CN118126120A - Inhibition of Abeta42Tripeptide compounds that aggregate and reduce their cytotoxic effects - Google Patents
Inhibition of Abeta42Tripeptide compounds that aggregate and reduce their cytotoxic effects Download PDFInfo
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Abstract
Tripeptide compounds are disclosed which inhibit Abeta 42 aggregation and reduce its cytotoxic effects, including ERW or WRR. According to the invention, a tripeptide database is constructed by using Python language, 5 tripeptides combined with Abeta 42 are screened out through molecular butt joint, interaction between 5 tripeptides (ERW, QRW, RRR, RRW and WRR) and Abeta 42 protein is analyzed by using a molecular dynamics method, and analysis of the structural rationality of a pull-type chart and the binding energy of umbrella-type sampling shows that the complexes Abeta 42 -WRR and Abeta 42 -ERW are effective in butt joint structure, and the WRR and ERW have an effective inhibition effect on Abeta 42 aggregation. The pharmacological experiment is designed and implemented, and the WRR, ERW and compound mixtures thereof are verified to have good inhibition effect on Abeta 42 aggregation, so that the cytotoxic effect of the secreted expressed Abeta 42 on SH-SY5Y nerve cell strains is lightened.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a tripeptide compound for inhibiting Abeta 42 from gathering and reducing the cytotoxic effect of Abeta 42.
Background
Alzheimer's disease is a neurodegenerative disease, the main pathological feature of which is the aggregation of beta-amyloid (Abeta) into amyloid plaques and hyperphosphorylation of Tau protein. Excessive activation of immune cells after aβ aggregation causes brain inflammation; disruption of neuronal synapses and neurons; causes necrosis of neurons, and spreads cell contents such as chromatin to peripheral brain tissues (unpublished data). Aβ (aβ 42) containing 42 amino acids is the main species forming amyloid plaques, not aβ 40. Alzheimer's disease, not only is a mental disability disorder, but is also a process of death that is harsh. The total number of Alzheimer's disease patients in the world is over 5000 ten thousand, and only the anti-amyloid Abeta 42 monoclonal antibody on the market is approved by the American FDA in 2021 through an urgent examination way at present for the pathological marker Abeta protein of Alzheimer's disease, however, a plurality of clinical experiments in the past show that Abeta 42 monoclonal antibody can cause encephalitis, and the development of new antibody medicines for a plurality of pharmaceutical factories is terminated in a clinical stage. The pathological complexity of Alzheimer's disease brings great challenges to the development of specific drug tasks for Alzheimer's disease treatment. Our earlier basic research shows that the aβ 42 monomer is globular protein, and as the degree of polymerization increases, an S-shaped tubular structure is formed, and the free energy of the system of the structure is the lowest. In this patent we propose tripeptide compounds and their compounds that inhibit aβ 42 aggregation and reduce their cytotoxic effects.
Disclosure of Invention
The present invention aims to provide tripeptide compounds and compounds for inhibiting aβ 42 from aggregation and reducing the cytotoxic effect thereof, so as to solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: tripeptide compounds, including ERW or WRR.
Preferably, the ERW is glutamic acid-arginine-tryptophan, and the WRR is tryptophan-arginine.
Preferably, the tripeptide compound is used to inhibit aβ 42 aggregation.
Preferably, the tripeptide compounds are used to reduce aβ 42 cytotoxicity.
Preferably, the preparation method of the tripeptide compound is characterized by comprising the following steps:
step one: writing CHEMSCRIPT scripts by using Python language, and creating a structural database of all tripeptides consisting of 20L-alpha amino acids;
Step two: screening from a protein database to obtain a protein molecular structure of five-membered oligomer Abeta 42 as a target protein conformation;
step three: taking penta oligomer Abeta 42 as target protein, and utilizing molecular docking software and a constructed tripeptide structure database to carry out molecular docking screening to obtain tripeptides with optimal docking states;
Step four: analyzing the binding free energy of the 5 tripeptides screened by butt joint and the pentad polymerization state Abeta 42 by utilizing GROMACS molecular dynamics software;
Step five: pharmacological experiments 5 tripeptide mixtures were analyzed to inhibit aβ 42 aggregation and to reduce the cytotoxic effects caused by aβ 42.
Preferably, the tripeptide compounds are used in medicaments and foods for the prevention and treatment of alzheimer's disease.
Preferably, the tripeptide compounds are used in medicine and food.
The invention has the technical effects and advantages that:
According to the invention, a tripeptide database is constructed by using Python language, 5 tripeptides which can be combined with Abeta 42 are screened out through molecular butt joint, and interaction of 5 tripeptides (ERW, QRW, RRR, RRW and WRR) and Abeta 42 proteins is analyzed by using a molecular dynamics method, and analysis of the rational analysis of a pull-type structure and umbrella-type sampling combined energy shows that the complexes Abeta 42 -WRR and Abeta 42 -ERW are effective in butt joint structure, on the basis, pharmacological experiments are designed and implemented, and pharmacological experiments are verified, the Abeta 42 aggregation of the WRR, ERW and compound mixtures thereof has a good inhibition effect on Abeta 42 aggregation, so that the cytotoxic effect of secreted expressed Abeta 42 on SH-SY5Y nerve cell lines is lightened, the main pathological characteristics of Alzheimer disease are that Abeta 42 in the brain of a patient is aggregated into plaques, and according to the pathogenesis, the compound mixtures composed of the WRR and the ERW are used for medicines or foods, the pathological processes such as Abeta 42 aggregation and the cytotoxicity caused by the pathological mechanism can be slowed down.
Drawings
FIG. 1 is a schematic diagram showing the binding of tripeptides obtained by molecular docking in accordance with the present invention to Abeta 42 penta-polymeric cores.
FIG. 2 is a drawing of a tripeptide ligand of the invention and Abeta 42 dimer complex.
FIG. 3 is a schematic diagram showing the free energy of binding between tripeptides and Abeta 42 five-membered aggregation core in the molecular dynamics analysis 5 according to the present invention.
FIG. 4 is a schematic diagram showing the effect of ThT method in detecting two tripeptides and compound thereof in inhibiting Abeta 42 aggregation.
FIG. 5 is a schematic diagram showing the effect of two tripeptides of the present invention on the aggregation morphology of Abeta 42 by electron microscopy.
FIG. 6 is a schematic diagram showing the effect of microscopy on SH-SY5Y cell survival to the analysis of Abeta 42 on WRR and ERW.
FIG. 7 is a schematic diagram showing the effect of flow cytometry on SH-SY5Y apoptosis secreting Abeta 42.
FIG. 8 is a schematic diagram showing the effect of flow cytometry on Reactive Oxygen Species (ROS) production by SH-SY5Y cells secreting Abeta 42.
FIG. 9 is a schematic diagram showing the effect of MTT (thiazole blue) method for detecting two tripeptides and compound thereof on reducing the cytotoxicity of Abeta 42.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The present invention provides tripeptide compounds as shown in fig. 1-9, comprising glutamic acid-arginine-tryptophan (ERW) or tryptophan-arginine (WRR), a compound mixture of tripeptide compounds for inhibiting aβ 42 aggregation and reducing aβ 42 cytotoxicity, and a tripeptide compound or a compound mixture thereof for preventing and treating diseases related to aβ 42 aggregation and cytotoxicity thereof or for affecting pathological processes of diseases related to aβ 42 aggregation and cytotoxicity thereof, wherein the composition comprises glutamic acid-arginine-tryptophan (ERW) or tryptophan-arginine (WRR).
Preferably, the method for researching the tripeptide compound or the compound mixture thereof comprises the following steps:
step one: writing CHEMSCRIPT scripts by using Python language, and creating a structural database of all tripeptides consisting of 20L-alpha amino acids;
step two: screening from a Protein Database (PDB) to obtain a protein molecular structure of five-membered oligomer Abeta 42 (No. 5 oqv) as a target protein conformation;
step three: the five-membered oligomer Abeta 42 is taken as target protein, molecular docking software and a tripeptide structure database constructed by us are utilized to carry out molecular docking screening to obtain tripeptides with optimal docking states;
Step four: analyzing the binding free energy of the butt-screened 5 tripeptides and the pentad polymerization state Abeta 42 (5 oqv) by utilizing GROMACS molecular dynamics software;
Step five: pharmacological experiments analyzed that two tripeptides (WRR and EWR) inhibited aβ 42 aggregation and reduced the cytotoxic effect caused by aβ 42.
Wherein, the 5 tripeptides selected in the step three comprise ERW, QRW, RRR, RRW and WRR, and the five tripeptides are the tripeptides with the best binding with Abeta 42.
Further, the analysis results obtained in the fourth and fifth steps show that the complexes Abeta 42 -WRR and Abeta 42 -ERW are effective for interfacing and the wrR and ERW play an effective role in inhibiting Abeta 42 aggregation, and the rationality analysis principle of the structure of the pull-type map is to evaluate the rationality of the bone conformation of two adjacent peptide units through the minimum contact distance between non-bonding atoms in the protein and express the allowed and disallowed conformation in the protein, and the number of amino acids in the disallowed area is less than 5% of the total number of amino acids. The free energy of binding of tripeptides to aβ 42 is known by analyzing the change in the free energy of the system during pulling of tripeptides away from the binding complex; the larger the change in free energy of the system, the higher the free energy of binding of tripeptide to aβ 42.
Further, the analysis result obtained in the pharmacological experiment in the step five verifies that WRR, ERW and compound mixtures thereof have an inhibition effect on Abeta 42 aggregation and can be used for relieving the cytotoxicity of secreted expressed Abeta 42 on SH-SY5Y nerve cell lines, and the pharmacological experiment comprises ELISA experiment, apoptosis experiment and cell ROS detection.
Furthermore, the tripeptide compound and the compound mixture thereof are applied to medicines and foods, and the medicines and the foods can be used for slowing down the aggregation of Abeta 42 and the cytotoxin pathological process caused by the aggregation, wherein the compound mixture refers to a compound formed by WRR and ERW. Because the main motive force of Abeta 42 aggregation is free energy reduced by hydrophobic interaction (comprehensive expression of three interactions), the tripeptide and the compound thereof are combined with Abeta 42 to inhibit excessive aggregation and cytotoxic effect thereof.
According to the invention, a tripeptide database is constructed by using Python language, 5 tripeptides which can be combined with Abeta 42 are screened out through molecular butt joint, and the interaction of the 5 tripeptides (ERW, QRW, RRR, RRW and WRR) with Abeta 42 protein is analyzed by using a molecular dynamics method, and analysis of the reasonability of a pulling-type structure and the umbrella-type sampling combination can show that the complexes Abeta 42 -WRR and Abeta 42 -ERW are effective in butt joint structure and the WRR and ERW have an effective inhibition effect on Abeta 42 aggregation, on the basis, pharmacological experiments are designed and implemented, and the pharmacological experiments are verified, so that the Abeta 42 aggregation is well inhibited by the WRR, ERW and compound mixtures thereof, the main pathological characteristics of the Abeta 42 secreted and expressed in the Abeta 42 are that Abeta 42 in the brain of a patient is aggregated into plaques, and the compound mixtures consisting of the WRR and ERW are used for medicines or foods according to the mechanism, the pathological processes of Abeta 42 aggregation and the pathological effects caused by the Abeta 42 aggregation and the Abeta 4326 aggregation are slowed down.
The first embodiment is as follows: a method for analyzing the interaction between the tripeptide compound and the penta-polymerized Abeta 42 by adopting information science and technology;
(1) Constructing a tripeptide compound structure database, writing CHEMSCRIPT scripts by using Python language, and constructing a sdf (Structure Data File) format structure database of all tripeptide compound molecular structures formed by random combination of 20L-alpha-amino acids;
(2) The protein structure database (PDB) of the american structural bioinformatics research co-organization (RCSB) was searched for, and the three-dimensional structure data of aβ 42 aggregation core (No. 5 oqv) was downloaded. The downloaded structure file is renamed as 5 oqvPentatmer.pdb and stored as a target protein structure file for molecular docking;
(3) Using molecular docking software, taking 5oqvPentamer as a docking receptor, carrying out molecular docking by using the constructed tripeptide compound structure database, sequencing docking results according to the optimized binding energy, selecting 5 tripeptide compounds with the optimal docking state for subsequent molecular dynamics and pharmacological analysis, and screening out 5 tripeptide compounds with ERW, QRW, RRR, RRW and WRR respectively by molecular docking;
(4) Molecular dynamics analysis the stability and interaction energy of binding of 5 tripeptides to aβ 42. The GROMACS molecular dynamics analysis software (2020.03 edition) is installed on Ubuntu (18.04 edition) Linux operating system, adopts a GPU mixed parallel operation mode supported by CUDA (Compute Unified Device Architecture), and analyzes the stability and the binding free energy of the binding of 5 tripeptide compounds with Abeta 42 by using an AMBER99SB force field and TIP3P clear water molecule model which are optimized for the de novo calculation of a protein structure. The change in free energy is a very important physical quantity that determines the direction of the reaction. The free energy is used as a basis for judging whether a process can be spontaneously performed or not in the second law of thermodynamics. The gibbs free energy (Δg) reflects the magnitude of binding energy, and the stability of tripeptide and pentapeptide compound substrates binding to aβ 42 monomers was investigated by the present study by calculating the gibbs free energy (Δg) change when the ligand was pulled away from the target protein along the reaction axis (ζ) using umbrella-type sampling (Umbrella sampling) and weighted histogram method (WHAM) by GROMACS (2020.03) software. First, after energy minimization and correction of the complex structure of the molecular docking-screened protein-ligand complex, a GROMACS topology file was generated by the pdb2gmx instruction. The GROMOS96 53a6 force field and SPC216 water molecule model solvent are selected. The topol _protein_chain_h.itp file contents are modified. A rectangular box with the size of 6.0x7.0x15.0nm was defined as the system analysis unit box, and the center position of the Abeta 42 protein complex combined with tripeptides and pentapeptides was 3.0,3.5, 12.5 (x, y, z). And the protein-ligand complex structure is placed on one side of the cuboid cell. The cell is filled with water molecules and 0.1M sodium chloride is added into the system to balance the system charge. The reference atoms are set to the carbonyl carbon atom of glycine at position 29 of Abeta 42 and the C alpha of amino acid 2 of tripeptide or the C alpha of amino acid 3 of pentapeptide. The pressure equalization shown above is employed prior to the pull and umbrella sampling phases. After system energy minimization and pressure equilibration, the ligand substrate was pulled away from the protein complex along the reaction axis (ζ) and serial configuration data were generated by applying a resonance force at a constant rate of 0.01nm/ps over 250,000 time steps, and umbrella-type sampling analysis was performed with a system configuration with a center distance of no more than 0.2nm as the analysis window, each of 5,000,000 time steps, followed by a weighted histogram analysis to obtain gibbs free energy change curves in the protein-ligand complex system.
The second embodiment is as follows: the action of ThT detection tripeptide compound and compound mixture thereof for inhibiting Abeta 42 aggregation is utilized, abeta 42 adopts beta-lamellar conformation to mutually aggregate to generate three hydrophobic regions, and hydrophobic amino acid is buried at the inner side of a protein compound, so that the free energy of a system is reduced. ThT is a benzothiazole fluorescent dye commonly used for aggregation state Abeta 42, can be specifically combined to a beta-lamellar structure of Abeta 42, after combination, fluorescence of the ThT is enhanced, the more the polymerized state Abeta lamellar structure of Abeta 42 in the system is, the stronger the fluorescence intensity of the system is, and the ThT is combined outside Abeta 42 polymerized state fibers and does not interfere Abeta 42 aggregation, so that the ThT is commonly used for analysis and judgment of the formation of Abeta 42 polymerized state fibers;
(1) Aβ 42 and tripeptide sample treatment aβ 42 powder was dissolved in 1mM stock solution with 1.0% nh 4 OH, dipeptide compound solid powder was dissolved in 1mM stock solution with 10mM phosphate buffer (ph 7.4) (PBS), and in subsequent experiments all stock solutions were diluted with 10mM PBS to working solutions of different concentrations immediately before use;
(2) Using thioflavine T (ThT), fluorescence detection of inhibition of Abeta 42 aggregation by tripeptide compound and compound mixture thereof, dissolving 0.0128mg ThT powder in PBS, stirring thoroughly to dissolve, fixing volume to 10mL, preparing 4mM ThT stock solution, sequentially adding Abeta 42 with final concentration of 10 μm, tripeptide compound with 10 μm or tripeptide compound mixture with tripeptide compound mixture (5 μm each) and ThT with thickness of 16 μm into 96-well plate with transparent black wall, reacting to obtain total volume of 125 μl, mixing 5 repeats each, incubating at 37deg.C for 24 hr, placing into a syngy H1 enzyme-labeled instrument, setting excitation wavelength of 450nm, and detecting fluorescence intensity at 485 nm.
And a third specific embodiment: observing the aggregation morphology of Abeta 42 by using a Transmission Electron Microscope (TEM), wherein an experimental group Abeta 42 is incubated by adding tripeptide WRR and ERW with the concentration being four times that of Abeta 42 respectively, and a control group only contains Abeta 42;
(1) The sample incubated for 48h at 37 ℃ with a constant temperature shaker is taken out and added dropwise onto a carbon support membrane deionized copper mesh, then 10 μl of 2% phosphotungstic acid solution is used for negative staining, the drying treatment is followed by observation with a transmission electron microscope system, the accelerating voltage is 200kv, the scale bar is 200nm, the final concentration of Abeta 42 is 10 μM, and the final concentrations of tripeptide WRR and ERW are 40 μM.
(2) Aβ 42 alone incubated for 48 hours showed that fibrils were tightly plaque and cross each other and aβ 42 incubated for 48 hours after addition of tripeptides WRR and ERW exhibited smaller amorphous aggregate particles, and transmission electron microscopy results showed that addition of tripeptide compounds prevented the formation of aβ 42 fibril aggregates to some extent, producing only small amounts of oligomers.
The specific embodiment IV is as follows: ethidium Bromide (EB) explored the effect of tripeptides on aβ42 cytotoxicity;
(1) After the SH-SY5Y cells are resuscitated, the cells which are in the logarithmic growth phase and have good conditions are inoculated to a 12-hole cell culture plate for overnight culture, and pcDNA3.1-Abeta 42 plasmids are transfected into the cells when the growth abundance reaches 75-80%, so that the SH-SY5Y cells can exocrine and express Abeta 42.
(2) Three replicates were set for each dosing group after transfection was completed and treated with two short peptides ERW and WRR at final concentrations of 10. Mu.M and 50. Mu.M for 24 hours. The cells of each group were added with ethidium bromide at a final concentration of 1mg/ml and transferred to a carbon dioxide incubator for incubation for 10 min.
(3) Cells were observed using a fully automated inverted fluorescence microscope (Axio bserver 7), setting 545nm excitation wavelength, and measuring emission wavelength at 590 nm. Dead cells showed red fluorescence. Dead cells were counted using Image J and the results showed: SH-SY5Y cells transfected with pcdna3.1-aβ42 plasmid were significantly reduced in number of dead cells and dose-dependent in the case of tripeptide pretreatment compared to the no oligopeptide-treated group.
Fifth embodiment: flow cytometry detection of tripeptide effects on Abeta 42 -induced apoptosis
SH-SY5Y cells in the logarithmic growth phase are selected to be digested by pancreatin and inoculated into 12-hole cell plates for culturing for 24 hours, pcDNA3.1-Abeta 42 plasmid is transfected into the SH-SY5Y cells by a Lipofectamine 2000 reagent, so that the cells can exogenously secrete and express Abeta 42, three repeats are arranged for each administration group after the transfection is finished, ERW and WRR short peptides with the final concentration of 10 mu M and 50 mu M are added for processing for 24 hours, apoptotic cells and necrotic cells are marked by using an apoptosis kit of Shanghai hundred-Sieve organisms, and the apoptosis level of the SH-SY5Y cells is detected by a flow cytometer.
Specific embodiment six: flow cytometry detection explored the effect of tripeptides on aβ 42 -stimulated cells to produce reactive oxygen species;
(1) Plating the pancreatin digested SH-SY5Y cells into 24-well cell plates, plating about 10 6 cells per cell plate, transferring into a carbon dioxide incubator for culturing for 24 hours, and then transfecting pcdna3.1-aβ 42 plasmid into the SH-SY5Y cells with a PEI 40K transfection kit, so that the SH-SY5Y cells can exogenously express aβ 42, adding WRR and ERW with final concentrations of 10 μm and 50 μm to an experimental group, and treating for 24 hours at 37 ℃, wherein each tripeptide treatment group is configured with three replicates;
(2) Observing the cell state under an inverted microscope and taking a picture;
(3) Cells were treated with active oxygen detection reagents, the FL1 channel of a flow cytometer was selected, excitation wavelength was set at 488nm, and emission wavelength at 530nm was measured. Microscopic observation shows that the tripeptide has no neurotoxic effect on SH-SY5Y cells, WRR and ERW can well inhibit the aggregation of Abeta 42 secreted by SH-SY5Y cells, eliminate the toxic effect of Abeta 42 aggregation on cells, and the flow type result shows that: SH-SY5Y cells transfected with pcdna3.1-aβ 42 plasmid had significantly reduced levels of induced ROS in the case of tripeptide pretreatment compared to the no oligopeptide-treated group and reduced dose-dependent ROS formation in aβ 42 -induced SH-SY5Y cells.
Seventh embodiment: MTT (thiazole blue) method for detecting influence of tripeptide and compound thereof on Abeta 42 cytotoxicity;
When SH-SY5Y cells are in the logarithmic growth phase, pancreatin digestion is carried out, the cells are uniformly inoculated into a 96-well plate by cell counting, the number of cells in each well is 5x 10 3, transfection is carried out after 24 hours of culture, WRR (10 mu M), EWR (10 mu M) or WRR/EWR (5 mu M) mixed solutions with different concentrations are added into SH-SY5Y cells secreting Abeta 42 protein, blank wells are set as cell culture solution, control wells are normal untransfected cells, zeroing wells are transfected cells added with phosphate buffer, 4 repeats are set, DPBS buffer is added into edge wells, solution volatilization in sample wells of an experimental group is prevented, solution in the 96-well plate is abandoned after 24 hours of culture, 90 mu L of serum-free medium and 10 mu L of MTT solution (5 mg/mL) are added into each well, 100 mu L of mixed solution is sucked out after 4 hours of treatment, a shaking speed of 100 mu L of light absorption enzyme is added into each well is set as a shaking table, the OD value of the control wells is calculated at 37 nm, the light absorption enzyme activity of the control wells is calculated, the OD value of the control wells is calculated, the shaking value of the control wells is calculated, the OD value of the control wells is calculated, and the shaking value of the control wells is calculated, and the OD value is calculated.
In fig. 1, the band-like structure represents the spatial conformation of the penta-polymeric state aβ 42, wherein a is glutamate-arginine-tryptophan (ERW); b is glutamine-arginine-tryptophan (QRW); c is arginine-arginine (RRR); d is arginine-tryptophan (RRW); e is tryptophan-arginine (WRR).
In fig. 2, a is aβ 42-Aβ42 dimer; b is ERW combined with Abeta 42; c is QRW and is combined with Abeta 42; d is the combination of RRR and Abeta 42; e is RRW combined with Abeta 42; f is the binding of WRR to Abeta 42.
Fig. 4, P <0.01, compared to aβ 42; $$ P <0.01, compared to Abeta 42 -WRR; ## P <0.01, compared to Abeta 42 -EWR; one-way analysis of variance (ANOVA), modified-Tukey test was used for multiple comparisons between groups, n=5, and repeated three times.
In fig. 5, a is a 10 μ M A β 42 transmission electron microscopy image incubated alone for 48 hours; b is a transmission electron microscope image of 10 mu M A beta 42 and 40 mu M WRR incubated together for 48 hours; transmission electron microscopy images of 10. Mu. M A β 42 and 40. Mu.M ERW incubated together for 48 hours.
In fig. 6, ethidium Bromide (EB) -stained dead cells showed red fluorescence. A is a blank control; b is transfected Abeta; c is non-transfected +50μM WRR; d is WRR transfected +10μm; e is WRR transfected +50. Mu.M; f is ERW of non-transfected +50. Mu.M; g is ERW transfected +10μm; h is ERW transfected +50μm; i is a quantitative analysis plot of the above observations (counted using Image J tool). * P <0.05, P <0.01, single factor analysis of variance (ANOVA) with aβ 42 group (aβ), modified-Tukey test was used for multiple comparisons between groups, n=6.
In fig. 7, a is a blank control; b is transfected Abeta; c is non-transfected +50μM WRR; d is WRR transfected +10μm; e is WRR transfected +50. Mu.M; f is ERW of non-transfected +50. Mu.M; g is ERW transfected +10μm; h is ERW transfected +50μm; i is a quantitative analysis chart of the above observation results. * P <0.05, P <0.01, single factor analysis of variance (ANOVA) with aβ42 group (aβ) using Modified-Tukey test for multiple comparisons between groups, n=3.
In FIG. 8, the effect A and H of two tripeptides on Reactive Oxygen Species (ROS) production by SH-SY5Y cells secreting Abeta 42 is examined as a blank for flow cytometry; b is non-transfected +50μM WRR; c is transfected Abeta; d is WRR transfected +10μm; e is WRR transfected +50. Mu.M; f is the overlap of A-E; i is non-transfected +50μm ERW; j is transfected Abeta; k is ERW transfected +10μm; l is ERW transfected +50μm; m is H-L overlapping; g and N are quantitative analysis graphs of experimental results of the WRR group and the ERW group. Results are expressed as mean ± SEM. * P <0.01, using one-way analysis of variance (ANOVA), multiple comparisons using Modified-Tukey test, n=3.
In fig. 9, P <0.01, compared to aβ 42; $ P <0.05, compared to Abeta 42 -WRR; # P <0.05, compared to Abeta 42 -EWR; one-way analysis of variance (ANOVA), modified-Tukey test was used for multiple comparisons between groups, n=5, and repeated three times.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements or changes may be made without departing from the spirit and principles of the present invention.
Claims (6)
1. Tripeptide compound, characterized in that it comprises WRR;
Application of tripeptide compound with WRR as amino acid sequence in preparing medicine for preventing and treating Alzheimer disease.
2. The tripeptide compound of claim 1, wherein the WRR is tryptophan-arginine.
3. The tripeptide compound of claim 1, wherein said tripeptide compound is used for inhibiting aβ 42 aggregation.
4. The tripeptide compound of claim 1, wherein said tripeptide compound is used for reducing aβ 42 cytotoxicity.
5. The method for producing a tripeptide compound according to claim 1, comprising the steps of:
step one: writing CHEMSCRIPT scripts by using Python language, and creating a structural database of all tripeptides consisting of 20L-alpha amino acids;
Step two: screening from a protein database to obtain a protein molecular structure of five-membered oligomer Abeta 42 as a target protein conformation;
step three: taking penta oligomer Abeta 42 as target protein, and utilizing molecular docking software and a constructed tripeptide structure database to carry out molecular docking screening to obtain tripeptides with optimal docking states;
Step four: analyzing the binding free energy of the 5 tripeptides screened by butt joint and the pentad polymerization state Abeta 42 by utilizing GROMACS molecular dynamics software;
Step five: pharmacological experiments 5 tripeptide mixtures were analyzed to inhibit aβ 42 aggregation and to reduce the cytotoxic effects caused by aβ 42.
6. The use of a tripeptide compound according to claims 1-5 for the preparation of a medicament.
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