CN118255843A - Tapelin targeting TEAD-VGL4 interaction and application thereof in skin repair - Google Patents
Tapelin targeting TEAD-VGL4 interaction and application thereof in skin repair Download PDFInfo
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- CN118255843A CN118255843A CN202410400732.3A CN202410400732A CN118255843A CN 118255843 A CN118255843 A CN 118255843A CN 202410400732 A CN202410400732 A CN 202410400732A CN 118255843 A CN118255843 A CN 118255843A
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention relates to the field of biological medicine, in particular to a targeting TEAD-VGL4 interaction stapling peptide and application thereof in preparing a medicine for promoting skin wound repair, wherein a PDB ID 4LN0 straight-chain peptide Ac-DPVVEEHFRRSLGK-NH 2 and Ac-SVDDHFAKALGDTWLQIKAA-NH 2 are used as peptide chain templates, and the i-th and i+4-th amino acids are replaced by 2-amino-2-methylhept-6-enoic acid (S 5) and cyclized. The stapling peptide can maintain a stable alpha helical conformation, improve the binding force of the stapling peptide to TEAD, promote the proliferation and migration capacity of various cells, and especially promote the proliferation of human dermal fibroblast HDF-alpha; has good serum stability and excellent cell penetrating capacity, can penetrate through cell membranes and enter cytoplasm, and targets target proteins in cells to exert biological functions.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a targeting TEAD-VGL4 interaction staple peptide and application thereof in preparing medicaments for promoting skin wound repair.
Background
Wound healing is a complex process of skin tissue repair, and for some chronic or wound with larger wound surface, the wound healing process is disordered due to more wound factors, and the wound healing process is difficult to heal by a body, so that normal function and structure recovery are affected, and medical treatment is needed. Promoting the regeneration of the function of damaged skin tissues is one of important ways for repairing the skin wound surface, can rapidly seal the wound surface, recover the defending function of the skin, and reduce the occurrence of complications such as infection. Skin wound repair healing treatments have been the focus of research in recent years.
The Hippo signaling pathway is one of the important regulatory pathways for cell growth, proliferation, and migration. Research shows that the YAP/TAZ complex which is a core element of the Hippo signal pathway 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 and apoptosis in cell proliferation. Activation of the Hippo signaling pathway relies on a series of kinase reactions, upon stimulation by upstream signals, MST1/2 is phosphorylated by upstream kinases, activated MST1/2 binds to regulatory protein SAV1, further activates LATS1/2 and MOB1A/B, which, when combined, induce downstream transcription cofactor YAP/TAZ phosphorylation, and then continues to bind to 14-3-3 protein, eventually remaining in the cytoplasm to degrade through ubiquitination, inhibiting the pro-proliferative and anti-apoptotic activity of YAP/TAZ, and further negatively regulating organ development, preventing cell proliferation and promoting apoptosis. If the Hippo signaling pathway is inactivated upstream, YAP/TAZ cannot be phosphorylated, and the non-phosphorylated YAP/TAZ migrates into the nucleus and combines with TEAD transcription factors to form a complex, activate transcription of cell cycle and cell survival genes (e.g., CTGF, CYR 61), initiate pro-proliferative processes, and promote cell proliferation, invasion, and metastasis.
YAP/TAZ does not have a DNA binding domain as a transcription coactivator and needs to bind to the transcription factor TEAD to regulate expression of downstream genes. It was found that VGL4 competes with YAP for binding to TEAD through the TDU domain, thereby inhibiting the activity of YAP-TEAD transcription complex, thereby inhibiting the expression of target gene downstream of YAP. VGL4 can regulate the stability of TEAD and the interaction of the TEAD and YAP. The TEAD-VGL4 interaction promotes degradation of TEAD1 by cysteine proteases, reducing TEAD1 protein levels, and thus reducing proliferation and survival of cells. A number of biological experiments indicate that inhibition of TEAD-VGL4 interactions can promote tissue repair and cell regeneration. There are few reports of effective inhibitors for this interaction, and thus this target is of great interest.
HER ne Adihou et al reported a protein mimetic 4E designed based on the double helix structure of the transcription co-repressor VGL4, aimed at binding TEAD transcription factors. The X-ray crystal structure verifies that the protein mimics maintain a certain conformational binding to TEAD, inhibiting TEAD-VGL4 interactions. Experiments find that the protein mimics stimulate the expression of TEAD target genes in human myocardial cells and promote YAP nuclear translocation and cell cycle activities in young rat myocardial cells, which are central features required for myocardial cell proliferation. However, the protein mimics have low membrane permeability, and can improve intracellular activity by linking TAT, so that the affinity with a target can be further improved, and the effect and mechanism of the protein mimics in other diseases related to cell proliferation and regeneration are still to be studied.
Disclosure of Invention
The invention aims to provide a novel stapler peptide targeting TEAD-VGL4 protein interaction, and further provides an application of the stapler peptide in preparing a cell proliferation promoting medicine.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The crystal structure of the mTEAD-VGL4 complex was analyzed, and the binding sequence of VGL4 to TEAD was mainly 3 fragments, including the N-terminal α1-helix (green), the C-terminal α2, α3 helix (blue) and the β -strand (yellow) connecting the two parts. The helix α1 of VGL4 can bind to TEAD monomer a, while the β chain, helices α2 and α3 can bind to TEAD monomer b. Studies have shown that full binding sequences have the highest affinity for TEAD, the affinity of the intermediate β chain is very low (Kd >100 μm), the N-terminal α1-helix sequence has moderate affinity, while the C-terminal α2, α3 helix structure has higher affinity, which α2, α3 helix structure is also reported to be critical for VGL4 binding to TEAD.
Modulation of the balance between VGL4, TEAD and YAP activity, or inhibition of TEAD degradation by cysteine proteases caused by TEAD-VGL4 interactions, may be useful for wound repair or promotion of fibroblast proliferation. Therefore, we performed structural simplification and optimization on the α2, α3 helical portion of VGL4, yielding the linear peptides Hip1 and Hip2; the alpha helicity, stability, membrane permeability and affinity with TEAD of a series of novel stapler peptides targeting TEAD-VGL4 interaction are designed and synthesized by introducing alpha-alkenyl unnatural amino acid S 5 (S-2-amino-2-methylhept-6-enoic acid) at the i and i+4 positions of the linear peptide, the proliferation capacity of promoting skin fibroblasts is examined, the stapler peptides with high proliferation promoting activity are preferentially examined, the influence and action mechanism of the stapler peptides on YAP target genes and downstream signal paths are further examined, and a lead compound for effectively promoting the proliferation and skin regeneration of the fibroblasts is found, so that a new thought and theoretical basis is provided for the regeneration and repair of damaged skin tissue functions.
Studies show that constructing perhydro-staple peptides at the i, i+4 positions of polypeptides through olefin metathesis reaction can stabilize the alpha helix structure of polypeptides, improving the stability, membrane permeability and binding affinity of polypeptides to target proteins. The currently developed polypeptide mainly aims at YAP/TEAD interaction, most of the polypeptide acts on tumor inhibition, and has the problems of poor stability, low permeability, low binding affinity and the like. With the intensive research on the Hippo signal pathway, it is important to develop new targeted drugs, discover new action mechanisms and apply the targeted drugs to new diseases. The work is expected to bring a new idea for skin tissue regeneration, especially for treatment of wound repair.
The invention takes a linear peptide Ac-DPVVEEHFRRSLGK-NH 2 (SEQ ID NO. 1) shown in PDB ID 4LN0 as a peptide chain template, replaces the ith and the (i+4) th amino acids with (S) -2-amino-2-methylhept-6-enoic acid (S 5) and cyclizes to obtain 5 SHip series of staple peptides; the linear peptide Ac-SVDDHFAKALGDTWLQIKAA-NH 2 (SEQ ID NO. 2) is used as a peptide chain template, and the ith and (i+4) th amino acids are replaced by (S) -2-amino-2-methyl-6-heptenoic acid (S 5) and cyclized to obtain 8 SHip series of staple peptides. 13 staples of peptides with the following structural formula:
In a first aspect of the invention there is provided a stapled peptide targeting a TEAD-VGL4 interaction, said stapled peptide being selected from one of the following:
SHip1-1: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 1 D and 5 E are replaced by S 5 and are cyclized;
SHip1-2: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 3 V and 7 H are replaced by S 5 and are cyclized;
SHip1-3: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 4 V and 8 F are replaced by S 5 and are cyclized;
SHip1-4: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 7 H and 11 S are replaced by S 5 and are cyclized;
SHip1-5: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 8 F and 12 L are replaced by S 5 and are cyclized;
SHip2-1: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 3 D and 7 A are replaced by S 5 and are cyclized;
SHip2-2: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 4 D and 8 K are replaced by S 5 and are cyclized;
SHip2-3: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 7 A and 11 G are replaced by S 5 and are cyclized;
SHip2-4: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 11 G and 15 L are replaced by S 5 and are cyclized;
SHip2-5: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 12 D and 16 Q are replaced by S 5 and are cyclized;
SHip2-6: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 13 T and 17 I are replaced by S 5 and are cyclized;
SHip2-7: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 14 W and 18 K are replaced by S 5 and are cyclized;
SHip2-8: ac-SVDDHFAKALGDTWLQIKAA-NH 2 was used as a peptide chain template, wherein 15 L and 19 A were replaced by S 5 and were cyclized.
In a second aspect of the present invention, there is provided a method for preparing a stapled peptide targeting TEAD-VGL4 interaction as described above, comprising the steps of:
a) Coupling the C-terminal of the first amino acid with a solid phase carrier under the action of a condensing agent;
b) Removing Fmoc protecting groups on the amino acids using a deprotection reagent;
c) Coupling the next amino acid under the action of a condensing agent;
D) Repeating deprotection-coupling operation to synthesize peptide chain according to amino acid sequence; wherein, S 5 is used for replacing amino acid at i and i+4 positions respectively at the cyclization site;
e) The last amino acid is acetylated after deprotection;
F) Under the action of a cyclization agent, carrying out olefin metathesis reaction on the amino acids at the positions i and i+4S 5, and cyclizing a peptide chain;
G) The peptide chain is cut off from the carrier by using a cutting reagent, and the corresponding staple peptide is obtained after purification.
In a third aspect of the invention there is provided the use of a stapled peptide targeting a TEAD-VGL4 interaction as described above in the preparation of a TEAD protein targeting medicament.
Cell experiment results show that the staple peptides SHip-2 can promote proliferation and migration of human skin fibroblasts in a dose-dependent manner; meanwhile, the cell membrane penetrating capability is excellent, and the cell membrane can penetrate into cytoplasm, so that target proteins in cells can be better targeted to exert biological functions.
In a fourth aspect of the invention, there is provided the use of a stapled peptide targeting the TEAD-VGL4 interaction as described above in the manufacture of a medicament for promoting skin wound repair.
In a fourth aspect of the invention, there is provided a medicament for promoting skin wound repair comprising an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient comprises a targeting TEAD-VGL4 interaction stapling peptide as described above as the sole active ingredient or comprises a targeting TEAD-VGL4 interaction stapling peptide as described above.
Further, the medicine can be prepared into various dosage forms with pharmaceutically commonly used auxiliary materials, for example, the medicine can be decoction, powder, pill, intravenous emulsion, liposome preparation, aerosol, precursor medicine preparation, injection, mixture, oral ampoule agent, tablet, capsule and the like. The administration mode is not limited to oral administration, injection, and the like.
The invention has the advantages and beneficial effects that:
The invention designs and synthesizes a series of novel stapled peptides targeting TEAD/VGL4 protein interaction by taking alpha helix fragment helix alpha 1 at the N end and C end double helix fragment helix alpha 2/3 of VGL4 protein as templates based on TEAD-VGL4 compound crystal structure (PDB ID:4LN 0), determines the secondary structure by circular dichroism, evaluates the membrane permeability by utilizing flow cytometry, and evaluates the proliferation promoting effect of the stapled peptides on various cells from the cellular level. The stapling peptide can maintain a stable alpha helical conformation, improve the binding force of the stapling peptide to TEAD, promote the proliferation and migration capacity of various cells, and especially promote the proliferation of human dermal fibroblast HDF-alpha; has good serum stability and excellent cell penetrating capacity, can penetrate through cell membranes and enter cytoplasm, and targets target proteins in cells to exert biological functions.
1. In the preparation aspect, amino resin is used as a carrier, ac-DPVVEEHFRRSLGK-NH 2 and Ac-SVDDHFAKALGDTWLQIKAA-NH 2 are used as peptide chain templates, peptide chains are synthesized through Fmoc solid-phase synthesis, S 5 is used for replacing original amino acid at a specific position on the basis of retaining key amino acid residues, and linear peptides coupled on the resin are subjected to olefin metathesis cyclization in 1, 2-dichloroethane solution of Grubbs 1 st reagent, and then target stapling peptides are cut off from the resin. After the obtained compound is purified, characterization analysis is carried out by adopting HPLC, HR-MS, circular dichroism and the like, the purity of the obtained staple peptide is more than 95 percent, and the typical alpha helix conformation is reserved.
2. Effect, the staples SHip are capable of promoting human skin fibroblast migration in a dose dependent manner; meanwhile, the cell membrane penetrating capability is excellent, and the cell membrane can penetrate into cytoplasm, so that target proteins in cells can be better targeted to exert biological functions. The stapler peptide can up-regulate YAP gene expression and the mRNA level of target genes hCCF and hCCYR 61 hANKRD downstream thereof.
3. The alpha helix of Hip2 is kept in a stable conformation by the stapling peptide strategy, so that the membrane permeability of the Hip2 is enhanced, the serum stability is improved, the binding force of the Hip2 to TEAD is further reserved or even improved, and the proliferation and migration promoting capability of various cells is exerted.
Drawings
FIG. 1 is a circular dichroism spectrum of the purified staple peptide in pure water;
FIG. 2 is a graph showing the proliferation of human skin fibroblasts by using CCK-8 assay;
FIG. 3 is a flow cytometry assay to determine the transmembrane ability of FITC-modified polypeptides; detecting the film penetration condition of the staple peptide by a fluorescence confocal experiment;
FIG. 4 shows the detection of YAP expression by dual luciferase reporter gene assay and the detection of hCCTGF, hCDER 61 and hANKRD1 expression of YAP downstream target genes by qRT-PCR assay;
FIG. 5 is a graph of cell scratch assay evaluating the ability of stapled peptides to promote cell migration;
FIGS. 6-7 are mass and high performance liquid chromatograms of Hip1 and Hip2, respectively;
mass spectrometry and high performance liquid chromatography analyses of figures 8-12 for SHip1-1, SHip1-2, SHip1-3, SHip1-4, SHip1-5, respectively;
Mass spectrometry and hplc analysis of figures 13-20 SHip2-1, SHip2-2, SHip2-3, SHip2-4, SHip2-5, SHip2-6, SHip2-7, SHip2-8, respectively;
FIGS. 21-24 are mass spectra and high performance liquid chromatography analyses of FITC-Hip1, FITC-Hip2-5, and FITC-Hip2-6, respectively.
Detailed Description
The following provides a detailed description of embodiments of the present invention with reference to examples. The following examples are given by way of illustration of the present invention, and the scope of the invention is not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified.
Example 1: tapelin synthesis targeting TEAD proteins
Taking the stapling peptide SHip as an example, specific synthesis steps are described as follows:
(1) Solid phase synthesis of polypeptide:
RINK AMIDE MBHA resin is weighed in a peptide connecting tube, and a reagent Dichloromethane (DCM) is added in the peptide connecting tube to soak the swelling resin for 30min. Removing Fmoc protecting group on the resin by using 20% (v/v) piperidine/N, N-Dimethylformamide (DMF), standing for 5min, removing the solvent, adding 20% (v/v) piperidine/DMF, and slowly shaking at room temperature for 10min in a shaking table to completely expose free amino group.
The washing was alternated 5 times with DMF/DCM, respectively. 5eq Fmoc-AA-OH, 5eq Oxyma, 10eq DIC were dissolved in N-methylpyrrolidone NMP reagent and poured into a peptide tube, which was placed in a 60℃incubator and shaken slowly for 20min. Then washed alternately 5 times with DMF/DCM etc. Then, 20% (v/v) piperidine/DMF was used to remove Fmoc protecting groups from the resin, the mixture was allowed to stand for 5min, the solvent was drained off, 20% (v/v) piperidine/DMF was added, and the mixture was slowly shaken at room temperature for 10min in a shaker. Then, an NMP solution containing 5eq Fmoc-protected amino acid but exposed carboxyl group, 5eq Oxyma, 10eq DIC was added and reacted at 60℃for 20min. This is repeated until the polypeptide is grafted. The reaction conditions of the specific amino acid (S 5) and the next amino acid in the peptide chain were slightly different, and an NMP solution containing 2.5eq Fmoc-S 5 -OH,2.5eq Oxyma, 5eq DIC was added to the resin, which was placed in a 60℃incubator and shaken slowly for 2 hours. The next amino acid of S 5 was used in the same amount as the other amino acids and reacted at 60℃for 2 hours. The acetylation of the side chain-NH 2 of the amino acid at the end of the polypeptide was carried out with acetic anhydride/DIEA/DMF in a volume ratio of 1/1/8 at room temperature for 10min.
(2) Olefin metathesis reactions
To the washed resin, 1, 2-dichloroethane solution of Grubbs 1 st at a concentration of 8mg/mL was added to conduct olefin metathesis reaction of S 5 side chain for 2 hours/time and 2 times. Then washed alternately 10 times with DMF/DCM, respectively. The polypeptide resin after the reaction is polycondensed with methanol for 10min and dried with nitrogen. The resin was soaked with the cleavage solution TFA/PhOH/H 2 O/TIPS (87.5:5:2.5, v/v/v) for 4H, after which the filtrate was collected and purged with argon for 30min. By utilizing the characteristic that polypeptide is not easy to dissolve in diethyl ether, other impurities are removed by precipitation with glacial diethyl ether, and the crude polypeptide is dissolved by 50% acetonitrile water.
And separating and purifying the crude polypeptide product by using preparative HPLC (high performance liquid chromatography) to obtain a pure polypeptide product, and identifying the molecular weight of the target product by using a high-resolution mass spectrometry technology. The structure of the stapled peptides is shown in table 1; the purity of the polypeptide product is identified by analytical High Performance Liquid Chromatography (HPLC) and mass spectrometry, wherein the HPLC spectrogram and the mass spectrogram are shown in figures 6-24, and the purity of the synthesized staple peptide polypeptide is more than 95%.
TABLE 1 sequences and molecular weights of all polypeptides
Hip1 (DPVVEEHFRRSLGK) was lyophilized as a white powder. Purity of :96.0923％.HR-MS:C75H120N24O22,Calc.for1708.9000,found(M+2H)2+:855.4606
Hip2 (SVDDHFAKALGDTWLQIKAA) was lyophilized as a white powder. Purity of :95.0744％.HR-MS:C101H155N27O30,Calc.for2226.14 found(M+2H)2+:1114.5832
SHip1-1 (S 5PVVS5 EHFRRSLGK), which is a white powder after lyophilization. Purity of :99.7852％.HR-MS:C80H130N24O18,Calc.for1714.9995,found(M+2H)2+:859.0145
SHip1-2 (DPS 5VEES5 FRRSLGK) as a white powder after lyophilization. Purity of :96.1205％.HR-MS:C78H126N22O22,Calc.for1722.9417,found(M+2H)2+:862.4807
SHip1-3 (DPVS 5EEHS5 RRSLGK), which are white powders after lyophilization. Purity of :99.6155％.HR-MS:C75H124N24O22,Calc.for1712.9322,found(M+2H)2+:857.4764
SHip1-4 (DPVVEES 5FRRS5 LGK) as a white powder after lyophilization. Purity of :98.7719％.HR-MS:C80H130N22O21,Calc.for1734.9781,found(M+2H)2+:869.0001
SHip1-5 (DPVVEEHS 5RRSS5 GK) as white powder after lyophilization. Purity of :97.5935％.HR-MS:C74H122N24O22,Calc.for1698.9166,found(M+2H)2+:850.4663
SHip2-1 (SVS 5DHFS5 KALGDTWLQIKAA) as a white powder after lyophilization. Purity of :97.3495％.HR-MS:C108H167N27O28,Calc.for2290.2474,found(M+2H)2+:1146.6399
SHip2-2 (SVDS 5HFAS5 ALGDTWLQIKAA) as a white powder after lyophilization. Purity of :97.7301％.HR-MS:C105H160N26O28,Calc.for2233.19,found(M+2H)2+:1116.0887
SHip2-3 (SVDDHFS 5KALS5 DTWLQIKAA), which after lyophilization is a white powder. Purity of :97.5935％.HR-MS:C110H169N27O30,Calc.for2348.2529,found(M+2H)2+:1175.6444
SHip2-4 (SVDDHFAKALS 5DTWS5 QIKAA), which after lyophilization is a white powder. Purity of :96.5342％.HR-MS:C107H163N27O30,Calc.for2306.2100,found(M+2H)2+:1154.6136
SHip2-5 (SVDDHFAKALGS 5TWLS5 IKAA), which is a white powder after lyophilization. Purity of :97.0400％.HR-MS:C106H164N26O27,Calc.for2233.2259,found(M+2H)2+:1118.1299
SHip2-6 (SVDDHFAKALGDS 5WLQS5 KAA) were lyophilized to a white powder. Purity of :95.8999％.HR-MS:C105H159N27O29,Calc.for2262.1797,found(M+2H)2+:1132.6059
SHip2-7 (SVDDHFAKALGDTS 5LQIS5 AA) as a white powder after lyophilization. Purity of :99.0433％.HR-MS:C98H155N25O30,Calc.for2162.1372,found(M+2H)2+1082.5847
SHip2-8 (SVDDHFAKALGDTWS 5QIKS5 A) as a white powder after lyophilization. Purity of :96.7048％.HR-MS:C106H161N27O30,Calc.for2292.19,found(M+2H)2+:1147.6135
FITC-Hip1 (FITC-. Beta.A-DPVVEEHFRRSLGK) was lyophilized as a white powder. Purity of :97.5935％.HR-MS:C97H134N26O27S Calc.for2126.96,found(M+2H)2+:1064.9938
FITC-Hip2 (FITC-. Beta.A-SVDDHFAKALGDTWLQIKAA) was lyophilized as a white powder. Purity of :98.0248％.HR-MS:C123H169N29O35S,Calc.for2644.21,found(M+2H)2+:1323.6183
FITC-SHip-5 (FITC-. Beta.A-SVDDHFAKALGS 5TWLS5 IKAA) was lyophilized as a white powder. Purity of :95.6105％.HR-MS:C128H178N28O32S,Calc.for2651.29 found(M+2H)2+:1327.1538
FITC-SHip-6 (FITC-. Beta.A-SVDDHFAKALGDS 5WLQS5 KAA) was lyophilized as a white powder. Purity of :96.2506％.HR-MS:C127H173N29O34S,Calc.for2680.24,found(M+2H)2+:1341.6308.
Experimental example 2: round two-chromatographic test of SHip polypeptides
The secondary conformation of the polypeptide in aqueous solution was determined by circular dichroism and a Circular Dichroism (CD) of the polypeptide was plotted. As a result, referring to fig. 1, it can be judged that the stapled peptide has a typical alpha helical conformation from the negative absorption peaks of the features around 208nm and 225nm and the positive absorption peak of the features around 195nm in the circular dichroism spectrum. CD experiment results show that the helicity of SHip series of staple peptides is improved to a certain extent relative to the helicity of the linear peptide Hip 1; SHip2-5 of the SHip2 series of staples showed a typical alpha helical conformation in its CD test.
Experimental example 3: stability test of SHip series of polypeptides
Polypeptide drugs are easily and rapidly degraded in vivo, and half-life of most natural active peptides in vivo is only within a few hours, so that the maintenance of serum stability is a key factor for the polypeptides to perform biological functions in organisms. The linear peptides Hip1, hip2 and the peptides SHip-SHip were selected as test subjects, and serum was used to simulate the degradation process of the polypeptides in vivo and to evaluate the serum stability of the full-hydrocarbon peptides.
The residual polypeptide content at different time points was monitored by HPLC tracking to obtain Hip1, hip2 and SHip HPLC graphs and degradation kinetics curves. The stapled peptides SHip showed considerable stability with a residual amount of intact polypeptide of 80% within 24 hours. Under the same conditions, the linear peptide Hip2 was rapidly degraded within the first 6 hours, and tended to be gentle after 6 hours, with about 40% degradation at 24 hours. The remaining amount of intact polypeptide of the linear peptide Hip1 after 24 hours was only 40%. In summary, the full hydrocarbon stapling modification can improve the proteolytic stability of the polypeptide, and the staple peptides SHip-SHip exhibit high serum stability.
Experimental example 4: SHip polypeptide affinity test with TEAD
In vitro binding forces between the linear peptide and the stapled peptide and TEAD protein respectively were detected by using SPR technique, and SPR fitting curves and results are shown in FIGS. 3-4. SPR experiment results show that the linear peptide Hip1 has no specific binding trend; the binding force between the linear peptide Hip2 and TEAD is low, and the K D value is more than 100 mu M. The interaction binding force between SHip series of staple peptides and TEAD proteins has no specific binding trend or weak affinity. SHip 2A series of staples, except SHip-1, SHip2-7 and SHip-8, all bind more strongly to TEAD than to the linear peptide Hip2, wherein SHip-4, SHip2-5 and SHip2-6 are more pronounced, the KD of SHip-5 interaction with TEAD is 3.502. Mu.M, and the binding force is increased by nearly 200 times, indicating that the alpha 2 helical domain plays an important role in VGL4-TEAD interaction.
Experimental example 5: CCK-8 assay to determine cell viability
To evaluate the effect of these polypeptides on cell proliferation, CCK-8 activity assays were performed to determine cell viability. The experimental group included 13 peptides of staples, and the effect of the 13 peptides on HDF- α proliferation was studied, with the non-administered group and the linear peptide-administered group as controls.
Cells HDF- α were seeded at 5000 cells/well in 96-well plates and incubated overnight in humidified sterile incubator at 37℃and 5% CO 2, then treated with polypeptide at the indicated concentration (40. Mu.M/20. Mu.M/10. Mu.M) for 24h or 48h, and the culture was gently aspirated. The CCK-8 reagent solution was dissolved in serum-free medium at a volume ratio of 1:10, 100. Mu.L was added to each well, incubated at 37℃for 1h, and absorbance was recorded at 450nm using an microplate reader.
As shown in FIG. 2, the CCK-8 experiment results show that the staple peptides SHip-2 can promote proliferation of cells, and SHip-6 has a certain proliferation promoting effect on cells, but has a weaker effect than SHip 2-5.
Example 6: flow cytometry experiments to determine the transmembrane ability of polypeptides
Because of the problems of molecular size, polarity, hydrophilicity, charging property and the like of the polypeptide, the polypeptide is difficult to quickly cross cell membranes like small molecular medicines, and the development of the polypeptide medicines is greatly limited due to the lack of the permeability of the cell membranes. Effective intracellular modulation requires efficient uptake of the stapled peptide by the cell and cytoplasmic localization. We selected HDF- α and examined the amount of Hip2 linear peptide and SHip series of stapled peptides into HDF- α cells using flow cytometry.
HDF-alpha cells were seeded at a density of 1x 10 6 per well in 6-well plates and cultured overnight in humidified, sterile incubator at 37 ℃ and 5% CO 2 until the cells were fully adherent. The initial medium was replaced with serum-free basal medium for 2h incubation. HDF-alpha cells were treated with 10 μm FITC-labeled SHip-5 and SHip2-6 in the dark for 24h, then washed with PBS to remove excess FITC-labeled polypeptide, trypsinized for 1min, cells were collected in a 1.5mL EP tube with cold basal medium and centrifuged at 500rpm for 10min at 4 ℃. Finally, removing the supernatant, adding PBS to resuspend the cells, and then carrying out flow cytometry analysis. As shown in FIG. 3A, the fluorescence intensities of the stapled peptides SHip-5 and SHip2-6 in HDF-. Alpha.were significantly higher than that of the linear peptide, SHip2-5, indicating that SHip2-5 most readily penetrated the cell membrane and was taken up by the cell. The fluorescence intensity of FITC-SHip2-5 treated HDF-alpha cell samples at different concentrations (10. Mu.M, 20. Mu.M, 40. Mu.M) was then further examined and found to be concentration dependent (FIG. 3C). Flow cytometry experiment results prove that SHip2-5 can efficiently penetrate through cell membranes and enter cells.
We observed the distribution of FITC-SHip in HDF-alpha cells directly with a fluorescence microscope. The blue portion of FIG. 3B is the nucleus stained with Hoechst 33342 and the green color is fluorescence labeled FITC-SHip2-5. Under a fluorescence microscope, the effective uptake SHip2-5 of the HDF-alpha cells into the cells can be clearly seen, and a foundation is laid for the accurate targeting TEAD protein of SHip-5 to exert the intracellular biological function in the next step. In summary, the all-hydrocarbon stapling strategy can increase the cell membrane penetration of polypeptides, and the staples SHip-2 can rapidly cross the HDF- α cell membrane and localize into the cytoplasm.
Example 7: tachypeptide up-regulates YAP expression and transcription levels of downstream target genes hTGF, hTYR 61 and hANKRD1
The Hippo signal pathway is inactivated upstream, YAP is not phosphorylated, enters the nucleus, combines with the transcription factor TEADs, induces and expresses a target gene, starts proliferation-promoting and survival-promoting genes, and promotes cell proliferation, invasion and metastasis.
YAP reporter plasmid and sv-40l plasmid were transfected together into HEK293T cells for expression, 3 multiplex wells were made in 96-well plates, luciferase activity in each well was measured after 6 hours of treatment with compound at a final concentration of 10. Mu.M, and relative luciferase activity in each well was calculated using the luciferase activity of DMSO-treated cells as a control. The results of the dual luciferase reporter experiments show that SHip-5 and SHip-6 can up-regulate YAP expression, and have statistical significance (FIG. 4A).
To further investigate how staplers affect the expression of target genes downstream of the Hippo signaling pathway, qRT-PCR methods were next chosen to test for further detection of whether staplers inhibitors affect transcription levels of target genes downstream of the Hippo signaling pathway (CTGF, CYR61, and ANKRD 1). Samples of SHip2-5 treated HDF- α cells at different concentrations (10 μM,20 μM,40 μM) were collected and the results of FIG. 4B indicate that SHip-5 and SHip2-6 up-regulated the levels of the downstream target genes hTGF, hCYR61 and hANKRD1 mRNA compared to the untreated group.
Example 8: cell scratch test for detecting migration promoting capability of polypeptide
We used a scratch assay to examine cell migration ability. The experimental groups were non-dosed, YAP activator (PY-60) and dosed. HDF- α cells in the logarithmic growth phase were taken, starved for 24 hours in serum-free medium, trypsinized and resuspended in cell suspension, gently blown into cell suspension, counted under a microscope, plated in 6-well plates with 5×105cells/ml cell density adjusted, and incubated for 24 hours at 37 ℃ for starvation overnight. The fused cells were scratched with a pipette tip. Image acquisition was performed 12h,24h after drug intervention. Cell migration was quantified as the difference in area of two cell-free wounds. All experiments were independently repeated three times.
The scratch test results are shown in FIG. 5, and the drug delivery SHip can promote the migration of HDF-alpha cells; and the migration distance becomes longer compared with YAP activator (PY-60) after treatment. Statistical differences were found by Image J software to account for migration distances, which were found to be statistically different in that SHip-5 promoted YAP expression followed by migration of HDF- α cells.
While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.
Claims (5)
1. A stapled peptide targeting TEAD-VGL4 interactions, wherein said stapled peptide is selected from one of the following:
SHip1-1: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 1 D and 5 E are replaced by S 5 and are cyclized;
SHip1-2: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 3 V and 7 H are replaced by S 5 and are cyclized;
SHip1-3: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 4 V and 8 F are replaced by S 5 and are cyclized;
SHip1-4: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 7 H and 11 S are replaced by S 5 and are cyclized;
SHip1-5: ac-DPVVEEHFRRSLGK-NH 2 is used as a peptide chain template, wherein 8 F and 12 L are replaced by S 5 and are cyclized;
SHip2-1: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 3 D and 7 A are replaced by S 5 and are cyclized;
SHip2-2: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 4 D and 8 K are replaced by S 5 and are cyclized;
SHip2-3: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 7 A and 11 G are replaced by S 5 and are cyclized;
SHip2-4: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 11 G and 15 L are replaced by S 5 and are cyclized;
SHip2-5: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 12 D and 16 Q are replaced by S 5 and are cyclized;
SHip2-6: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 13 T and 17 I are replaced by S 5 and are cyclized;
SHip2-7: ac-SVDDHFAKALGDTWLQIKAA-NH 2 is used as a peptide chain template, wherein 14 W and 18 K are replaced by S 5 and are cyclized;
SHip2-8: ac-SVDDHFAKALGDTWLQIKAA-NH 2 was used as a peptide chain template, wherein 15 L and 19 A were replaced by S 5 and were cyclized.
2. Use of a stapled peptide targeting a TEAD-VGL4 interaction according to claim 1 for the preparation of a medicament targeting a TEAD protein.
3. Use of a stapled peptide targeting the TEAD-VGL4 interaction according to claim 1 for the preparation of a medicament for promoting skin wound repair.
4. A medicament for promoting skin wound repair, comprising an active ingredient and a pharmaceutically acceptable auxiliary material, wherein the active ingredient takes the stapled peptide for targeting the TEAD-VGL4 interaction as the only active ingredient or comprises the stapled peptide for targeting the TEAD-VGL4 interaction as claimed in claim 1.
5. A method of preparing a stapled peptide targeting a TEAD-VGL4 interaction according to claim 1, comprising the steps of:
a) Coupling the C-terminal of the first amino acid with a solid phase carrier under the action of a condensing agent;
b) Removing Fmoc protecting groups on the amino acids using a deprotection reagent;
c) Coupling the next amino acid under the action of a condensing agent;
D) Repeating deprotection-coupling operation to synthesize peptide chain according to amino acid sequence; wherein, S 5 is used for replacing amino acid at i and i+4 positions respectively at the cyclization site;
e) The last amino acid is acetylated after deprotection;
F) Under the action of a cyclization reagent, carrying out olefin metathesis reaction on the S 5 at the i and i+4 positions, and cyclizing the peptide chain;
G) The peptide chain is cut off from the solid phase carrier by using a cutting reagent, and the corresponding staple peptide is obtained after purification.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410400732.3A CN118255843A (en) | 2024-04-03 | 2024-04-03 | Tapelin targeting TEAD-VGL4 interaction and application thereof in skin repair |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410400732.3A CN118255843A (en) | 2024-04-03 | 2024-04-03 | Tapelin targeting TEAD-VGL4 interaction and application thereof in skin repair |
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| Publication Number | Publication Date |
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| CN118255843A true CN118255843A (en) | 2024-06-28 |
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| CN202410400732.3A Pending CN118255843A (en) | 2024-04-03 | 2024-04-03 | Tapelin targeting TEAD-VGL4 interaction and application thereof in skin repair |
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| Country | Link |
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| CN (1) | CN118255843A (en) |
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