Cyclic nonapeptide with barrier repair effect and use thereof

Abstract

The application discloses a cyclic nonapeptide with a skin barrier repair effect and application thereof, and the amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-phenylalanine-lysine-valine-lysine-lysine). The cyclic nonapeptide can obviously improve the gene expression of FLG and AQP3, and reduce the gene expression of TNF-alpha and NF-kappa B, thereby having the skin barrier repair effect, and can be applied to skin barrier repair cosmetics, drugs or health foods as an effective component.

Description

Technical Field

[0001] This invention relates to a cyclic peptide, particularly a cyclic nonapeptide with barrier repair function and its applications. Background Technology

[0002] Impaired skin barrier function is often associated with decreased expression of filaggrin (FLG) and aquaporin 3 (AQP3), and increased expression of TNF-α and NF-κB. Filagrin (FLG) is a crucial molecule in the stratum corneum of human skin that binds and aggregates keratin fibers. With the assistance of filaggrin monomers, keratin fibers aggregate regularly, forming a robust physical barrier on the outermost layer of the epidermis, thus preventing water loss and the invasion of external substances. Aquaporin 3 (AQP3) is a key water-glycerol channel in the epidermis, primarily maintaining and repairing the skin barrier through multiple dimensions, including regulating hydration status, lipid synthesis, keratinocyte proliferation and differentiation, and the rate of barrier repair. Tumor necrosis factor α (TNF-α) and NF-κB are important inflammatory chemokines; inflammation can also damage the skin barrier.

[0003] In the field of skin anti-aging and barrier repair, traditional treatment drugs have provided solutions for improving skin aging and alleviating barrier dysfunction in a certain period of time. However, they often have problems such as obvious side effects, single biological activity, and limited mechanism of action. Some drugs may cause adverse reactions such as skin irritation, dryness, and allergies. Moreover, they mostly focus on a single target and are difficult to fully cover the complex pathological process of skin barrier damage. Long-term use may also lead to decreased skin tolerance and affect the sustainability of treatment effects.

[0004] There are currently few research reports on cyclic nonapeptides with barrier repair effects. Summary of the Invention

[0005] The purpose of this invention is to provide a cyclic nonapeptide with barrier repair function and its application. This invention discovers a novel cyclic nonapeptide with skin barrier repair function.

[0006] The technical solution of the present invention is a cyclic nonapeptide with barrier repair function, wherein the amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-phenylalanine-lysine-valine-lysine-lysine).

[0007] The aforementioned cyclic nonapeptide with barrier repair function has the following structural formula: .

[0008] In the aforementioned cyclic nonapeptide with barrier repair function, the cyclic nonapeptide is prepared by resin solid-phase synthesis using a fluorene methoxycarbonyl N-terminal protection strategy.

[0009] This invention also provides the application of the above-mentioned cyclic nonapeptide in the preparation of skin barrier repair products.

[0010] In the aforementioned applications, the cellular concentration of the cyclic nonapeptide is ≤160ppm.

[0011] In the aforementioned applications, the cellular concentration of the cyclic nonapeptide is 160 ppm.

[0012] In the aforementioned applications, the product is a cosmetic, pharmaceutical, and / or health food.

[0013] A skin barrier repair cosmetic comprising the aforementioned cyclic nonapeptide and a cosmetically acceptable carrier.

[0014] A skin barrier repair drug comprising the aforementioned cyclic nonapeptide and a pharmaceutically acceptable carrier.

[0015] A health food for skin barrier repair includes the aforementioned cyclic nonapeptide and a food-grade acceptable carrier.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The cyclic nonapeptide RGDSFKVKK of this invention was prepared by resin solid-phase synthesis. After cytotoxicity and barrier repair experiments, it was demonstrated that the cyclic nonapeptide RGDSFKVKK was non-toxic to HaCaT cells at a concentration ≤160ppm, and could significantly increase the gene expression of FLG and AQP3, and decrease the gene expression of TNF-α and NF-κB, thereby having the effect of skin barrier repair. It can be used as an effective ingredient in cosmetics, pharmaceuticals or health foods for skin barrier repair. Attached Figure Description

[0018] Figure 1 This is a simulation diagram of the docking of the cyclic nonapeptide with integrin aVβ3 in this invention.

[0019] Figure 2 This is a simulation diagram of the docking of the cyclic nonapeptide and integrin a5β1 molecules in this invention.

[0020] Figure 3 This is a statistical graph showing the effect of the cyclic nonapeptide in this invention on the viability of HaCaT cells.

[0021] Figure 4 This is a statistical graph showing the expression of FLG gene in HaCaT cells in each experimental group; in the graph, * indicates a significant difference compared to the NC group, "*" indicates P<0.05, and "**" indicates P<0.01.

[0022] Figure 5This is a statistical graph showing the expression of AQP3 gene in HaCaT cells in each experimental group; * in the graph indicates a significant difference compared to the NC group, "*" indicates P<0.05, and "***" indicates P<0.001.

[0023] Figure 6 This is a statistical graph showing the expression of TNF-α gene in HaCaT cells in each experimental group; * in the graph indicates a significant difference compared to the NC group, and "*" indicates P<0.05.

[0024] Figure 7 This is a statistical graph showing the expression of NF-κB gene in HaCaT cells in each experimental group; * in the graph indicates a significant difference compared to the NC group, "*" indicates P<0.05, and "**" indicates P<0.01. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0026] Example:

[0027] Structural simulation and screening of cyclic nonapeptides:

[0028] This invention designs cyclic nonapeptides as the sequence RGDS-X1-KVK-X2, where X1 and X2 are one of 20 L-amino acids and one of 20 D-amino acids, respectively, for a total of 40×40=1600 cyclic nonapeptide candidate sequences.

[0029] The three-dimensional structures of the 1600 candidate cyclic peptides were simulated using structural simulation software. Then, the energy differences between the 1600 candidate cyclic peptides and their corresponding linear peptide sequences were calculated using molecular docking software. A structural stability score (Total_score) < 0 indicates that the cyclic peptide structure has low steric hindrance between amino acid residues, exhibits better structural stability than its linear peptide form, and can stably form a cyclic peptide structure, making it suitable for further experimental verification. The structural stability scoring table is shown in Table 1.

[0030] Table 1. Structural stability scores of different cyclic peptide sequences

[0031] Candidate sequences Total score RGDSFKVKK -3.134 RGDS-DAR-KVKK 7.025 RGDSFKVK-DTH 6.494 RGDSWKVKW 9.251 RGDS-DPR-KVK-DAR 15.563

[0032] Based on the above screening criteria, this invention screened out structurally stable candidate cyclic nonapeptide RGDSFKVKK.

[0033] Molecular docking simulations were performed between the RGDSFKVKK cyclic nonapeptide structure and integrin αVβ3. Based on the crystal structure (αVβ3_PDB ID: 4MMX) in the Protein Data Bank, the RGDS sequence from the cyclic peptide was docked to the RGDS sequence in the αVβ3 complex crystal structure. The specific docking structure simulation diagram is shown below. Figure 1 As shown. Figure 1 In the diagram, the red linear structures represent the positions of the RGDS peptide segments in the known 4MMX crystal structure, while the green and blue rod-shaped structures represent cyclic nonapeptides.

[0034] The docking fraction between the cyclic nonapeptide RGDSFKVKK and integrin αVβ3 was -11.2579, which is less than -7, indicating a strong binding interaction. The docking results showed that the RMSD of the RGDS sequence of the cyclic nonapeptide and the RGDS sequence in the 4MMX crystal structure was 1.560 Å, and the positions of the amino acids and the direction of the side chain extension were very close, which is conducive to the binding of the cyclic nonapeptide to integrin αVβ3.

[0035] Subsequently, molecular docking simulations were performed between the RGDSFKVKK cyclic nonapeptide structure and integrin α5β1. Based on the crystal structure (α5β1_PDB ID: 4WK2) in the Protein Data Bank, the RGDS sequence in the cyclic peptide was docked to the RGDS sequence in the α5β1 complex crystal structure. The specific docking structure simulation diagram is shown below. Figure 2 As shown. Figure 2 In the diagram, the red linear structures represent the position of the RGDS peptide in the known crystal structure 4WK2, while the green and blue rod-shaped structures represent cyclic nonapeptides.

[0036] The docking fraction between the cyclic nonapeptide RGDSFKVKK and integrin α5β1 was -12.1568, which is less than -7, indicating a strong binding interaction. The docking results showed that the RMSD of the RGDS sequence of the cyclic nonapeptide and the RGDS sequence in the crystal structure 4WK2 was 1.556 Å, and the positions of the amino acids and the direction of the side chain extension were very close, which is conducive to the binding of the cyclic nonapeptide to integrin α5β1.

[0037] The above molecular docking simulation results indicate that the structure of the cyclic nonapeptide is conducive to binding integrin αVβ3 and α5β1.

[0038] The amino acid sequence of the cyclic nonapeptide of the present invention is: cyclic (arginine-glycine-aspartic acid-serine-phenylalanine-lysine-valine-lysine-lysine), i.e., Cyclo(Arg-Gly-Asp-Ser-Phe-Lys-Val-Lys-Lys), and the cyclic sequence is shown in SEQ ID NO.1. The structural formula is: .

[0039] The preparation method of cyclic nonapeptide is as follows:

[0040] S1. Using natural amino acids arginine-glycine-aspartic acid-serine-phenylalanine-lysine-valine-lysine-lysine as starting materials, and employing a fluorene methyloxycarbonyl (Fmoc) N-terminal protection strategy, the corresponding amino acids are sequentially linked according to the resin solid-phase synthesis method, and the Fmoc- protecting groups are sequentially removed during the process to obtain a resin containing linear nonapeptide chains.

[0041] The specific steps are as follows:

[0042] 1) Swelling resin:

[0043] 0.6 g of 2-Cl Trt-Lys resin (degree of substitution SD = 0.39 mmol / g) was added to the reactor, and DCM (10 mL / g) was added as a swelling agent to allow swelling for 5 min.

[0044] 2) Resin deprotection:

[0045] Vacuum-dry the swollen reagent DCM, add 20% piperidine (Pip) / DMF (10 mL / g) as a deprotection agent, stir for 5 min, dry under vacuum, and then add 20% piperidine (Pip) / DMF (10 mL / g) and stir for 5 min.

[0046] 3) Remove protective washing:

[0047] The protective reagent Pip / DMF was removed by vacuum drying, and the resin was washed 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time and then dried for 20 seconds to obtain resin-1.

[0048] 4) Deprotection detection:

[0049] Take about 20 resin-1 particles and put them into a test tube. Add 1 mL of ninhydrin detection reagent to the test tube, and then put the test tube into a metal bath at 120°C or above for 2 minutes. Take it out and observe the color of the resin. If the resin color becomes darker, it is a positive result, indicating that the deprotection was successful.

[0050] 5) Condensation of the second amino acid:

[0051] Add 3 equivalents of Fmoc-Val-OH and 3 equivalents of Oxyma to resin-1, dissolve in 10 mL of DMF, add 3 equivalents of DIC, activate for 5 min, pour into a reactor, and stir to react for 1 h.

[0052] 6) Reaction washing:

[0053] The reaction reagent DMF was dried under vacuum, and the mixture was washed 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time and then dried under vacuum for 20 seconds to obtain resin-2.

[0054] 7) Reaction detection:

[0055] Take about 20 resin-2 particles and put them into a test tube. Add 1 mL of ninhydrin test reagent to the test tube, and then put the test tube into a metal bath at 120°C or above for 2 minutes. Take it out and observe the color of the resin. If there is no obvious change in the color of the resin, it indicates that the reaction condensation is successful.

[0056] 8) Repeat steps 2)-7), condensing the subsequent amino acids in the sequence from right to left according to the polypeptide sequence, namely Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH until Fmoc-Phe-OH, to obtain resin-3.

[0057] 9) Deprotection:

[0058] Add 20% Pip / DMF (10 mL / g) by volume to resin-3 as a deprotection agent, stir for 5 min and then dry under vacuum. Add 20% Pip / DMF (10 mL / g) by volume again and stir for 5 min.

[0059] 10) Remove protective wash:

[0060] The protective reagent Pip / DMF was removed by vacuum drying, and then washed 5 times with DMF (10 mL / g), stirring for 20-30 s each time and drying for 20 s to obtain resin-4.

[0061] 11) Deprotection detection:

[0062] Place approximately 20 resin-4 particles into a test tube, add 1 mL of ninhydrin detection reagent, place the test tube in a metal bath at 120°C or higher for 2 minutes, remove and observe the resin color. A darker resin color indicates a positive result, signifying successful deprotection.

[0063] 12) Washing:

[0064] The resin was washed 5 times with 10 mL of methanol and then vacuum dried for 10 min to complete solid-phase condensation, yielding a resin containing linear nonapeptide chains.

[0065] S2. Cut the resin containing the linear nonapeptide chain, cut the linear nonapeptide chain off the resin, remove the remaining protecting groups of the peptide chain, collect the cutting fluid containing the crude linear nonapeptide chain, and obtain a fully protected polypeptide solid to be cyclic.

[0066] The specific steps are as follows:

[0067] 13) Resin-protected pyrolysis:

[0068] The resin containing the linear nonapeptide chain was loaded into a boat-shaped reactor, and 10 mL / g lysis buffer (TFE:DCM volume ratio of 30:70) was added. The mixture was lysed three times at room temperature for 1 hour each time. The reaction solution was filtered out, and the solvent was concentrated and evaporated using a rotary evaporator. After evaporation, the sample was dissolved in 30% acetonitrile / water solution and lyophilized to obtain a fully protected peptide solid.

[0069] S3. The fully protected polypeptide solid to be cyclized is mixed with polypeptide coupling agent, activator, etc., and then cyclized to obtain a cyclic peptide containing a protecting group.

[0070] The specific steps are as follows:

[0071] 14) Modification cyclization reaction:

[0072] Weigh out the fully protected polypeptide solid to be cyclic, dissolve it in AR grade DMF to obtain a polypeptide solution with a concentration of 1 mM; after dissolution, weigh out 2 eq PyBOP using an electronic balance, and transfer 4 eq DIEA to the polypeptide solution, stir at room temperature for 10 h to obtain a cyclic peptide containing a protecting group.

[0073] S4. The cyclic peptide containing the protecting group was purified by preparative HPLC to obtain cyclic nonapeptide.

[0074] The specific steps are as follows:

[0075] 15) Rotary freeze-drying:

[0076] After the reaction was complete, the cyclic peptide reaction solution containing the protecting group was concentrated using a rotary evaporator. After concentration, it was dissolved in 30% acetonitrile / water solution, then freeze-dried on the wall, and finally purified by preparative HPLC to obtain the cyclic (arginine-glycine-aspartic acid-serine-phenylalanine-lysine-valine-lysine-lysine).

[0077] Experimental example:

[0078] Experimental consumables: Biosafety cabinet (Nuaire, USA, NU-543-600s), CO2 incubator (Panasonic Corporation, MCO-18AIC), centrifuge (Eppendorf AG, Germany, Centrifuge 5804R), mini cryogenic centrifuge (Sigma, Germany, 1-14K), analytical balance (Sartorius Group, Germany, BCE224-1CCN), microscope (Carl Zeiss Microscopes (Germany) GmbH, Axio Observer3), quantitative real-time PCR instrument (Thermo Fisher Scientific, QuantStudio 3).

[0079] Experimental reagents: Phosphate-buffered saline (PBS, Beijing Lanjieke Technology Co., Ltd., BL1425A), high-glucose DMEM medium (Thermo Fisher Scientific, Gibco, 11965118), 0.25% trypsin solution (Thermo Fisher Scientific, Gibco, 25200056), penicillin-streptomycin (Thermo Fisher Scientific, Gibco, 15070063), fetal bovine serum (Shanghai Beyotime Biotechnology Co., Ltd., C0234).

[0080] Preparation of cyclic nonapeptide solution: Reagent grade, which is a water-soluble raw material. Dissolve the cyclic nonapeptide powder directly in an EP tube of deionized water, shake to mix, and use this as the stock solution concentration after complete dissolution. Dilute with deionized water to the appropriate concentration during the experiment.

[0081] 1. Cytotoxicity test:

[0082] 1.1 Experimental Groups: A blank control group, a sample group, and a zero-adjustment group were set up. Blank control group: Contains only cells and culture medium; Sample group: Contains cells and a certain concentration of sample; Zero-adjustment group: Contains no cells, only culture medium.

[0083] 1.2 Experimental Methods: HaCaT cells were incubated at 37℃ at a rate of 1×10⁻⁶ cells per well. 4 Cells were evenly seeded into 96-well plates at a uniform concentration and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The supernatant was discarded, and the cells were treated according to the protocol in Table 2. The blank control group was treated with complete culture medium for 24 hours, and the sample groups were treated with different concentrations of cyclic nonapeptide for 24 hours.

[0084] Following the manufacturer's instructions, the CCK-8 working solution was prepared using a colorimetric CCK-8 kit (Beyotime). 100 μL of CCK-8 working solution was added to each well of the zeroing group, blank control group, and sample group. The cells were incubated at 37°C with 5% CO2, and the OD450 readings were recorded using a microplate reader. The effect of its cyclic nonapeptide on the survival rate of HaCaT cells was calculated.

[0085] The specific design scheme is shown in Table 2.

[0086] Table 2. Cytotoxicity Experimental Design

[0087]

[0088] The formula for calculating the survival rate of HaCaT cells by cyclic nonapeptide is: Cell viability (%) = (OD of sample well - OD of zeroing well) / (OD of blank control well - OD of zeroing well) × 100%.

[0089] 1.3 Experimental Results:

[0090] The results of the cytotoxicity assay for cyclic nonapeptide are shown in Table 3 and Figure 3 .

[0091] Table 3. Effects of cyclic nonapeptide on HaCaT cytotoxicity

[0092] Based on the toxicity test results, from Table 3 and Figure 3 It can be seen that when HaCaT cells are treated with cyclic nonapeptide, the cell viability reaches more than 90% when the concentration is ≤160ppm, compared with the blank control group, and it is non-toxic to HaCaT cells.

[0093] 2. Barrier repair experiment:

[0094] 2.1 Experimental grouping: Blank control group, negative control group, and sample group were set up.

[0095] 2.2 Experimental Methods: Human immortalized keratinocytes in good growth condition were cultured at 3.5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured at 37°C in a 5% CO2 incubator. Drug administration was performed according to the design in Table 4. The blank control group was incubated with culture medium. The negative control group and sample group were incubated with the drug (SDS + sample) for 1 hour, after which the liquid was discarded, and the cells were washed three times with HBSS. The blank control group and negative control group were replaced with complete culture medium, while the sample group was incubated with different concentrations of cyclic peptides for another 24 hours. RNA extraction and reverse transcription experiments were then performed, and the expression of FLG / AQP3 / TNF-α / NF-κB genes was detected by quantitative real-time PCR.

[0096] Table 4. Barrier Repair Design Scheme

[0097]

[0098] 2.3 Experimental Results:

[0099] The barrier repair experimental results of cyclic nonapeptide RGDSFKVKK are shown in Table 5. Figures 4-7 As shown.

[0100] Table 5. Gene expression results of cyclic nonapeptide in HaCaT cells

[0101] The experimental results showed that, compared with the BC group, the expression of AQP3 and FLG genes in the NC group was significantly reduced, indicating successful model establishment. Compared with the negative control group, AQP3 was significantly increased at a concentration of 160 ppm after treatment with the sample (cyclic nonapeptide RGDSFKVKK), and FLG was significantly increased at concentrations of 40, 80, and 160 ppm. Compared with the BC group, the expression of TNF-α and NF-κB genes in the NC group was significantly increased, indicating successful model establishment. Compared with the negative control group, TNF-α was significantly reduced at a concentration of 160 ppm after treatment with the sample (cyclic nonapeptide RGDSFKVKK), and NF-κB was significantly reduced at concentrations of 40, 80, and 160 ppm, indicating that the sample (cyclic nonapeptide RGDSFKVKK) has a barrier repair effect.

[0102] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Those skilled in the art can modify the technical solutions described in the above embodiments, or make equivalent substitutions for some of the technical features; and all such modifications and substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A cyclic nonapeptide with barrier repair function, characterized in that: The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-phenylalanine-lysine-valine-lysine-lysine).

2. The cyclic nonapeptide with barrier repair function according to claim 1, characterized in that: The structural formula of the cyclic nonapeptide is: .

3. The cyclic nonapeptide with barrier repair function according to claim 1 or 2, characterized in that: The cyclic nonapeptide was prepared by resin solid-phase synthesis using a fluorene methyloxycarbonyl N-terminal protection strategy.

4. The use of the cyclic nonapeptide according to any one of claims 1-2 in the preparation of skin barrier repair products.

5. The application according to claim 4, characterized in that: The cellular concentration of the cyclic nonapeptide is ≤160ppm.

6. The application according to claim 4, characterized in that: The cellular concentration of the cyclic nonapeptide is 160 ppm.

7. The application according to claim 4, characterized in that: The products mentioned are cosmetics and / or pharmaceuticals.

8. A skin barrier repair cosmetic, characterized in that: It includes the cyclic nonapeptide as described in any one of claims 1-2.

9. A medicine for repairing the skin barrier, characterized in that: It includes the cyclic nonapeptide as described in any one of claims 1-2.

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