Cyclic nonapeptide with anti-glycation efficacy and use thereof

Abstract

The application discloses a cyclic nonapeptide with anti-glycation efficacy and application thereof, and the amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-lysine-lysine-valine-lysine-phenylalanine). The cyclic nonapeptide can obviously inhibit the formation of CML and has the anti-glycation effect, so that the cyclic nonapeptide can be applied in anti-glycation cosmetics or medicines.

Description

Technical Field

[0001] This invention relates to a cyclic peptide, particularly a cyclic nonapeptide with anti-glycation effects and its applications. Background Technology

[0002] Glycation damage is a significant factor contributing to skin aging. When reducing sugars (such as glucose) react with amino acids in proteins (such as lysine and arginine) in a non-enzymatic reaction, glycation intermediates are produced. These intermediates further undergo rearrangement and oxidation reactions to form advanced glycation end products (AGEs). AGEs accumulate with skin aging, particularly in areas exposed to ultraviolet radiation, which is the core cause of skin glycation damage.

[0003] Glycation produces small AGEs (Advanced Glycation End Products) covalently bound proteins, forming AGE adducts. AGE adducts cause protein discoloration, turning colorless or transparent proteins yellow after glycation. Moreover, this glycation damage is not limited to the epidermis, causing yellowing of the skin; it can also occur in the dermis. For example, glycation of extracellular elastin and collagen, as well as intracellular cytoskeletal proteins and vimentins, leads to decreased skin elasticity, sagging skin, and increased wrinkles. Therefore, glycation damages the skin structure in multiple dimensions, affecting all tissue layers comprehensively.

[0004] In existing technologies, anti-glycation methods mainly include two aspects: on the one hand, protective methods can be used to inhibit the production of glycation reactions, such as protecting collagen and elastin, clearing reducing sugars, or using antioxidants to remove free radicals; on the other hand, methods can be used to reduce the formation of advanced glycation end products (AGEs), such as Nε-carboxymethyl lysine (CML), or even reverse the initial glycation of collagen, in order to inhibit the production of glycation reactions and thus achieve anti-glycation effects.

[0005] There are currently no research reports on cyclic nonapeptides with anti-glycation effects. Summary of the Invention

[0006] The purpose of this invention is to provide a cyclic nonapeptide with anti-glycation effects and its applications. This invention discovers a novel cyclic nonapeptide with anti-glycation effects.

[0007] The technical solution of the present invention is a cyclic nonapeptide with anti-glycation effect, wherein the amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-lysine-lysine-valine-lysine-phenylalanine).

[0008] The aforementioned cyclic nonapeptide with anti-glycation effects has the following structural formula: .

[0009] In the aforementioned cyclic nonapeptide with anti-glycation effects, the cyclic nonapeptide is prepared by resin solid-phase synthesis using a fluorene methoxycarbonyl N-terminal protection strategy.

[0010] The present invention also provides the application of the above-mentioned cyclic nonapeptide in the preparation of anti-glycation products.

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

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

[0013] An anti-glycation cosmetic comprising the aforementioned cyclic nonapeptide and a cosmetically acceptable carrier.

[0014] An anti-glycation drug comprising the aforementioned cyclic nonapeptide and a pharmaceutically acceptable carrier.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The cyclic nonapeptide RGDSKKVKF of this invention was prepared by resin solid-phase synthesis. After cytotoxicity and anti-glycation experiments, it was demonstrated that the cyclic nonapeptide RGDSKKVKF was non-toxic to HDF cells at a concentration ≤160ppm and could significantly inhibit the formation of CML, thus having an anti-glycation effect. It can be used as an effective ingredient in anti-glycation cosmetics and pharmaceuticals. Attached Figure Description

[0017] Figure 1 This is a simulation diagram of the docking of the cyclic nonapeptide with integrin aVβ3 in this invention.

[0018] Figure 2 This is a simulation diagram of the docking of the cyclic nonapeptide and integrin a5β1 molecules in this invention.

[0019] Figure 3 This is a statistical graph showing the effect of the cyclic nonapeptide in this invention on the cell viability of human primary fibroblasts.

[0020] Figure 4 This is a statistical graph showing the effect of each experimental group on the carboxymethyl lysine content of human primary fibroblasts; in the graph, # indicates a significant difference compared to the BC group, "###" indicates P<0.001; * indicates a significant difference compared to the NC group, and "***" indicates P<0.001.

[0021] Figure 5 This is an immunofluorescence image showing the effect of cyclic nonapeptide on carboxymethyl lysine in human primary fibroblast cells. Detailed Implementation

[0022] 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.

[0023] Example:

[0024] Structural simulation and screening of cyclic nonapeptides:

[0025] This invention designs cyclic nonapeptides as 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.

[0026] Subsequently, the three-dimensional spatial structures of the aforementioned 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 steric hindrance between the amino acid residues of the candidate cyclic peptide structure is small, and its structural stability is superior to its linear peptide form, enabling it to stably form a cyclic peptide structure suitable for further experimental verification. The structural stability scoring table is shown in Table 1.

[0027] Table 1. Structural stability scores of different cyclic peptide sequences

[0028]

[0029] Based on the above screening criteria, this invention screened out structurally stable candidate cyclic nonapeptide RGDSKKVKF.

[0030] Molecular docking simulations were performed between the RGDSKKVKF 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.

[0031] The docking fraction between the cyclic nonapeptide RGDSKKVKF and integrin αVβ3 was -9.8298, 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 1.306 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.

[0032] Subsequently, molecular docking simulations were performed between the RGDSKKVKF 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.

[0033] The docking fraction between the cyclic nonapeptide RGDSKKVKF and integrin α5β1 was -10.449, 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 1.196 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.

[0034] The above molecular docking simulation results indicate that the structure of the cyclic nonapeptide is conducive to binding integrin αVβ3 and α5β1.

[0035] The amino acid sequence of the cyclic nonapeptide of the present invention is: cyclic (arginine-glycine-aspartic acid-serine-lysine-lysine-valine-lysine-phenylalanine), i.e., Cyclo(Arg-Gly-Asp-Ser-Lys-Lys-Val-Lys-Phe), and the cyclic sequence is shown in SEQ ID NO.1. The structural formula is: .

[0036] The preparation method of cyclic nonapeptide is as follows:

[0037] S1. Using natural amino acids arginine-glycine-aspartic acid-serine-lysine-lysine-valine-lysine-phenylalanine as starting materials, a fluorene methyloxycarbonyl (Fmoc) N-terminal protection strategy is adopted, and the corresponding amino acids are sequentially linked according to the resin solid-phase synthesis method, during which the Fmoc- protecting group is sequentially removed to obtain a resin containing a linear nonapeptide chain.

[0038] The specific steps are as follows:

[0039] 1) Swelling resin:

[0040] 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 induce swelling for 5 min.

[0041] 2) Resin deprotection:

[0042] 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.

[0043] 3) Remove protective washing:

[0044] 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.

[0045] 4) Deprotection detection:

[0046] 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.

[0047] 5) Condensation of the second amino acid:

[0048] 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.

[0049] 6) Reaction washing:

[0050] 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.

[0051] 7) Reaction detection:

[0052] 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.

[0053] 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-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH until Fmoc-Lys(Boc)-OH ends, to obtain resin-3.

[0054] 9) Deprotection:

[0055] 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.

[0056] 10) Remove protective wash:

[0057] 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.

[0058] 11) Deprotection detection:

[0059] 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.

[0060] 12) Washing:

[0061] 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.

[0062] 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.

[0063] The specific steps are as follows:

[0064] 13) Resin-protected pyrolysis:

[0065] 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.

[0066] 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.

[0067] The specific steps are as follows:

[0068] 14) Modification cyclization reaction:

[0069] 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.

[0070] S4. The cyclic peptide containing the protecting group was purified by preparative HPLC to obtain cyclic nonapeptide.

[0071] The specific steps are as follows:

[0072] 15) Rotary freeze-drying:

[0073] 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-lysine-lysine-valine-lysine-phenylalanine).

[0074] Experimental example:

[0075] Experimental consumables: Biosafety cabinet (Nuaire, USA, NU-543-600s), CO2 incubator (Panasonic Corporation, MCO-18AIC), high-speed refrigerated centrifuge (Eppendorf AG, Germany, Centrifuge 5804R), small low-temperature refrigerated centrifuge (Sigma, Germany, 1-14K), analytical balance (Sartorius Group, Germany, BCE224-1CCN), microscope (Carl Zeiss Microscopes (Germany) GmbH, Axio Observer3), microplate reader (Tecan (Shanghai) Trading Co., Ltd., SPARK), integrated fluorescence microscopy imaging system (KEYENCE, Japan, BZ-X810).

[0076] Experimental reagents: Phosphate-buffered saline (PBS, Beijing Lanjieke Technology Co., Ltd., BL1425A), DMEM (1×, Gibco Life Technologies, 11885084), Australian fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, 10099141C), bovine serum albumin (Bovine serum albumin, ... BSA (Sigma-Aldrich (Shanghai) Trading Co., Ltd., A9418), Immunofluorescence Fixative (Shanghai Beyotime Biotechnology Co., Ltd., P0098), Goat Serum (Shanghai Beyotime Biotechnology Co., Ltd., C0265), Triton X-100 (Sigma-Aldrich (Shanghai) Trading Co., Ltd., X100), Pyruvaldehyde (Sigma-Aldrich (Shanghai) Trading Co., Ltd., W296902), Aminoguanidine Sulfate (Shanghai Maclean Biochemical Technology Co., Ltd., A824549), Antifluorescence Quenching Mounting Tablet (Shanghai Beyotime Biotechnology Co., Ltd., AC28L532), Carboxymethyl Lysine Antibody (Abogen (Shanghai) Trading Co., Ltd., ab125145).

[0077] Preparation of cyclic nonapeptide solution: Reagent grade, which is a water-soluble raw material. Dissolve the cyclic nonapeptide powder directly in an EP tube containing 1 mL 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.

[0078] 1. Cytotoxicity test:

[0079] 1.1 Experimental Groups: A blank control group, a sample group, and a zeroing group were set up. Blank control group: contained only cells and culture medium; Sample group: contained cells and a certain concentration of sample; Zeroing group: contained no cells and only culture medium containing CCK-8 was added.

[0080] 1.2 Experimental Methods: HDF cells were incubated at 37℃ at a density of 8 × 10⁸ cells per well. 3 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 washed once with 200 μL of PBS. The blank control group was treated with complete culture medium for 24 hours, while the sample groups were treated with different concentrations of cyclic nonapeptide for 24 hours. Cell viability was measured using a colorimetric CCK-8 assay kit (Beyotime) according to the manufacturer's instructions, and the effect of cyclic nonapeptide on HDF cell survival was calculated.

[0081] The specific design scheme is shown in Table 2.

[0082] Table 2. Cytotoxicity Experimental Design

[0083]

[0084] The formula for calculating the survival rate of HDF cells by cyclic nonapeptide is: Cell viability (%) = (OD of sample wells - OD of zeroing wells) / (OD of blank control wells - OD of zeroing wells) × 100%.

[0085] 1.3 Experimental Results:

[0086] The results of the cytotoxicity assay for cyclic nonapeptide are shown in Table 3 and Figure 3 .

[0087] Table 3. Effects of cyclic nonapeptide on HDF cytotoxicity

[0088]

[0089] Based on the toxicity test results, from Table 3 and Figure 3 It can be seen that when HDF 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 HDF cells.

[0090] 2. Anti-glycation experiment:

[0091] As a highly active α-dicarbonyl compound, acetone aldehyde (MGO) can overcome the detoxification capacity of the cell's own glyoxalase system when exogenously stimulating human primary skin fibroblasts (HDF). MGO reacts with lysine residues of proteins in HDF cells through non-enzymatic glycosylation. After a series of reactions including Schiff base formation, Amadori rearrangement, oxidation, and dehydration, it finally generates a stable and irreversible glycosylation end product—Nε-carboxymethyl lysine (CML).

[0092] CML, as a specific marker of glycosylation damage, is positively correlated with the degree of glycosylation damage induced by acetone aldehyde in HDF cells. Quantitative determination of intracellular CML content using specific detection techniques (such as ELISA and immunofluorescence) can accurately reflect the glycosylation damage effect of acetone aldehyde on HDF cells, providing in vitro experimental evidence for subsequent related technology development and efficacy evaluation.

[0093] 2.1 Experimental grouping: Blank control group, negative control group, positive control group, and sample group were set up.

[0094] 2.2 Experimental Methods: Cells were cultured at 8.0 × 10⁻⁶. 4Cells were evenly seeded into 12-well plates at a rate of [number] cells / well and incubated overnight in an incubator (37°C, 5% CO2). After overnight incubation, drug treatment was administered according to the specific design protocol in Table 4. After drug treatment, the 12-well plates were incubated for 3 hours. The blank control group received no treatment. After 3 hours of incubation, the original solution in the wells of the negative control group, positive control group, and sample group was aspirated, and 1.0 mM acetone aldehyde stimulant and drug were added for co-incubation overnight. After overnight incubation, the cells were washed and fixed with immunofluorescence fixative. Subsequent detection was performed according to the specific immunofluorescence procedure.

[0095] Statistical analysis and graphing were performed using data analysis software. All values ​​are expressed as mean ± SD. Statistical comparisons were conducted using t-tests or one-way ANOVA, with P < 0.05 considered statistically significant.

[0096] Table 4. Anti-glycation efficacy design scheme

[0097]

[0098] 2.3 Experimental Results:

[0099] The anti-glycation experimental results of cyclic nonapeptide RGDSKKVKF are shown in Table 5. Figure 4 and Figure 5 As shown.

[0100] Table 5. Effect of cyclic nonapeptide on CML content in HDF cells

[0101]

[0102] The results showed that CML expression was significantly upregulated in the negative control group compared with the blank control group. CML content was significantly downregulated in the sample group compared with the negative control group. Specifically, the inhibition rate of CML content was 74.08% at 160 ppm, 62.38% at 80 ppm, and 54.41% at 40 ppm, indicating that cyclic nonapeptide-RGDSKKVKF has the ability to inhibit CML production, thereby achieving an anti-glycation effect.

[0103] Therefore, it is a cyclic nonapeptide with anti-glycation properties.

[0104] 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 anti-glycation effects, characterized in that: The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-lysine-lysine-valine-lysine-phenylalanine).

2. The cyclic nonapeptide with anti-glycation effect according to claim 1, characterized in that: The structural formula of the cyclic nonapeptide is: .

3. The cyclic nonapeptide with anti-glycation effect 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 cyclononapeptide according to any one of claims 1-2 in the preparation of anti-glycation cosmetics.

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

6. An anti-glycation cosmetic, characterized in that: It includes the cyclic nonapeptide as described in any one of claims 1-2.

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