁺ : 1323.6183

FITC-SHip2-5 (FITC-βA-SVDDHFAKALGS ₅ TWLS ₅ IKAA), white powder after freeze-drying. Purity: 95.6105%. HR-MS: C ₁₂₈ H ₁₇₈ N ₂₈ O ₃₂ S, Calc. for 2651.29 found (M+2H) ²⁺ : 1327.1538

FITC-SHip2-6 (FITC-βA-SVDDHFAKALGDS ₅ WLQS ₅ KAA), white powder after freeze-drying. Purity: 96.2506%. HR-MS: C ₁₂₇ H ₁₇₃ N ₂₉ O ₃₄ S, Calc. for 2680.24, found (M+2H) ²⁺ : 1341.6308.

Experimental Example 2: Circular dichroism test of SHip peptide

The secondary conformation of the peptide in aqueous solution was determined by circular dichroism spectrometer, and the circular dichroism spectrum (CD) of the peptide was drawn. The results are shown in Figure 1. According to the characteristic negative absorption peaks near 208nm and 225nm and the characteristic positive absorption peak near 195nm in the circular dichroism spectrum, it can be judged that the stapled peptide has a typical α helical conformation. The CD experimental results show that the helicity of the SHip1 series stapled peptides has been improved to a certain extent compared with the straight-chain peptide Hip1; SHip2-5 in the SHip2 series stapled peptides showed a typical α helical conformation in its CD test.

Experimental Example 3: Stability test of SHip series peptides

Peptide drugs are easily degraded in vivo, and the half-life of most natural active peptides in vivo is only a few hours. Therefore, maintaining serum stability is a key factor for peptides to exert their biological functions in vivo. The linear peptides Hip1, Hip2 and the stapled peptides SHip2-5 were selected as test objects, and serum was used to simulate the degradation process of peptides in vivo to evaluate the serum stability of all-carbon stapled peptides.

HPLC was used to track and monitor the remaining content of the peptide at different time points, and the HPLC curves and degradation kinetic curves of Hip1, Hip2 and SHip2-5 were obtained. The stapled peptide SHip2-5 showed considerable stability, and the remaining amount of the complete peptide reached 80% within 24 hours. Under the same conditions, the straight-chain peptide Hip2 was rapidly degraded within the first 6 hours, tended to be stable after 6 hours, and was degraded by about 40% in 24 hours. The straight-chain peptide Hip1 had only 40% of the complete peptide remaining after 24 hours. In summary, the all-carbon-hydrogen stapled ring modification can improve the protease hydrolysis stability of the peptide, and the stapled peptide SHip2-5 showed high serum stability.

Experimental Example 4: Affinity test of SHip peptide and TEAD

The SPR technique was used to detect the in vitro binding forces between straight-chain peptides and stapled peptides and TEAD proteins, respectively. The SPR fitting curves and results are shown in Figures 3-4. The results of the SPR experiment showed that the straight-chain peptide Hip1 had no specific binding tendency; the binding force between the straight-chain peptide Hip2 and TEAD was low, with a _KD value of >100μM. The binding forces between the SHip1 series stapled peptides and TEAD proteins had no specific binding tendency or weak affinity. Except for SHip2-1, SHip2-7 and SHip2-8, the binding forces of all stapled peptides in the SHip2 series with TEAD proteins were higher than those of the straight-chain peptide Hip2, among which SHip2-4, SHip2-5 and SHip2-6 were particularly obvious. The KD value of SHip2-5 interacting with TEAD was 3.502μM, and the binding force was increased by nearly 200 times, indicating that the α2 helical domain played an important role in the VGL4-TEAD interaction.

Experimental Example 5: CCK-8 assay to determine cell viability

In order to evaluate the effect of these peptides on cell proliferation, CCK-8 activity assay was performed to determine cell survival rate. The untreated group and the linear peptide-treated group were used as controls. The experimental group included 13 stapled peptides to study the effect of the above 13 peptides on HDF-α proliferation.

The cells HDF-α were seeded in a 96-well plate at a density of 5000 cells/well and cultured overnight in a humidified sterile incubator at 37°C and 5% CO ₂ , and then treated with the specified concentration (40μM/20μM/10μM) of peptide for 24h or 48h, and the culture medium was gently aspirated. The CCK-8 reagent solution was dissolved in serum-free culture medium at a volume ratio of 1:10, 100μL was added to each well, incubated at 37°C for 1h, and the absorbance was recorded at 450nm using an enzyme reader.

The results of the CCK-8 experiment are shown in Figure 2. The stapled peptide SHip2-5 can promote cell proliferation, and SHip2-6 also has a certain effect on promoting cell proliferation, but the effect is weaker than that of SHip2-5.

Example 6: Flow cytometry experiment to determine the membrane-penetrating ability of polypeptides

Due to the molecular size, polarity, hydrophilicity and charge of peptides, it is difficult for them to cross the cell membrane as quickly as small molecule drugs. The lack of cell membrane permeability has greatly limited the development of peptide drugs. Effective intracellular regulation requires effective cell uptake and cytoplasmic localization of stapled peptides. We selected HDF-α and used flow cytometry to detect the number of Hip2 linear peptides and SHip series stapled peptides entering HDF-α cells.

HDF-α cells were seeded in 6-well plates at a density of 1×10 ⁶ cells/well and cultured overnight in a humidified sterile incubator at 37°C and 5% CO ₂ until the cells were completely attached. The initial culture medium was replaced with serum-free basal medium for incubation for 2 hours. HDF-α cells were treated with 10μM FITC-labeled SHip2-5 and SHip2-6 in the dark for 24 hours, then washed with PBS to remove excess FITC-labeled peptides, trypsinized cells for 1 minute, and collected cells with cold basal medium into 1.5mL EP tubes, centrifuged at 4°C and 500rpm for 10 minutes. Finally, the supernatant was removed, PBS was added to resuspend the cells, and flow cytometry analysis was performed immediately. The experimental results showed that, as shown in Figure 3A, the fluorescence intensity of stapled peptides SHip2-5 and SHip2-6 in HDF-α was significantly higher than that of linear peptides, and SHip2-5 was the most obvious, indicating that SHip2-5 was most likely to pass through the cell membrane and be taken up by cells. Then, the fluorescence intensity of HDF-α cell samples treated with FITC-SHip2-5 at different concentrations (10 μM, 20 μM, 40 μM) was further detected, and it was found to be concentration-dependent (Figure 3C). The results of flow cytometry experiments proved that SHip2-5 can efficiently pass through the cell membrane and enter the cell.

We used a fluorescence microscope to directly observe the distribution of FITC-SHip2-5 in HDF-α cells. The blue part in Figure 3B is the cell nucleus stained with Hoechst 33342, and the green part is the fluorescently labeled FITC-SHip2-5. Under the fluorescence microscope, it can be clearly seen that HDF-α cells effectively take up SHip2-5 into the cells, laying the foundation for the next step of SHip2-5 to accurately target TEAD proteins to exert intracellular biological functions. In summary, the all-carbon-hydrogen stapled cyclization strategy can improve the cell membrane penetration of peptides, and the stapled peptide SHip2-5 can quickly pass through the HDF-α cell membrane and localize in the cytoplasm.

Example 7: Staple peptides upregulate YAP expression and transcription levels of its downstream target genes hCTGF, hCYR61 and hANKRD1

When the upstream of the Hippo signaling pathway is inactivated and YAP is not phosphorylated, it will enter the cell nucleus and bind to the transcription factor TEADs, inducing the expression of target genes, activating pro-proliferation and pro-survival genes, and promoting cell proliferation, invasion, and metastasis.

The YAP reporter plasmid and sv-40l plasmid were transfected into HEK293T cells for expression. Three replicate wells were made in a 96-well plate. The luciferase activity of each well was detected after treatment with the compound at a final concentration of 10 μM for 6 hours. The luciferase activity of cells treated with DMSO was used as a control to calculate the relative luciferase activity of each well. The results of the dual luciferase reporter gene experiment showed that SHip2-5 and SHip2-6 could upregulate the expression of YAP, which was statistically significant (Figure 4A).

In order to further study how the stapled peptide affects the expression of downstream target genes of the Hippo signaling pathway, the qRT-PCR method was used to further detect whether the stapled peptide inhibitor has an effect on the transcription level of downstream target genes of the Hippo signaling pathway (CTGF, CYR61 and ANKRD1). HDF-α cell samples treated with SHip2-5 at different concentrations (10μM, 20μM, 40μM) were collected. The results in Figure 4B showed that compared with the untreated group, SHip2-5 and SHip2-6 could upregulate the mRNA levels of downstream target genes hCTGF, hCYR61 and hANKRD1.

Example 8: Cell scratch assay to detect the ability of polypeptides to promote migration

We used the scratch test to detect cell migration ability. The experimental groups were divided into the non-drug group, the YAP activator (PY-60) group, and the drug group. HDF-α cells in the logarithmic growth phase were taken. After starvation treatment with serum-free medium for 24 hours, the cells were trypsinized and resuspended in a cell suspension. The cell suspension was gently blown and counted under a microscope. The cell density was adjusted to 5×105cells/ml and inoculated in a 6-well plate. The cells were incubated at 37°C for 24 hours and starved overnight. The fused cells were scratched with a pipette tip. Images were collected 12 hours and 24 hours after drug intervention. Cell migration was quantified as the difference between the two cell-free wound areas. All experiments were repeated three times independently.

The results of the scratch test are shown in Figure 5. Administration of SHip2-5 can promote the migration of HDF-α cells, and the migration distance is longer than that after treatment with the YAP activator (PY-60). The migration distance was statistically analyzed using Image J software, and it was found that SHip2-5 can promote the migration of HDF-α cells after promoting YAP expression, with statistical significance.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims (5)

1. A stapled peptide targeting TEAD-VGL4 interaction, characterized in that the stapled peptide is selected from one of the following: SHip1-1: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 1 _D and 5 _E were replaced by S ₅ and cyclized; SHip1-2: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 3 _V and 7 _H were replaced by S ₅ and cyclized; SHip1-3: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 4 _V and 8 _F were replaced by S ₅ and cyclized; SHip1-4: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 7 _H and 11 _S were replaced by S ₅ and cyclized; SHip1-5: Ac-DPVVEEHFRRSLGK-NH ₂ was used as the peptide chain template, in which 8 _F and 12 _L were replaced by S ₅ and cyclized; SHip2-1: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 3 _D and 7 _A were replaced by S ₅ and cyclized; SHip2-2: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 4 _D and 8 _K were replaced by S ₅ and cyclized; SHip2-3: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 7 _A and 11 _G were replaced by S ₅ and cyclized; SHip2-4: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 11 _G and 15 _L were replaced by S ₅ and cyclized; SHip2-5: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 12 _D and 16 _Q were replaced by S ₅ and cyclized; SHip2-6: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 13 _T and 17 _I were replaced by S ₅ and cyclized; SHip2-7: Ac-SVDDHFAKALGDTWLQIKAA-NH ₂ was used as the peptide chain template, in which 14 _W and 18 _K were replaced by S ₅ and cyclized; SHip2-8: Ac-SVDDHFAKALGDTWLQIKAA- _NH2 was used as the peptide chain template, in which _15L and _19A were replaced by _S5 and cyclized. 2. Use of the stapled peptide targeting TEAD-VGL4 interaction as claimed in claim 1 in the preparation of a drug targeting TEAD protein. 3. Use of the stapled peptide targeting TEAD-VGL4 interaction as claimed in claim 1 in the preparation of a drug for promoting skin wound repair. 4. A drug for promoting skin wound repair, characterized in that it comprises an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the stapled peptide targeting the TEAD-VGL4 interaction as claimed in claim 1 as the only active ingredient, or comprises the stapled peptide targeting the TEAD-VGL4 interaction as claimed in claim 1. 5. A method for preparing a stapled peptide targeting TEAD-VGL4 interaction as claimed in claim 1, characterized in that it comprises the following steps: A) coupling the C-terminus of the first amino acid to a solid phase carrier under the action of a condensing agent; B) removing the Fmoc protecting group on the amino acid using a deprotection reagent; C) coupling the next amino acid under the action of a condensing agent; D) Repeating the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence; wherein the cyclization site is replaced by S ₅ to replace the amino acid at position i and i+4 respectively; E) The last amino acid is deprotected and then acetylated; F) Under the action of the cyclization reagent, the i and i+4 positions S ₅ undergo olefin metathesis reaction to cyclize the peptide chain; G) Using a cleavage agent to cut the peptide chain from the solid phase support, the corresponding stapled peptide is obtained after purification.