A polypeptide inhibitor targeting Keap1-Nrf2 complex and its application

Abstract

The present invention discloses a peptide inhibitor targeting the Keap1-Nrf2 complex and its application, belonging to the fields of molecular biology and pharmaceutical technology. The peptide inhibitor contains an ETGE active motif, is amidated at its C-terminus, and has an amino acid sequence of DVETGEV, DEETGEY, or DWETGEE. At a concentration of 4 to 64 μM, the peptide inhibitor can eliminate ultraviolet-induced damage to cells, significantly protecting cells from phototoxicity. The peptide inhibitor can be used to prepare anti-photoaging products or drugs to protect against ultraviolet radiation.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and medical technology, and in particular to a polypeptide inhibitor targeting a Keap1-Nrf2 complex and an application thereof. Background Art

[0002] Skin aging is the result of a combination of exogenous (ionizing radiation, toxic chemicals, pollutants and chronic light exposure) and endogenous (genetically encoded hormones, cells and metabolic processes) factors, leading to a decline in skin structural integrity and physiological function, as well as a progressively aged appearance. It has become the most concerning issue among many skin health problems.

[0003] Skin aging is largely influenced by the cumulative damage of ultraviolet radiation. Human skin is frequently exposed to ultraviolet radiation from sunlight, which penetrates deep into the dermis and induces oxidative stress mediated by endogenous photosensitization in skin cells, leading to early signs of photoaging and photodamage. Photoaging accounts for over 80% of facial aging and is the primary external cause of skin aging. Symptoms include rough, dry skin, sagging, loose skin, wrinkles, loss of skin tone, hyperpigmentation, and melanoma. Therefore, providing adequate photoprotection to prevent photoaging and other skin diseases caused by UVR is crucial.

[0004] Ultraviolet radiation leads to excessive production of reactive oxygen species (ROS) in the skin and reduces antioxidant enzymes, including catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), triggering a series of oxidative damage events in the skin. UV irradiation induces activation of mitogen-activated protein kinases (MAPKs) and activator protein 1 (AP-1). MAPK proteins, such as extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK), lead to the expression of AP-1 (c-Jun and c-Fos) transcription factors. Furthermore, increased AP-1 activity induces the activation of matrix metalloproteinases (MMPs), which hydrolyze types I, III, and V collagen and degrade the extracellular matrix (ECM). UV-induced ROS production stimulates the expression of cell surface cytokines and MAPKs. Cytokines such as cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β recruit neutrophils in the dermis and produce MMPs, leading to ECM degradation. Simultaneously, nuclear transcription factor kappa B (NF-κB) is activated, ultimately participating in the induction of MMP activation. Therefore, blocking these signaling pathways and cytokines can inhibit MMP activation and achieve anti-photoaging effects.

[0005] Skin cells are equipped with a complex enzymatic and nonenzymatic antioxidant defense system to neutralize oxidative stress caused by ultraviolet radiation (UVR) and maintain cellular homeostasis. In addition to major free radical scavenging enzymes, several antioxidants or phase II detoxification enzymes, such as heme oxygenase 1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and γ-glutamate-cysteine ligase (γ-GCLC), participate in antioxidant defense and detoxification mechanisms through their anti-inflammatory, anti-apoptotic, and anti-proliferative properties in various cell and tissue types and are crucial for protecting skin cells from ROS-mediated oxidative stress and carcinogenesis. Therefore, activation of antioxidant enzymes is an important cytoprotective strategy that can enhance protection against ROS generated by environmental stress or endogenous metabolism.

[0006] Basal expression and induction of genes encoding phase II / antioxidant enzymes are mediated by the transcription of cis-acting enhancers, antioxidant response elements (AREs). The cytoplasmic Keap-1 / Nrf2 (Keap: Kelch-like ECH-associated protein 1; Nrf2: nuclear erythroid-related factor 2) regulatory complex plays a central role in protecting cells from oxidative stress, exogenous stress, and maintaining cellular homeostasis. Nrf2 is a specific redox transcription factor, considered a central hub for neutralizing reactive oxygen species (ROS) and restoring cellular redox balance. It regulates the expression of various downstream enzymes and is negatively regulated by its inhibitor, Keap-1. Under normal physiological conditions, Nrf2 activity is strictly restricted by binding to Keap1 in the cytoplasm. Mechanistic studies have revealed that Keap1, acting as a substrate scaffold for the Cul3-containing E3 ubiquitin ligase, can induce the ubiquitin-proteosomal degradation of Nrf2. Only a small fraction of Nrf2 is immune to Keap1-Cul3-mediated ubiquitination and degradation, contributing to basal antioxidant maintenance. However, under conditions of oxidative stress, the interaction between Nrf2 and DLG-Keap-1 is disrupted, while the interaction between Nrf2 and ETGE-Keap-1 remains intact. Disruption of the former interaction ultimately inactivates the E3 ligase, inhibiting Keap-1-mediated Nrf2 ubiquitination. This rapidly reduces Nrf2 degradation, leading to Keap-1 protein saturation. Newly synthesized Nrf2 can escape Keap1 and translocate to the nucleus, where it binds to the AREs and induces the promoter regions of antioxidant / II detoxification enzymes, activating the transcription of downstream protective genes such as NADPH quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HMOX1), and ferritin heavy polypeptide 1 (FTH1), thereby inducing a potent anti-toxic response. Therefore, inducing Nrf2 activation is considered key to the transcriptional activation of various genes.

[0007] The proper conformation of the Nrf2-Keap1 dimer complex is essential for the ubiquitination process. Disrupting the Nrf2-Keap1 dimer complex interaction hinders Nrf2 ubiquitination, leading to Nrf2 accumulation and activation. Therefore, the discovery of inhibitors that directly and competitively disrupt the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as a novel approach to activating Nrf2. Compared to classic electrophilic Nrf2 activators, PPI inhibitors can competitively and selectively bind to Keap1, thereby reducing uncertain safety risks. Based on this, the discovery of candidate PPI inhibitors that activate Nrf2 is being actively pursued, with the goal of developing highly active anti-photoaging products or drugs to protect against UV radiation.

[0008] Bioactive peptides can interact well with cell receptors and improve the physiological functions of the skin to a certain extent, such as enhancing skin permeability and stability. In addition, peptides have high safety, low sensitization, and controllable production costs. Therefore, it is urgent to design a bioactive peptide that contains all key features based on the structural information of the Keap1-Nrf2 complex, and use computer virtual screening strategies (including molecular docking, molecular dynamics simulation, MM / GBSA binding free energy decomposition) and anti-photoaging activity evaluation to discover the PPI inhibitor peptides with the greatest anti-photoaging potential, prevent and delay skin aging, and reduce age-related skin damage and diseases. Summary of the Invention

[0009] In view of this, one object of the present invention is to provide a polypeptide inhibitor targeting the Keap1-Nrf2 complex, a second object of the present invention is to provide a nucleic acid encoding the polypeptide inhibitor, a third object of the present invention is to provide a biomaterial comprising the nucleic acid, a fourth object of the present invention is to provide a use of the polypeptide inhibitor or the nucleic acid or the biomaterial in the preparation of a medicine or cosmetic for preventing or treating photoaging of the skin; a fifth object of the present invention is to provide a use of the polypeptide inhibitor in the preparation of an inhibitor of UVA-induced damage.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] 1. A peptide inhibitor targeting the Keap1-Nrf2 complex, wherein the peptide inhibitor contains an ETGE active motif and has the general formula:

[0012] DA1ETGEB-NH2

[0013] Wherein A1 is V or I or E or G or P or W, and B is V or F or Y or E or L.

[0014] Preferably, in the present invention, when A1 is V, B is V.

[0015] In the present invention, preferably, when A1 is E, B is Y.

[0016] Preferably, in the present invention, when A1 is W, B is E.

[0017] 2. A nucleic acid encoding the polypeptide inhibitor.

[0018] 3. A biological material comprising the nucleic acid, wherein the biological material is an expression cassette, a vector, a host cell or a transgenic cell line.

[0019] 4. Use of the polypeptide inhibitor in the preparation of medicines or cosmetics for preventing or treating skin photoaging.

[0020] Preferably, the polypeptide inhibitor is used at a concentration lower than 100 μM.

[0021] Preferably, the polypeptide inhibitor is used at a concentration of 4 to 64 μM.

[0022] 5. Use of the polypeptide inhibitor in the preparation of inhibitors of UVA-induced damage

[0023] Preferably, the inhibitor of the present invention is to inhibit the expression of MMP-1 and MMP-9 proteins induced by UVA.

[0024] Preferably, the inhibitor of the present invention inhibits UVA-induced MAPK / AP-1 activation.

[0025] The present invention provides a peptide inhibitor targeting the Keap1-Nrf2 protein-protein interaction, comprising an ETGE active motif and an amino acid sequence of DVETGEV as shown in SEQ ID No. 1, DEETGEY as shown in SEQ ID No. 3, or DWETGEE as shown in SEQ ID No. 6. The peptide inhibitor disrupts the interaction of the Nrf2-Keap1 dimer complex, thereby hindering Nrf2 ubiquitination and leading to Nrf2 accumulation and activation. The peptide inhibitor exhibits antioxidant activity, reducing UVA-induced intracellular ROS accumulation, attenuating UVA-induced expression of matrix metalloproteinases (MMP-1 and MMP-9), and significantly inhibiting UVA-induced phosphorylation of JNK, P38, and ERK, thereby inhibiting AP-1 activation. At concentrations of 4 to 64 μM, the peptide inhibitor eliminates UV-induced cell damage, significantly protecting cells from phototoxicity, and can be used to prepare anti-photoaging products or drugs to protect against UV radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0027] Figure 1 is the RMSD change between Keap1 and the peptide inhibitor main chain;

[0028] Figure 2 This is the docking binding mode of pep18 and Keap1;

[0029] Figure 3 100 ns MD simulation analysis of pep18 and Keap1, (a) Alignment analysis of the pep18 / Keap1 complex before (dark blue ribbon, light blue sticks) and after (green ribbon, orange sticks) MD simulation; (b) Global pattern of the pep18 / Keap1 complex; (c) Specific binding mode of pep18 and Keap1; (d) Binding free energy contribution of key amino acid residues calculated by the MM / GBSA method;

[0030] Figure 4 MST analysis of Keap1 and Nrf2;

[0031] Figure 5 MST analysis of Keap1 and peptide inhibitors;

[0032] Figure 6 The effect of different concentrations of peptide inhibitors on 293T cell viability;

[0033] Figure 7 The effect of the screened peptides on the luciferase reporter gene activity in HEK293-ARE cells;

[0034] Figure 8 CCK-8 assay was used to analyze the protective effect of anti-photoaging peptide inhibitors on UV-damaged HaCat cells (results from three or more experiments are expressed as mean ± SD, and compared with the untreated control group, the statistical significance is: #### p < 0.0001; compared with the UVA-irradiated control group, the statistical significance is indicated by *p < 0.05, **p < 0.01, and ****p < 0.0001);

[0035] Figure 9 Effects of Seq1 and Seq3 on ROS levels in UVA-irradiated HaCat cells (results from 3 or more experiments are expressed as mean ± SD, ****p < 0.0001 vs. unirradiated control group; ####p < 0.0001 vs. UVA-irradiated untreated control group);

[0036] Figure 10 The effects of Seq4 and Seq5 on ROS levels in HaCat cells irradiated by UVA;

[0037] Figure 11 The effect of Seq6 on ROS levels in HaCat cells irradiated by UVA;

[0038] Figure 12 Effects of Seq1 and Seq3 on the expression of MMPs proteins in HaCat cells irradiated by UVA (the results are expressed as the mean ± SD of three independent experiments, #### p < 0.0001 vs. untreated control group; * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. UVA irradiated control group);

[0039] Figure 13 The effects of Seq4 and Seq5 on the expression of MMPs proteins in HaCat cells irradiated by UVA;

[0040] Figure 14 The effect of Seq6 on the expression of MMPs proteins in HaCat cells irradiated by UVA;

[0041] Figure 15 Effects of Seq1 and Seq3 on UVA-induced MAPK / AP-1 signal transduction in HaCat cells (results are expressed as the mean ± SD of three independent experiments, #### p < 0.0001 vs. untreated control group; ** p < 0.01, **** p < 0.0001 vs. UVA-irradiated control group);

[0042] Figure 16 The effects of Seq4 and Seq5 on UVA-induced MAPK / AP-1 signal transduction in HaCat cells;

[0043] Figure 17 This is the effect of Seq6 on UVA-induced MAPK / AP-1 signal transduction in HaCat cells. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0045] Example 1: In silico screening strategy for PPI inhibitor peptides

[0046] 1. Establishment of screening peptide library

[0047] Structural information on the Keap1-Nrf2 interaction suggests that the secondary structure formed by the ETGE motif is crucial for Keap1 binding. Due to its high binding capacity and short sequence length, ETGE-containing motifs provide a promising starting point for the discovery of PPI inhibitors. For the development of a screening peptide library, we used the ETGE active motif as a template sequence. The new PPI inhibitor sequence has the core sequence DA1ETGEB-NH2. Based on conformational relationships, the A1 position is replaced by Ala(A) / Pro(P) / Ile(I) / Val(V) / Glu(E) / Gly(G) / Trp(W), and the B position is introduced with Phe(F) / Trp(W) / Leu(L) / Tyr(Y) / Cys(C) / Val(V) / Glu(E).

[0048] 2. Preparation of Receptor Ligands

[0049] The X-ray crystal structure of the Keap1-Nrf2 complex was obtained from the Protein Data Bank (https: / / www.rcsb.org / ) and used as the starting structure for molecular docking. Before docking calculations, the protein was subjected to necessary structural preparation, such as ligand extraction, removal of water molecules and metal ions, hydrogenation, and residue repair. Protomol files were created using the "Ligand" method, docking pockets were set, and then used for docking of candidate molecules. Energy minimization calculations were performed on all peptides using the Tripos force field to optimize the structure, generate the lowest energy conformation of the ligand, and hydrogenate it. The prepared ligand files were further used for virtual screening.

[0050] 3. Screening based on molecular docking

[0051] In this study, the docking program Surflex-Dock GeomX (SFXC) in SYBYL-X 2.1 was used to construct receptor-ligand complexes, where the docking score was calculated using the scoring function -log 10 (Kd) indicates that this scoring function is based on the binding affinity of the protein-ligand complex, taking into account hydrophobicity, entropy, polarity, repulsion, and solvation effects. During the docking process, the following docking parameters were used: (1) the number of starting conformations for each ligand was set to 10, and the maximum number of conformations for each fragment was set to 20; (2) the maximum number of rotatable bonds for each molecule was set to 100; (3) in the Flag region, "pre-docking energy minimization", "post-docking energy minimization", "molecular fragmentation", and "soft grid treatment" were activated; (4) the spin alignment method was activated, and the search density was set to 9.0; (5) the number of spins per alignment was set to 12.

[0052] In this study, a library of 2,394 peptides was constructed using a permutation and combination approach for molecular docking-based virtual screening to identify promising PPI inhibitors. The Keap1 substrate-binding cavity was defined as the docking pocket, with the central groove formed by key amino acid residues Tyr334, Ser363, Arg380, Asn414, Arg415, Phe478, Arg483, Ser508, Tyr525, Gln530, Tyr572, Asp573, and Phe577 as the active pocket. Receptor-based virtual screening was performed using the Surflex-dockGeomX method via molecular docking. The docking results were ranked from high to low by Total-Score. Based on a comprehensive evaluation of Total-Score, Crash, and Polar scores, peptides with high docking scores, good spatial fit, and reasonable binding profiles were selected as potential PPI modulators. Finally, the top six peptides were selected for bioassays (docking scores are listed in Table 1).

[0053] Table 1 Sequences and docking scores of inhibitors

[0054]

[0055] 4. Molecular Dynamics Simulation

[0056] Molecular dynamics (MD) simulations were performed using AMBER16 to evaluate the validity of the binding modes of the selected compounds. MD simulations were performed based on the initial conformations of the protein receptor-ligand complexes generated by GeomX docking. The Antechamber tool loaded the GAFF force field for the ligands, and the AMBER ff99SB force field was selected for the loaded Keap1 receptor. Each protein-inhibitor complex system was immersed in a truncated octahedral box, with TIP3P water molecules extending from the complex. The whole system is neutralized by adding counter ions (Cl - Or Na + ).

[0057] Molecular dynamics (MD) simulations were performed using the PMEMD.mpi and PMEMD.cuda modules in AMBER16. Before the start of the MD simulation, several consecutive energy minimization steps were performed on the system using the steepest descent and conjugate gradient algorithms in the Sander module to avoid possible molecular collisions. The force field fixed the protein skeleton, and the simulation system was optimized using 5000 steps of the steepest descent method and 5000 steps of the conjugate gradient method. Then, the constraints on the protein heavy atoms were released and the energy optimization was continued for 10,000 steps. Each system was then gradually heated from 0K to 300K in the heating phase and maintained at 300K in the subsequent equilibrium and production phases. A time step of 2fs was used in the heating phase, the equilibrium phase and the entire production phase. Periodic boundary conditions were used to maintain a constant temperature and pressure (NPT) set. The pressure was set to 1atm and controlled by an anisotropic (x-, y-, z-) pressure scaling protocol with a pressure relaxation time of 1ps. The temperature was adjusted using the Langevin temperature control method with a collision frequency of 2ps. -1 The particle mesh Ewald (PME) method is used to calculate the long-range electrostatic interactions under periodic boundary conditions, and the A cutoff of 0.000 was used to treat non-bonded interactions. The SHAKE algorithm was applied to constrain all covalent bonds involving hydrogen atoms. MD simulations were performed for 100 ns for each system, and trajectory files for the simulations were saved every 100 ps.

[0058] After MD analysis, all six peptides were able to form stable structures with Keap1. Figure 1 As shown, the root mean square deviation (RMSD) of the protein backbone atoms of the peptide inhibitor complex was found to be The dynamic convergence of these systems was achieved in simulations of 85 ns (pep18), 45 ns (pep64), 10 ns (pep90), 40 ns (pep111), 60 ns (pep121), and 40 ns (pep144), respectively. These results indicate that these complexes undergo reasonable conformational changes.

[0059] Taking pep18 as an example, Figure 2 The docking pattern of the crystal structure of pep18 and Keap1 is shown. The important residues involved in the binding pocket include Ser363, Arg380, Asn382, Arg408, Arg415, Ser508, Gln530, Gly574, and Ser602. All the residues described here are basically consistent with the information obtained from the Keap1 crystal structure. 2D docking pattern diagram ( Figure 2 ) showed that the above-mentioned important amino acids mainly interacted with Keap1 through hydrogen bonds. After 100ns of MD simulation, the peptide inhibitor structure underwent some conformational changes ( Figure 3 ,a, docking: blue; MD simulation: orange), making it fit better and enter deeper into the active pocket of Keap1 ( Figure 3 ,b). The specific binding mode of pep18 and Keap1 revealed by MD simulation is shown in Figure 2. Figure 3As shown in Figure ,c, the carboxyl group on the polypeptide chain is bound to Tyr334, Ser363, Arg380, Asn382, Ser383, Arg415, Gln530, Ser555, Gly574, and Ser602 through hydrogen bonds. In order to explore the contribution of key amino acids, the binding free energy decomposition was performed using the MM / GBSA method. Figure 3 ,d shows that Arg415, Arg483, Ser508, and Ser602 can stabilize the interaction with the alkyl chain due to the contribution of electrostatic interaction, however, polar solvation is not conducive to the interaction.

[0060] 5. MM / GBSA Calculation

[0061] The binding free energy and energy decomposition calculations of the complex were performed using the MM / GBSA method in AMBER16. Based on the energy decomposition analysis of key amino acids, the binding mode was theoretically predicted. For the saved MD simulation trajectory, the molecular mechanics / generalized Born surface area (MM / GBSA) method was used to calculate the binding energy of the receptor under different ligand treatments. For each system, the stable conformation generated from 90 to 100 ns was extracted 50 snapshots every 200 ps to calculate the average binding energy. The formula used is as follows:

[0062] ΔE bind =ΔE MM +ΔE SOL =ΔE MM +ΔE GB +ΔE SA

[0063] where ΔE bind is the binding energy, ΔE MM represents the sum of the mechanical energy of molecules in vacuum, which can be further divided into electrostatic energy (E ELE ), van der Waals energy (E VDW ) and the contribution of internal energy. ΔE SOL is the solvation energy, including the polar solvation energy (ΔE GB ), and the nonpolar fraction (ΔE) obtained by fitting the solvent accessible surface area (SASA) with the linear combination of pairwise overlaps (LCPO) model. SA ). In addition, the energy of each residue was decomposed into main chain and side chain atoms, and the contribution of important amino acid residues to receptor-ligand binding was analyzed by energy decomposition.

[0064] Binding free energy analysis results for the peptide inhibitors are shown in Table 2. Pep90 and Pep18 exhibit stronger binding free energies for their interactions with Keap1. For all peptide inhibitors, electrostatic interactions play a more significant role than van der Waals forces. Furthermore, polar interactions are greater than nonpolar interactions in all systems, indicating that nonpolar interactions favor the binding of peptides to Keap1.

[0065] Table 2. Binding free energy ΔE of the interaction between peptide inhibitors and Keap1 bind (Kcal / mol)

[0066]

[0067]

[0068] Note: ΔE bind =△G TOT

[0069] 6. Microscale Thermophoresis (MST)

[0070] The interaction between PPI inhibitors and Keap1 was investigated using microthermophoresis. Keap1 protein was labeled with Monolith His-Tag Labeling Kit RED tris-NTA secondary dye. Various concentrations of PPI inhibitors (15.25 nM to 500 μM) were titrated against labeled Keap1 (50 nM) in PBS (supplemented with 0.05% Tween). The samples were aspirated into capillaries and thermophoresis was measured at ambient temperature using 40% LED / infrared laser power and Medium MST power. Data were processed using MO.Affinity Analysis software (NanoTemper Technologies GmbH, Munich, Germany) and binding constants were analyzed.

[0071] The MST method was used to detect whether the above peptide inhibitors interacted with Keap1 protein. The results showed that ( Figure 5 ) The peptides screened had different degrees of affinity with Keap1. Steady-state analysis was performed based on the recorded data. The dissociation constants (Kd) calculated from the fitted saturation binding curves were 6.73 μM (Seq1), 81.3 nM (Seq3), 481 μM (Seq4), 115 nM (Seq5), and 2.78 μM (Seq6), respectively. The Kd for the interaction between Keap1 and Nrf2 was 33.1 nM ( Figure 4 ), the results showed that nanomolar peptides have very strong binding ability with Keap1.

[0072] Example 2: Evaluation of the anti-photoaging activity of PPI inhibitor polypeptides

[0073] 1. Cell Viability Assay

[0074] 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and HaCat cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, all in a cell culture incubator at 37°C and 5% CO2.

[0075] The cytotoxicity of PPI inhibitors was assessed using CCK-8 assay. 293T cells were plated at 5 × 10 4 Cells were inoculated into 96-well plates (100 μL) at 100 μL / ml and incubated overnight. The drug was dissolved in DMSO, and the drug solution was diluted to a final concentration (3.125-100 μM) in complete DMSO medium. The cells were incubated for 24 hours, and the cells were treated with the same concentration of DMSO (0.1%) as a control group, and the culture medium without cells (same drug treatment) was used as a blank group. After incubation, 10 μL of CCK-8 solution was added to each well and incubated in the incubator for another 0.5-4 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability was assessed as follows:

[0076] Survival rate (%) = [(Ae-Ab) / (Ac-Ab)] × 100%

[0077] Ae is the absorbance of treated cells; Ac is the absorbance of untreated cells; Ab is the absorbance of culture medium without cells. IC50 values were calculated by nonlinear regression fitting.

[0078] CCK8 detected the effects of anti-photoaging peptide inhibitors on cell survival and growth. Using the Control group as the reference ratio, a cell activity detection experiment was carried out on HEK293 cells. The measured OD value was proportional to the cell activity. Figure 6 The figure shows the effect of the screened peptide inhibitors on the activity of HEK293 cells. The results show that after 24 hours of treatment with peptide inhibitors, compared with the Control (0μM) group, the peptide inhibitors were not toxic to cells within 100μM and could significantly promote cell proliferation. However, there was no significant difference in cell viability and proliferation after peptide treatment at 100μM. Therefore, based on the comprehensive significance analysis, concentrations below 100μM were selected for subsequent experiments.

[0079] 2. Dual-luciferase reporter gene assay

[0080] A 293T-ARE cell screening model for antioxidant drugs was constructed for further screening of PPI inhibitors. The Dual-Luciferase Reporter Assay System (Promega, UK) was used to measure the reporter gene activity in transiently transfected cells. 293T cells were cultured at 2×10 4 The cells were seeded at a density of 100 cells / well in a 96-well white cell culture plate. On the day of transfection, the cells in each well were confluent to 50-70%. The pARE-luc and pRL-TK plasmids were co-transfected into cells at a 5:1 ratio using Transfection Reagent. Renilla luciferase was encoded and used as an internal control for transfection efficiency. Twelve hours after transfection, the transfection reagent was removed, and the test sample was added for 12 hours before luciferase activity was measured. Specifically, the cultured cell plates were removed and equilibrated to room temperature. Substrate I was added to each well of the plate at a volume equal to the culture medium in the well. The cells were incubated at room temperature for at least 10 minutes, and luminescence of the firefly luciferase was measured. Subsequently, an equal volume of substrate II was added to each well, and the cells were incubated at room temperature for at least 10 minutes, and luminescence of the Renilla luciferase was measured.

[0081] The experiment included a blank control group (untransfected), a control group (transfected cells without drug treatment), and a positive control group (transfected cells treated with tBHQ). Each group had six replicates. Data were analyzed in triplicate and expressed as fold induction relative to the control.

[0082] Experimental group ratio = (experimental group F-background F) / (experimental group R-background R)

[0083] Control group ratio = (control group F - background F) / (control group R - background R)

[0084] Expression fold = experimental group ratio / control group ratio

[0085] Background F: untransfected cells + firefly luciferase detection reagent; Background R: untransfected cells + firefly luciferase detection reagent + Renilla luciferase detection reagent; Experimental group: transfected cells were treated with experimental compounds (i.e., experimental group F and experimental group R); Control group: transfected cells were not treated to normalize the results (i.e., control group F and control group R).

[0086] The Nrf2 / ARE luciferase reporter assay was used to evaluate the cellular activation effect of the screened peptides on Nrf2. The transfected HEK293-ARE cells were treated with 20 μM of peptide inhibitors, and the same concentration of t-BHQ was used as a positive control. Figure 7As shown, all six peptide inhibitors exhibited varying degrees of activity, with luciferase activity significantly increasing by more than 5-fold after treatment with Seq1, Seq3, Seq4, Seq5, and Seq6. Dual-luciferase reporter gene assays demonstrated that these peptide inhibitors promoted Nrf2 transcriptional activity at the cellular level and significantly enhanced the activation of the Nrf2-ARE system.

[0087] 3. UVA Radiation and Cell Viability Assay

[0088] To detect the protective effect of peptide inhibitors on UVA-induced HaCat cell damage, CCK8 reagent was used to determine cell viability. HaCat cells were cultured at a rate of 1×10 4 Cells / well were seeded in 96-well plates and cultured overnight until 70% confluence was achieved. Cells were then pretreated with different concentrations of PPI peptide inhibitors (4μM, 8μM, 16μM, 32μM, 64μM) or control (0.1% DMSO) for 1-2 hours. After incubation, cells were washed with phosphate buffered saline (PBS) and supplemented with PBS to cover the cells. 5J / cm 2 HaCat cells were irradiated with or without UVA. After irradiation, cells were cultured for another 24 h in fresh complete medium containing the above-mentioned concentrations of peptide inhibitors. After the incubation period, cells were washed with ice-cold PBS and cell viability was determined using CCK8 as described above.

[0089] The results are as follows Figure 8 As shown in the results, UVA irradiation significantly reduced the cell viability of HaCat cells compared with untreated control cells, while pretreatment with different concentrations (4μM, 8μM, 16μM, 32μM, 64μM) of peptide inhibitors alleviated radiation-induced cell death (except Seq4 and Seq5). 2 After UVA irradiation, the cell viability decreased by 75%. Pretreatment with the peptide restored the cell viability. Moreover, at a concentration of 32 μM, it completely eliminated the effects of UV-induced cell damage, greatly protecting the cells from phototoxicity.

[0090] 4. DCFH2-DA determination of intracellular ROS production levels

[0091] To observe whether the peptide inhibitor has antioxidant capacity to reduce UVA-mediated ROS production in HaCat cells, 2',7'-dichlorofluorescein diacetate (DCFH2-DA) fluorescence staining was used to detect the fluorescence intensity of cells irradiated by UVA after treatment with different concentrations of peptide inhibitor. The experiment was divided into three groups: UVA+Control, UVA+drug, and no irradiation+Control, and three replicates were set up. HaCaT cells were cultured at 1×10 4Cells / well were seeded in a 96-well plate with a black transparent bottom and incubated overnight. After the cells grew to 70% confluence, they were incubated with peptide inhibitors at the specified concentrations (8μM, 16μM, 32μM) for 24 hours. The culture medium was then removed and the cells were washed. 100μL PBS was added and the cells were exposed to UVA (5J / cm 2 ) irradiation. After washing, the cells were treated with 20 μM DCFH2-DA diluted in serum-free medium 1640 at 37°C in the dark for 30 minutes. Finally, the cells were washed three times with serum-free medium to fully remove the DCFH2-DA that had not entered the cells. Fluorescence intensity was measured using a multifunctional microplate reader at excitation and emission wavelengths of 488 nm and 525 nm, respectively. Fluorescence emitted by the cells was imaged using an Olympus fluorescence microscope (200x magnification).

[0092] The results are as follows Figure 9-11 As shown in the results, compared with control cells, UVA irradiation alone caused intracellular ROS levels to be approximately 10-fold higher than that of the blank control group. This difference was highly statistically significant (P < 0.0001), suggesting that UVA irradiation can induce ROS production in HaCat cells. However, treatment with 8μM, 16μM, and 32μM of the peptide inhibitor significantly decreased intracellular ROS levels in a dose-dependent manner, with statistically significant differences compared to the UVA irradiation control group. Therefore, peptide inhibitor treatment can reduce UVA-induced ROS accumulation in HaCat cells, demonstrating antioxidant activity.

[0093] 5. Western blot detection of matrix metalloproteinases (MMPs) protein levels and phosphorylation levels of JNK, P38, and ERK

[0094] To investigate the effects of anti-photoaging peptide inhibitors on UV-induced photoaging, the phosphorylation levels of MAPKs were evaluated. The experiment was divided into three groups: a control group without UVA irradiation, a control group with UVA irradiation, and a drug group with UVA irradiation. The control group was cultured in complete medium containing 0.1% DMSO; the drug group was cultured in medium containing different concentrations (8 μM, 16 μM, and 32 μM) of peptide inhibitors. HaCat cells were cultured at 3×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and incubated overnight. When the cells grew to 70% confluence, the cells were pretreated with or without peptide inhibitors for 2 hours. After removing the culture medium, 1 mL of PBS was added and 5 J / cm 2Following UVA irradiation, cells were incubated for 24 hours, replacing PBS with the indicated concentrations of inhibitors. After incubation, cells were washed with ice-cold PBS and lysed on ice by adding RIPA lysis buffer containing the protease inhibitor PMSF to each well. Total cellular protein was extracted by centrifugation at 12,000 g for 10 minutes at 4°C, and the protein content of the supernatant was determined using a BCA protein quantification kit. Equal amounts of protein were collected and separated by SDS-PAGE western blotting.

[0095] Specifically, add 5× SDS Loading Buffer to the protein sample obtained in the above steps, mix thoroughly, and set aside. Prepare the SDS-PAGE gel according to conventional methods, place it in an electrophoresis tank, add an appropriate amount of 1× running buffer, remove the comb, load the prepared sample and protein marker into each lane of the SDS-PAGE gel, and run the gel at 80V for 30 minutes. Then adjust the voltage to 120V and run the gel for 100 minutes. After electrophoresis, wet-transfer to a PVDF membrane. The membrane was blocked with rapid blocking buffer at room temperature for 15–30 min. The blocked membrane was cut into strips according to protein size and incubated overnight at 4°C with primary antibodies (rabbit anti-human MMP-1, rabbit anti-human MMP-9, rabbit anti-human JNK, rabbit anti-human β-JNK, rabbit anti-human β-P38, rabbit anti-human β-P38, rabbit anti-human ERK, rabbit anti-human β-ERK, rabbit anti-human GAPDH, and mouse anti-human α-Tubulin). The next day, the membrane was washed four times in 1× TBST for 5 min each, followed by incubation with secondary antibodies for 1 h at room temperature. After secondary antibody incubation, the membrane was washed four times for 5 min each. The blot was analyzed using enhanced chemiluminescence (ECL) substrate and imaged using a LAS-4000 luminescence image analyzer. The grayscale of the target protein bands was quantified using ImageJ software, and relative protein expression was calculated using an internal reference gene as correction. Statistical analysis was performed using GraphPad.

[0096] Anti-photoaging inhibitors downregulate UVA-induced MMP-1 and MMP-9 protein expression

[0097] like Figure 12-14 As shown in the figure, compared with the blank control group without UVA irradiation, the MMP-1 and MMP-9 protein levels in the UVA irradiated control group were significantly increased, indicating that UVA irradiation can induce the expression of MMP-1 and MMP-9 proteins in HaCat cells. After UVA irradiated cells were treated with peptides, the secretion of MMP-1 and MMP-9 was significantly reduced, among which Seq4 and Seq5 had the most significant inhibitory effect, indicating that peptide inhibitors can attenuate UVA-induced matrix metalloproteinase (MMP) protein expression.

[0098] Anti-photoaging peptide inhibitors inhibit UVA-induced MAPK / AP-1 activation

[0099] like Figure 15-17 As shown in the figure, compared with the unirradiated control group, UVA irradiation significantly increased the phosphorylation levels of JNK, P38, and ERK. UV-induced reactive oxygen species generation activated the MAPK signaling pathway. However, after UV irradiation, treatment with a peptide inhibitor significantly inhibited the phosphorylation of JNK, P38, and ERK, thereby inhibiting AP-1 activation, without affecting the overall expression of JNK, P38, and ERK. Among them, the treatment with the highest concentration showed the greatest inhibition of phosphorylation. This shows that the anti-photoaging peptide inhibitor downregulates UVA-stimulated MAPK phosphorylation.

[0100] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A polypeptide inhibitor targeting the Keap1-Nrf2 complex, characterized in that The polypeptide inhibitor contains an ETGE active motif, and its general formula is: DA1ETGEB-NH2 When A1 is V, B is V; or when A1 is E, B is Y; or when A1 is W, B is E.

2. A nucleic acid encoding the polypeptide inhibitor according to claim 1.

3. The biological material comprising the nucleic acid according to claim 2, characterized in that The biological materials are expression cassettes, vectors, and host cells.

4. Use of the polypeptide inhibitor according to claim 1 in the preparation of medicines or cosmetics for preventing or treating skin photoaging.

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